Application of visceral fat-derived UCHL5 as myocardial fibrosis biomarker and drug target

Abstract

The invention discloses application of visceral fat-derived UCHL5 as a myocardial fibrosis biomarker and a drug target, and belongs to the technical field of cardiovascular disease detection, prevention and treatment. From the perspective of fat-cardiac axis, it is proved for the first time that deubiquitinating enzyme UCHL5 from visceral fat (VAT) and entering circulation is a key promoting factor of angiotensin II (Ang II) related myocardial fibrosis, and it is proved that UCHL5 can be used as a body fluid biomarker for monitoring myocardial fibrosis and also is an anti-fibrosis target with medicinal properties. On the basis, UCHL5 can be applied to diagnosis, prevention and treatment of myocardial fibrosis as a biomarker and a drug target of myocardial fibrosis.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of cardiovascular disease detection and prevention, and particularly relates to application of UCHL5 from visceral adipose tissue as a biomarker and drug target for myocardial fibrosis. BACKGROUND

[0002] Myocardial fibrosis runs through the occurrence and development of multiple types of cardiovascular diseases. Its pathological core is the activation of fibroblasts and the phenotypic conversion of myofibroblasts, which leads to excessive deposition of extracellular matrix (ECM) such as type I and type III collagen, and further causes matrix network remodeling, decreased myocardial compliance, impaired diastolic-systolic coupling, and electrical-mechanical remodeling, accelerating the evolution of heart failure. The early stage of fibrosis is mostly occult, but is closely related to the poor prognosis of HFpEF and HFrEF. Previous studies have shown that the renin-angiotensin-aldosterone system (RAAS), the TGF-β / Smad signaling pathway, inflammation and oxidative stress, mechanical stretching, endothelial dysfunction, and metabolic abnormalities / lipotoxicity, and other pathways cooperatively shape the fibrosis microenvironment. However, most of these pathways focus on the local effects of myocardium, and the understanding of how the upstream and distal tissues continuously drive fibrosis is still incomplete.

[0003] With the concept of "heart-fat axis", adipose tissue is considered to remotely regulate myocardial interstitial homeostasis through the secretion of multiple types of signaling molecules (adipokines, free fatty acids, inflammatory mediators, exosomal nucleic acids and proteins, matrix regulatory molecules, etc.). Compared with subcutaneous fat and epicardial fat, visceral adipose tissue (VAT) has higher metabolic and inflammatory activity, can release more factors related to pro-inflammatory and ECM remodeling, and is closely related to systemic insulin resistance, endothelial dysfunction, and myocardial structural remodeling. Imaging and population studies have shown that VAT increment is positively correlated with impaired cardiac diastolic function, increased myocardial stiffness, and diffuse fibrosis indicators of cardiac magnetic resonance (CMR). However, starting from "specific VAT secreted molecules", the closed-loop evidence of linking their delivery through circulation or exosomes, activating pro-fibrotic signals at the level of myocardial fibroblasts, and driving quantitative fibrosis load and functional outcome deterioration has not been established.

[0004] In the clinical evaluation level, the current evaluation of myocardial fibrosis mainly relies on CMR (such as LGE, T1, ECV), ultrasound structure and function parameters, and serum fibrosis markers (collagen precursor fragments, MMP / TIMP ratio, Galectin-3, sST2, etc.). But the above means still have deficiencies in cost and accessibility, organ source specificity, sensitivity to early activation, and tolerance to inflammation or renal function interference. The current heart failure or remodeling management drugs are mostly indirect inhibition of fibrosis; and the direct inhibition of TGF-β, broad-spectrum MMP, CTGF and other pathways is limited by selectivity, safety window and potential inhibition of physiological repair, which is difficult to meet the long-term precise myocardial fibrosis control requirements. Further, existing research has not provided: a complete causal evidence chain starting from VAT-specific secreted factors that can be repeatedly verified; source-specific markers that can distinguish "visceral fat-driven" from other etiological myocardial fibrosis; a stratification system integrating source differences, fibrosis activity and prognosis risk; candidate targets with etiology regulation, high specificity and safety; and "diagnosis-treatment integrated" molecules and detection tools that can support disease stratification, efficacy monitoring and intervention decision-making simultaneously. At the same time, there is also a lack of multi-center, prospective framework to verify the independent contribution of VAT-specific secreted molecules to myocardial fibrosis progression and clinical outcomes.

[0005] Therefore, the current technical gap is mainly reflected in that: no drugable molecule with visceral fat source indication, intervention reversibility, stable quantification in body fluid, and direct regulation of key links of myocardial fibrosis has been identified. The development of such molecules has an urgent need to achieve precise risk identification, individualized intervention and dynamic efficacy evaluation. SUMMARY

[0006] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide the application of visceral fat-derived UCHL5 as a biomarker and drug target for myocardial fibrosis, aiming to solve the technical problem that the existing myocardial fibrosis-related diseases lack "source-specific, intervention, and monitoring" molecular targets in risk assessment, stratification and intervention.

[0007] The technical solution of the present application is as follows: In a first aspect, the application provides the use of a reagent for detecting the expression level of UCHL5 in the preparation of a product for diagnosing myocardial fibrosis.

[0008] In a second aspect, the application provides the use of a reagent for inhibiting the expression level of UCHL5 in the preparation of a drug for preventing myocardial fibrosis.

[0009] In a third aspect, the application provides the use of a reagent for inhibiting the expression level of UCHL5 in the preparation of a drug for treating myocardial fibrosis.

[0010] Optionally, the agent for inhibiting the expression level of UCHL5 is a small molecule, a nucleic acid, a gene editing system, a protein, or a polypeptide.

[0011] Optionally, the agent for inhibiting the expression level of UCHL5 is b-AP15.

[0012] In a fourth aspect, a kit for diagnosing myocardial fibrosis is provided, the kit comprising: an agent for detecting the expression level of UCHL5.

[0013] Optionally, the kit detects UCHL5 in plasma, serum, visceral adipose tissue, or exosomes.

[0014] In a fifth aspect, a drug for preventing myocardial fibrosis is provided, the drug comprising: an agent for inhibiting the expression level of UCHL5.

[0015] In a sixth aspect, a drug for treating myocardial fibrosis is provided, the drug comprising: an agent for inhibiting the expression level of UCHL5.

[0016] Optionally, the drug further comprises one or more of an angiotensin-converting enzyme inhibitor, an angiotensin II receptor blocker, an angiotensin receptor-neprilysin inhibitor, a beta-adrenergic receptor blocker, a mineralocorticoid receptor antagonist, and a sodium-glucose co-transporter 2 inhibitor.

[0017] Compared with the prior art, the present application has the following advantages: (1) Source specificity: for the first time, UCHL5 secreted by adipocytes in visceral fat is defined as a core driving node for trans-tissue promotion of myocardial fibrosis, improving the ability to analyze the source of fibrosis.

[0018] (2) Integration of diagnosis and treatment: UCHL5 has both etiological participation and quantifiable characteristics in plasma, shortening the link between "mechanism discovery" and "clinical application".

[0019] (3) Multi-level intervention window: UCHL5 can be selected from multiple nodes, including production (expression / secretion), circulation, receptor binding blockage, and downstream signal regulation, reducing single-point tolerance and escape.

[0020] (4) Closed loop verification chain: visceral fat resection, exogenous UCHL5 supplementation, and Ang II model multi-directional mutual verification strengthen the reliability of causal inference and extrapolation.

[0021] (5) Risk advance: UCHL5 elevation can indicate the trend of myocardial fibrosis-related adverse remodeling before significant structural fixation (such as extensive collagen deposition or significant decrease in cardiac ejection fraction EF), which is beneficial for early strengthening of treatment.

[0022] (6) Multi-scenario detection adaptation: UCHL5 detection can adopt point-of-care rapid chromatography (primary screening), routine laboratory ELISA / chemiluminescence (batch), high-sensitivity digital immunoassay or multi-index microfluidic (precise stratification) to adapt to hierarchical medical care.

[0023] (7) Screening efficiency improvement: Fat secretion model + fibroblast activation model dual module combination, shortening the drug lead screening and mechanism confirmation cycle.

[0024] (8) Safety potential: Visceral fat targeted delivery strategy is expected to reduce adverse effects on systemic matrix homeostasis and wound repair.

[0025] (9) Precise stratification capability: UCHL5 dynamic level change (ΔUCHL5) combined with imaging / function parameters to construct a composite score can improve the prediction accuracy and incremental value of adverse cardiac remodeling and MACE.

[0026] (10) Synergistic effect and burden reduction: Inhibitors of UCHL5 combined with standard heart failure / hypertension / metabolic management drugs may achieve dose reduction or efficacy enhancement, enriching individualized combination strategies. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the experimental result of Example 1, showing that removal of visceral fat improves Ang II-induced cardiac function impairment and myocardial fibrosis.

[0028] Figure 2 is the experimental result of Example 2, which screened UCHL5 as a candidate adipokine for Ang II-induced myocardial fibrosis through combined analysis of VAT secretome and myocardial tissue proteomics.

[0029] Figure 3 is the experimental result of Example 3, showing that Ang II induces UCHL5 elevation in the VAT-heart axis and primary adipocytes, and VAT removal reduces UCHL5 accumulation in the circulation and heart.

[0030] Figure 4 is the experimental result of Example 4, showing that UCHL5 recombinant protein induces cardiac function impairment and myocardial fibrosis, and UCHL5 inhibitor b-AP15 reduces this pathological damage.

[0031] Figure 5 is the experimental result of Example 5, showing that plasma UCHL5 levels are elevated in hypertensive patients, UCHL5 has discriminative value and is significantly correlated with Ang II and myocardial fibrosis markers. DETAILED DESCRIPTION

[0032] The application relates to application of UCHL5 from visceral fat as a myocardial fibrosis biomarker and a drug target.

[0033] The application defines, from the perspective of a fat-heart axis, deubiquitinase UCHL5 from visceral adipose tissue and entering circulation as an active promoting factor of angiotensin II (Ang II) related myocardial fibrosis and adverse ventricular remodeling, instead of a simple passive damage marker. The following causal chain is constructed and demonstrated: Ang II stimulation → up-regulation of UCHL5 synthesis and secretion in VAT → increase of circulating UCHL5 → activation of fibrosis-promoting signals of myocardial fibroblasts and ECM remodeling → deterioration of myocardial structure and function. The pathogenicity and interventional property are supported by experiments of bidirectional intervention of inhibition / excision at the VAT level and exogenous UCHL5 supplementation. Compared with traditional NT-proBNP, Gal-3, sST2, PICP and other end or passive reflection indicators, UCHL5 provides an upstream promoting node that can be used for early risk identification and targeted inhibition. At the current data level, the application completes the correlation analysis of baseline (single time point) blood UCHL5 quantification and myocardial structure / function, fibrosis related indicators, simultaneously introduces a multi-marker panel combined with Ang II, sST2, PICP and the like and a derived ratio (for example, UCHL5 / Ang II) to improve the baseline risk discrimination ability. The longitudinal change of UCHL5 can be investigated in a prospective follow-up framework for efficacy evaluation and early progress warning (this part is a proposed implementation content and does not constitute the verified conclusion of this time). In terms of treatment concept, the application proposes a UCHL5 inhibition strategy and plans multiple inhibition forms (small molecules, antibodies, nucleic acids, PROTAC and the like) to construct a potential transformation path. It is also proposed that a software / model can be developed to predict the risk of adverse remodeling by combining baseline UCHL5 with other markers and image parameters.

[0034] The application provides the following contents: (1) UCHL5 signal axis from VAT is used as a drug target for preparing a drug for preventing or treating myocardial fibrosis and related diseases (adverse ventricular remodeling, heart failure (including HFrEF, HFmrEF, HFpEF), early myocardial remodeling caused by hypertension, metabolism related (obesity / metabolic syndrome caused) myocardial remodeling); (2) UCHL5 level and dynamic change (Delta UCHL5) thereof are used as a body fluid biomarker for identifying the risk of adverse remodeling, patient stratification, prognosis (including MACE risk) prediction and efficacy monitoring; (3) Single-index UCHL5 or multi-index (UCHL5 combined with Ang II, TGF-β1, sST2, PICP, ± NT-proBNP, etc.) integrated detection reagent / kit and risk score platform; (4) Prognosis, risk, and efficacy response determination algorithm: including UCHL5 threshold (such as quantile or ROC determination), ΔUCHL5 decline amplitude (such as ≥15-20%) combined with comprehensive determination of imaging / functional parameters (LVEF, GLS, E / E', etc.).

[0035] (5) Inhibitors / antagonists / modulators (antibodies, aptamers, nucleic acid drugs, small molecules / peptide segments, ligand traps / fusion proteins, etc.) targeting the UCHL5 signaling axis and pharmaceutical compositions thereof; (6) Animal and cell function verification and drug screening system: Ang II infusion + VAT resection model, exogenous recombinant UCHL5 administration model.

[0036] The core purpose of the present application is: (1) To reveal the cross-tissue fibrosis driving chain of "VAT adipocytes → UCHL5 → myocardial fibroblasts"; (2) To prove that targeted inhibition of VAT-derived UCHL5 can reduce or reverse Ang II-induced myocardial collagen deposition and systolic / diastolic function deterioration; (3) To build a continuous transformation path from mechanism discovery to function verification to clinical population detection to companion diagnosis to intervention strategy; (4) To construct a forward risk stratification and individualized intervention framework based on UCHL5, and to improve the early prediction ability of adverse cardiac remodeling and cardiovascular events.

[0037] The technical solutions of the present application include: (1) Target and use VAT and its derived UCHL5 signaling axis as a drug target for preventing or treating adverse cardiac remodeling diseases related to myocardial fibrosis (including hypertensive early myocardial remodeling, metabolic / obesity-related myocardial remodeling, and various phenotypes of heart failure). The signaling axis includes: upregulation and secretion of UCHL5 in VAT adipocytes, the existence form of UCHL5 in circulation (free, vesicle / exosome encapsulation, etc.), and its functional interaction with myocardial fibroblast membrane receptors or co-receptors (integrin family subunits, CD36, etc.) and potential TGF-β activation complexes.

[0038] (2) Inhibitor / antagonist / modulator categories (i) Antibodies: neutralizing antibodies, humanized or fully human monoclonal antibodies, bispecific antibodies (simultaneously binding UCHL5 and integrin specific sites, etc.) against UCHL5.

[0039] (ii) Nucleic acids: siRNA, shRNA, antisense oligonucleotide (ASO), CRISPRi system targeting UCHL5 mRNA, preferably enhanced VAT-targeted delivery by lipid nanoparticles (LNP), polymeric nanocarriers, or engineered exosomes.

[0040] (iii) Small molecules / peptides: cyclic peptides, peptidomimetics or structure-optimized small molecules that block the UCHL5-receptor binding interface (occupy hydrophobic / aromatic pockets), and polypeptide-PEG modified long-acting versions.

[0041] (iv) Ligand trap / fusion proteins: protein domains containing receptor binding regions or functional binding sites fused to immunoglobulin Fc as ligand trap / fusion proteins that circulate to "capture" or neutralize UCHL5.

[0042] (v) Aptamers: high-affinity oligonucleotide aptamers chemically modified with 2'-F, 2'-OMe, LNA, phosphorothioation (PS), or cyclization that bind to key functional surfaces of UCHL5.

[0043] (vi) Combination strategies: any of the above categories combined with standard-of-care treatments (ACEI, ARB, ARNI, beta blockers, MRA, SGLT2 inhibitors, etc.), or multiple UCHL5 inhibitory modalities stacked (e.g., siRNA + ligand trap).

[0044] (3) Pharmaceutical compositions and dosage forms / administration (i) Pharmaceutical compositions contain effective amounts of inhibitors / modulators and pharmaceutically acceptable carriers (buffers, fillers / excipients, surfactants, stabilizers, preservatives, cryoprotectants, etc.).

[0045] (ii) Dosage forms: injectable solutions, lyophilized powders, sustained / controlled release formulations, liposomes, polymeric or inorganic nanoparticles, microspheres, transdermal patches, oral solid formulations (for small molecules or specific stable peptide segments), inhalation or mucosal delivery formulations, implantable sustained release formulations.

[0046] (iii) Routes of administration: intravenous, subcutaneous, oral, VAT-directed (e.g., nanoparticles modified with adipose tissue-homing ligands / peptides), local delivery to pericardial cavity, synchronized delivery with catheter interventions or surgical procedures (e.g., vagal nerve modulation / ablation procedures).

[0047] (4) Biomarkers and diagnostics / monitoring (i) Biomarker: Plasma / Serum UCHL5 concentration; AUCHL5 = (Follow-up value - Baseline value) / Baseline value x 100%; can be combined with Ang II, TGF-β1, sST2, PICP, NT-proBNP, LVEF, GLS, E / E', etc. to form a composite remodeling risk index.

[0048] (ii) Use: Identify high-risk early remodeling individuals; adverse remodeling progression prediction; re-stratification of heart failure patients and MACE risk assessment; companion diagnosis and enrollment screening (clinical trials); efficacy and biological response monitoring.

[0049] (5) Detection reagent / kit (i) Form: ELISA, chemiluminescence immunoassay, immunochromatography (bedside rapid), digital single molecule immunoassay (SiMoA), electrochemical aptamer chip, multi-index microfluidic chip, mass spectrometry PRM / MRM quantification.

[0050] (ii) Composition: Capture antibody or aptamer, detection antibody / aptamer (labeled HRP, ALP, fluorescence or electrochemical tag), recombinant UCHL5 standard, high / medium / low quality control, diluent, washing solution, blocking solution, color developing / light emitting substrate, instruction manual; optionally containing multi-index pre-coated plate or chip.

[0051] (iii) Calibration and algorithm module: standard curve fitting software / algorithm, supporting AUCHL5 calculation and risk stratification output.

[0052] (5) Drug and inhibitor screening / evaluation method (i) In vitro adipocyte secretion model: after primary or differentiated mature adipocytes are treated with Ang II or pro-inflammatory / metabolic stimulation, the candidate compound is tested for its inhibition rate of UCHL5 secretion.

[0053] (ii) Fibroblast activation model: recombinant UCHL5 (rhUCHL5) induces upregulation of ACTA2, COL1A1, FN1, POSTN in cardiac fibroblasts; after adding candidate inhibitors, the inhibition rate of the above indicators is detected (qPCR) / protein (Western / IF).

[0054] (6) Animal models: (i) Ang II osmotic pump infusion ± VAT resection; (ii) Exogenous rhUCHL5 intravenous administration at different doses; (iii) Combined with hypertension, heart failure, etc. background treatment, to evaluate the synergistic or dose reduction effect.

[0055] (7) Prognostic risk and efficacy response prediction (i) By determining the baseline and follow-up (e.g. 3, 6, 12 months) plasma UCHL5 and combining clinical and imaging parameters, using statistical (Cox, LASSO) or machine learning models (random forest, gradient boosting, penalized regression) to output adverse remodeling or MACE risk grades, to evaluate incremental value (C-statistics, NRI, IDI).

[0056] (ii) Recheck 1-6 months after UCHL5 targeted intervention or comprehensive management: if ΔUCHL5 decreases and is accompanied by improvement in LVEF or GLS, decrease in E / E', or reduction in left ventricular mass index, it is determined as an early biological response for treatment adjustment and stratified optimization.

[0057] The specific scheme of the present application can be as follows: The embodiment of the present application provides application of a reagent for detecting UCHL5 expression level in preparation of a product for diagnosing myocardial fibrosis. Wherein, the product for diagnosing myocardial fibrosis detects the expression level of UCHL5 from visceral fat.

[0058] The embodiment of the present application provides application of a reagent for inhibiting UCHL5 expression level in preparation of a medicine for preventing myocardial fibrosis. Wherein, the medicine for preventing myocardial fibrosis inhibits the expression level of UCHL5 from visceral fat.

[0059] The embodiment of the present application provides application of a reagent for inhibiting UCHL5 expression level in preparation of a medicine for treating myocardial fibrosis. Wherein, the medicine for preventing myocardial fibrosis inhibits the expression level of UCHL5 from visceral fat.

[0060] In some embodiments, the reagent for inhibiting UCHL5 expression level is a small molecule, a nucleic acid, a gene editing system, a protein or a polypeptide.

[0061] In some embodiments, the reagent for inhibiting UCHL5 expression level is b-AP15.

[0062] The embodiment of the present application provides a kit for diagnosing myocardial fibrosis, the kit comprising: a reagent for detecting UCHL5 expression level.

[0063] In some embodiments, the detection object of the kit comprises UCHL5 in plasma, serum, visceral fat tissue or exosome.

[0064] The embodiment of the present application provides a medicine for preventing myocardial fibrosis, the medicine comprising: a reagent for inhibiting UCHL5 expression level.

[0065] The embodiment of the present application provides a medicine for treating myocardial fibrosis, the medicine comprising: a reagent for inhibiting UCHL5 expression level.

[0066] In some embodiments, the drug further comprises one or more of an angiotensin-converting enzyme inhibitor (ACEI), an angiotensin II receptor blocker (ARB), an angiotensin receptor-neprilysin inhibitor (ARNI), a beta-adrenergic receptor blocker, a mineralocorticoid receptor (MRA) antagonist, and a sodium-glucose cotransporter 2 (SGLT2) inhibitor.

[0067] The myocardial fibrosis described in the above examples is caused by an increase in Ang II, which is an Ang II-induced impairment of cardiac (contractile) function, which can lead to hypertensive heart failure.

[0068] The application is further described below through specific examples.

[0069] Example 1: VAT ectomy (VAT-E) improves Ang II-induced cardiac dysfunction and myocardial fibrosis.

[0070] This example establishes a model of myocardial remodeling caused by continuous angiotensin II (Ang II) infusion. The results show that, compared with the control group (Control), the left ventricular ejection fraction (EF) and fractional shortening (FS) of the Ang II group decreased, the end-diastolic / end-systolic internal diameter (LVIDd / LVIDs) significantly increased, and the interstitial collagen deposition increased. In the Ang II+VAT-E group, which was subjected to VAT (peritesticular fat of mice) removal at the same period, the above-mentioned functional and structural abnormalities were significantly improved (P<0.05 or P<0.01), indicating that VAT participates in the process of Ang II-induced myocardial fibrosis, and removal of VAT has a protective effect.

[0071] The specific implementation process is as follows: (1) Animals and grouping: 24 6-8-week-old C57BL / 6J male mice were randomly divided into Control, Ang II, and Ang II+VAT-E groups (n=8 / group).

[0072] (2) Continuous Ang II infusion: Under 3% (v / v) isoflurane inhalation anesthesia, ALZET 1004 osmotic pumps (DURECT) were implanted subcutaneously on the back to release Ang II (2 mg / kg / day) continuously for 4 weeks; the control group was implanted with empty pumps.

[0073] (3) VAT removal: In the Ang II+VAT-E group, during the same anesthesia process as pump implantation, a 1 cm incision was made in the midline of the abdomen, and the peritesticular white fat pad was bluntly separated and removed in its entirety, with care taken to protect the blood supply of the testis; the Ang II group was subjected to sham operation (after the abdominal midline incision was made, the tissue was returned without removing the VAT).

[0074] (4) Echocardiography: On day 28, short-axis M-mode images were acquired at the level of the papillary muscles using a Vevo 2100 imaging system under 1.0-1.5% (v / v) isoflurane anesthesia to measure EF, FS, LVIDd, and LVIDs, and the average of three consecutive heartbeats was calculated for each animal.

[0075] (5) Tissue collection and histology: After the heart was removed, it was flushed with PBS, and the left ventricle was transversely sectioned and fixed in 4% (w / v) paraformaldehyde overnight, paraffin-embedded, and prepared into 5-μm serial sections. Masson and Sirius Red staining were performed according to the kit instructions, and the threshold segmentation and calculation of the fibrosis area percentage (the average of ≥3 fields for each animal) were performed using ImageJ.

[0076] (6) Statistical analysis: The data are expressed as x ± SEM, and one-way ANOVA + Tukey post-hoc test was used for inter-group comparisons; P < 0.05 was considered statistically significant.

[0077] Specific results are shown in Figure 1 , indicating that the removal of visceral fat improves Ang II-induced impairment of cardiac function and myocardial fibrosis. Figure 1 Figure 1A is a schematic diagram of the experimental procedure: the Control group was implanted with an empty pump; the Ang II group and the Ang II+VAT-E group were continuously infused with Ang II subcutaneously for 4 weeks using a pump; the Ang II+VAT-E group was synchronously removed of the perirenal fat pad on the day of infusion; serum and hearts were collected on day 28. Figure 1 Figures 1B-E are the results of ultrasonic quantification, showing that Ang II reduces EF and FS and increases LVIDd and LVIDs, and VAT-E improves systolic function and reduces chamber / volume dilation, thereby significantly reversing the effects of Ang II; specifically, Figure 1 Figure 1B is the result of EF, Figure 1C is the result of FS, Figure 1D is the result of LVIDd, and Figure 1E is the result of LVIDs. Figure 1F is the result of Masson trichrome staining and quantification, showing that Ang II increases interstitial collagen (blue / cyan), and VAT-E reduces the percentage of fibrosis. Figure 1G is the result of Sirius Red staining and quantification, showing that Ang II increases collagen deposition (red), and VAT-E significantly reduces the deposition.

[0078] Example 2: UCHL5 is screened as a candidate adipokine for Ang II-induced myocardial fibrosis by combining VAT secretome proteomics and myocardial tissue proteomics.

[0079] The present embodiment will be short-term in vitro culture of visceral adipose tissue (VAT) of control group and Ang II group mice, collect the secretome for secretome detection; at the same time, the whole proteomics analysis of heart tissue of two groups. The results show that, compared with the control group, Ang II group appears a large number of differential proteins in VAT secretion and myocardial tissue; these differential proteins are significantly enriched in fibrosis related extracellular matrix (ECM) remodeling pathway in two types of group; further integration and protein interaction network analysis of differential proteins enriched in fibrosis / ECM remodeling pathway in two groups, found that deubiquitination related protein UCHL5 is located in the core position of the cross network, so it is determined as the preferred verification of candidate myocardial fibrosis adipose-derived factors.

[0080] The specific implementation process is as follows: (1) Sample acquisition: according to embodiment 1, after the end of animal treatment at the 4th week, the peritesticular VAT and left ventricular tissue of control group and Ang II group mice were rapidly stripped under sterile conditions. VAT was gently washed with ice-cold physiological saline to remove blood; myocardial tissue was quickly frozen in liquid nitrogen for testing. The whole process was kept at low temperature to reduce protein degradation.

[0081] (2) VAT collagenase digestion and short-term in vitro culture (secretome preparation): cut the VAT into about 1mm 3 small pieces, use 1mg / mL Collagenase I to digest at 37℃, 160rpm, gently for 30min; 300g centrifugation for 5min to remove supernatant, PBS washing 2 times. The tissue was evenly distributed in 6-well plate according to the wet weight of about 100mg per hole, and the basic DMEM (containing 2% (v / v) exosome removal treated FBS) was added, 37℃, 5% (v / v) CO2 culture for 48h, and the supernatant was collected for secretory protein analysis.

[0082] (3) Conditioned medium (CM) treatment: culture supernatant was centrifuged at 300g for 5min to remove cell debris, filtered through 0.22μm filter membrane; 10kDa molecular weight cutoff centrifugal concentration was used; BCA method was used to quantify total protein. The obtained secretory protein was used for subsequent trypsin digestion and quantitative mass spectrometry analysis.

[0083] (4) Myocardial tissue protein extraction and enzymolysis (tissue proteomics preparation): Take the left ventricular tissue and grind it in liquid nitrogen, add lysis buffer (8M urea, 50mM Tris-HCl, pH 8.0, containing protease and phosphatase inhibitors), and perform ice bath ultrasonic lysis; centrifuge at 14000g, 4°C for 10 min to take the supernatant, and then perform BCA quantification. After reduction (5mM DTT, 56°C, 30min) and alkylation (15mM IAA, avoid light, room temperature for 30min), perform trypsin digestion (enzyme: substrate = 1:50, w / w) at 37°C overnight (if necessary, add 1:100 for 2h of additional digestion).

[0084] (5) TMT labeling and LC-MS / MS: Perform TMT labeling on VAT secretomics and myocardial tissue proteomics respectively (randomly assign labels to ensure balance between groups; if multiple TMT batches are involved, set up a mixed internal standard channel). After labeling, mix and perform high-pH reversed-phase fractionation, and use nanoscale reversed-phase liquid chromatography combined with high-resolution mass spectrometry for acquisition. Typical parameters: MS1 resolution ≥ 60000; dynamic exclusion 30-45s; each cycle contains ≥8-10 MS / MS.

[0085] (6) Data analysis and difference determination: Use a mainstream search engine to search the mouse database (containing common contaminants), and set: trypsin specificity, missed cut ≤2; fixed modification: Carbamidomethyl (C), TMT (N-terminal and K); variable modification: oxidation (M), protein N-terminal acetylation. Peptide and protein FDR are both <1%. Only keep (i) at least 2 unique peptides, (ii) proteins that are quantified in ≥70% samples. Perform statistics on the reporter ion intensity after total ion or median normalization: use two-tailed t-test or linear model and perform Benjamini-Hochberg correction for two-group comparison. Difference threshold: |log2 FoldChange| ≥ 1.2 and FDR < 0.05. Display the difference distribution in a volcano plot.

[0086] (7) Functional enrichment and interaction network integration: (i) Perform GO (CC / BP), KEGG, etc. enrichment analysis on VAT up-regulated (secreted) proteins and myocardial differential proteins respectively, and screen FDR <0.05 pathways.

[0087] (ii) Predefine a set of “fibrosis-related pathways” (including ECM remodeling, collagen biosynthesis / crosslinking, matrix receptor interaction, MAPK signaling, etc.), and extract the corresponding differential protein set.

[0088] (iii) The VAT fibrosis-related upregulated secretome proteins were combined with the cardiac fibrosis-related differential proteins (and labeled by source), and high-confidence interactions (score≥0.7) were obtained from STRING, and the topological parameters such as Degree, Betweenness, etc. were calculated in Cytoscape.

[0089] (iv) UCHL5 showed high connectivity and betweenness in the network, and was a key node of VAT upregulation and protein homeostasis / deubiquitination and ECM-related pathways, located in the core position of the comprehensive network.

[0090] As shown in Figure 2 , UCHL5 was screened as a candidate adipokine for Ang II-induced myocardial fibrosis by joint analysis of VAT secretome and myocardial proteome. Figure 2 Figure 2A is a VAT secretome volcano plot showing the quantification results of the control group (Control) and Ang II group mouse peritesticular VAT short-term in vitro adherent culture (48h) after enzymolysis of the conditioned medium (secretome); the significantly different secreted proteins (statistical threshold: |log2 Fold Change|≥1.2, FDR<0.05) are colored up / down. Figure 2 Figure 2B is the functional / pathway enrichment analysis result of VAT secreted differential proteins, and the fibrosis and ECM remodeling related pathways are significantly enriched and marked in red. Figure 2 Figure 2C is a heart tissue overall proteome volcano plot showing the distribution of differential proteins in the left ventricular tissue of the control group (Control) and Ang II group mice. Figure 2 Figure 2D is a myocardial differential protein functional / pathway enrichment analysis showing that the fibrosis and extracellular matrix remodeling related pathways are also significantly enriched (marked in red). Figure 2 Figure 2E is a high-confidence protein interaction network (STRING, example threshold score≥0.7) constructed by integrating the differential proteins significantly enriched in fibrosis / ECM remodeling related pathways in the two types of omics; the nodes are identified by source (VAT secretion / cardiac / intersection) and topological degree; UCHL5 is located in the core topological position of the network and is highlighted. Figure 2 Figure 2F is a mechanism schematic diagram showing that Ang II stimulates visceral adipose tissue, induces changes in its secretion profile and upregulates the secretion of UCHL5; UCHL5 can enter the circulation to reach the myocardial microenvironment, and may promote ECM deposition and myocardial fibrosis by regulating deubiquitination / protein homeostasis and fibrosis-related signaling pathways.

[0091] Example 3: Ang II induces UCHL5 elevation in VAT-cardiac axis and primary adipocytes; VAT ablation reduces circulating and cardiac UCHL5 accumulation.

[0092] This embodiment verifies the expression changes of UCHL5 from two levels of tissue and cells, and finds that Ang II treatment significantly increases the content of UCHL5 in VAT tissue, serum UCHL5 and myocardial tissue UCHL5 levels; and VAT resection (VAT-E) can significantly reduce the enrichment of UCHL5 in serum and heart. Moreover, the mRNA, intracellular protein and secretion of UCHL5 in primary adipocytes stimulated by Ang II are synchronously up-regulated, confirming that UCHL5 is a stress secretory factor from visceral adipose tissue.

[0093] The specific implementation process is as follows: (1) Tissue and serum: follow the animal treatment process of Example 1, collect VAT, serum and heart at the end; homogenate the tissue (PBS + protease inhibitor), determine the total protein and normalize it by μg / g wet weight.

[0094] (2) UCHL5 quantification: use commercial ELISA kit to detect UCHL5 in tissue homogenate or serum, and calculate the concentration according to the standard curve.

[0095] (3) Isolation of primary adipocytes: digest perirenal adipose tissue with collagenase I according to the method described in the methodological part, obtain adipocytes and inoculate them in 6-well plates; after the cells are moderately spread, synchronize them with serum-free basal medium for 4h.

[0096] (4) Stimulation treatment: add Ang II (final concentration 1 μM) and incubate for 24h; control group add equal volume of solvent (PBS).

[0097] (6) RNA extraction and qPCR: use Trizol to extract total RNA, after reverse transcription, use SYBR Mix for qPCR, normalize with internal reference gene GAPDH, and calculate the relative expression by 2^-ΔΔCt.

[0098] (6) Protein and secretion detection: collect cell lysate for Western blot (internal reference GAPDH); after removing debris by centrifuging the culture supernatant at 300g for 5min, use ELISA to detect UCHL5 content.

[0099] (7) Statistical analysis: t test is used for comparison between two groups; single factor ANOVA is used for comparison among multiple groups; P<0.05 is considered statistically significant.

[0100] The specific results are shown in Figure 3 Ang II induces the increase of UCHL5 levels in VAT-heart axis and primary adipocytes, and VAT resection reduces the accumulation of UCHL5 in circulation and heart. Figure 3 Figure A shows the content of UCHL5 in VAT. Figure 3Figure 3B shows the serum UCHL5 content. Figure 3 Figure 3C shows the UCHL5 content in heart tissue, suggesting that Ang II increases UCHL5 in the VAT-heart axis, and VAT resection reduces the extent of the increase. Figure 3 Figure 3D shows the mRNA level of UCHL5 in primary adipocytes (qPCR, relative expression). Figure 3 Figure 3E shows the protein level of UCHL5 in primary adipocytes (Western blot, relative quantification). Figure 3 Figure 3F shows the secretion level of UCHL5 in primary adipocyte culture supernatant (ELISA quantification).

[0101] Example 4: Recombinant UCHL5 (rhUCHL5) administration in vivo promotes myocardial fibrosis and worsens systolic function, and UCHL5 inhibitor (b-AP15) combination can partially reverse it.

[0102] Long-term intermittent intravenous injection of rhUCHL5 leads to a decrease in the ejection fraction (EF) and fractional shortening (FS) of mice, an increase in the left ventricular internal diameter at end-diastole and end-systole (LVIDd, LVIDs), and a significant increase in myocardial collagen deposition; administration of b-AP15 at the same time can significantly improve the above indicators, indicating that targeting UCHL5 can be used as an anti-fibrosis intervention strategy.

[0103] The specific implementation process is as follows: (1) Grouping and administration: 8-week-old C57BL / 6 male mice were randomly divided into 3 groups (n=8-10): PBS, rhUCHL5, rhUCHL5+b-AP15. rhUCHL5 was dissolved in sterile buffer (dose range 0.1-2 mg / kg, preferred dose 0.5 mg / kg in this example), and was injected intravenously every other day for a total of 14 times.

[0104] (2) Inhibitor: b-AP15 (UCHL5 / USP14 deubiquitinase inhibitor, CAS: 1009817-63-3) was dissolved in PBS containing 5% (v / v) DMSO, and was injected intraperitoneally 30 min after each rhUCHL5 injection (5 mg / kg, every other day). The control group and the rhUCHL5 group were given the same volume of solvent control of the same formula.

[0105] (3) Cardiac ultrasound evaluation: cardiac ultrasound was performed 24 h after the last administration (method same as in Example 1), and parameters such as EF, FS, LVIDd, LVIDs were obtained.

[0106] (4) Histology: after heart sampling and fixation, Masson and Sirius Red staining and fibrosis area quantification were performed (method same as in Example 1).

[0107] (5) Statistical analysis: One-way ANOVA + Tukey post-hoc test; P < 0.05 is significant.

[0108] (6) Safety observation: Body weight and general activity were recorded; no significant body weight loss (change < 5%) was observed at the dose of b-AP15.

[0109] Specific results are shown in Figure 4 that rhUCHL5 induced cardiac dysfunction and myocardial fibrosis, and that the UCHL5 inhibitor b-AP15 attenuated this pathological injury. Figure 4 Figure 2A is a schematic diagram of the treatment protocol: the control group was given PBS; the UCHL5 group was injected with rhUCHL5 via the tail vein every other day from day 0, for a total of 28 days of treatment; the UCHL5 + b-AP15 group was given b-AP15 in combination with rhUCHL5 at the same schedule; after the last treatment, cardiac ultrasound detection and tissue sampling were performed. Figure 4 Figures 2B-E are ultrasound quantification results showing that UCHL5 reduced EF, FS, and increased LVIDd and LVIDs; b-AP15 improved systolic dysfunction and reduced chamber / volume dilation; specifically, Figure 2B is the result of EF, Figure 2C is the result of FS, Figure 2D is the result of LVIDd, and Figure 2E is the result of LVIDs. Figure 4 Figure 2F is the result of Masson's trichrome staining and quantification, showing that rhUCHL5 increased interstitial collagen (blue / cyan), and that b-AP15 reduced the percentage of fibrosis. Figure 4 Figure 2G is the result of Sirius Red staining and quantification, showing that rhUCHL5 increased collagen deposition (red), and that b-AP15 significantly reduced this deposition.

[0110] Example 5: Elevated plasma UCHL5 levels in a clinical cohort, with discriminative ability to distinguish hypertensive / early structural remodeling individuals, and positively correlated with Ang II and myocardial fibrosis-related markers.

[0111] This example evaluated plasma UCHL5 levels in a hypertensive and / or early cardiac structural remodeling population, analyzed its correlation with vasoactive factors and myocardial fibrosis / stress markers, and verified its discriminative ability through ROC curves. The results showed that the plasma UCHL5 of the patient group was significantly higher than that of the healthy controls (P < 0.001); UCHL5 was positively correlated with Ang II, PICP, and sST2 at a moderate intensity (Spearman r about 0.55-0.61, all P < 0.001). ROC analysis showed that UCHL5 had good overall performance in distinguishing patients from healthy controls, with AUC = 0.763 (95% CI 0.667-0.861, p < 0.001), supporting its feasibility as an auxiliary screening or risk stratification indicator.

[0112] The implementation process is as follows: (1) Source of subjects: consecutive recruitment of outpatients or inpatients with hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg, or receiving antihypertensive treatment) and early cardiac structural remodeling (evidence of left ventricular hypertrophy or mild diastolic dysfunction imaging) in adult subjects n=60; at the same time, gender and age matched healthy controls n=40 were recruited. The evidence of early cardiac structural remodeling is defined as: LVMI > 115 g / m 2 or female > 95 g / m 2 , or RWT > 0.42; or mild diastolic dysfunction (E / A < 0.8 and average E / e' 8-14) and any of the following: LAVI > 34 mL / m 2 , TRV > 2.8 m / s, septal e' < 7 cm / s or lateral e' < 10 cm / s. Exclusion criteria: active inflammation, acute cardiovascular events (within 3 months), malignant tumors, severe liver and kidney dysfunction and other factors that may significantly affect inflammation or metabolic status.

[0113] (2) Specimen collection and processing: 5 mL of peripheral venous blood was collected in the morning on an empty stomach (EDTA anticoagulation tube), centrifuged at 3000 rpm for 10 min (4°C), the supernatant plasma was aliquoted and stored at -80°C for standby, avoiding freeze-thaw cycles.

[0114] (3) Detection method: UCHL5, Ang II, PICP, sST2 were detected by commercial ELISA kit and according to the instruction. Blank wells, standard product gradient, internal quality control wells were set; OD was read at 450 nm (reference wavelength 570 / 620 nm optional), four-parameter Logistic (4PL) model was used to fit the standard curve to calculate the concentration. Each sample was repeated ≥ 2 wells in parallel, and the coefficient of variation CV was controlled < 15%.

[0115] (4) Data preprocessing and statistical analysis: (i) Distribution test: normality test was performed on the original data (Shapiro-Wilk). If necessary, log or Box-Cox transformation was performed on the skewed distribution data to meet the assumptions of parametric test. (ii) Comparison between groups: independent sample t test was used when data were normally distributed and homoscedastic, otherwise Mann-Whitney U test was used. (iii) Correlation: Spearman rank correlation was used to evaluate the correlation between UCHL5 and Ang II, PICP, sST2. (iv) ROC analysis: AUC was calculated by Graphpad10; 95% confidence interval and significance (two-sided) were obtained by DeLong method. The optimal cutoff value was determined by Youden index (J = Sensitivity + Specificity - 1). At this cutoff value (97.04 ng / L), the sensitivity (86.67%) and specificity (65.63%) were recorded, and the UCHL5 concentration range of 0-97.04 ng / L was predicted to be free of early structural remodeling of the heart (i.e. no myocardial fibrosis), and the UCHL5 concentration > 97.04 ng / L was predicted to be the presence of early structural remodeling of the heart (i.e. myocardial fibrosis). (v) Significance criterion: statistical significance was determined by two-sided P < 0.05.

[0116] (5) Ethical statement: all subjects signed a written informed consent; the study was approved by the Ethics Committee of the University of Hong Kong-Shenzhen Hospital.

[0117] The specific results are shown in Figure 5 The plasma UCHL5 level in hypertensive patients was elevated and had a differential value, and was significantly correlated with Ang II and myocardial fibrosis markers. Figure 5 Figure 2A shows that the plasma UCHL5 level in hypertensive patients was significantly higher than that in healthy controls (***, p < 0.001). Figure 5 Figure 2B is the ROC curve of plasma UCHL5 in differentiating hypertensive patients from healthy controls (AUC = 0.763, 95% CI: 0.667-0.861, p < 0.001). Figure 5 Figure 2C shows that UCHL5 is positively correlated with plasma Ang II content (Spearman r = 0.572, p < 0.001). Figure 5 Figure 2D shows that UCHL5 is positively correlated with fibrosis marker PICP (Spearman r = 0.611, p < 0.001). Figure 5 Figure 2E shows that UCHL5 is positively correlated with fibrosis / myocardial stress marker sST2 (Spearman r = 0.547, p < 0.001).

[0118] In summary, the present application proposes a "visceral adipose tissue (VAT)-UCHL5-cardiac fibrosis" pathological axis and its diagnosis and treatment applications. We found that under angiotensin II (Ang II) stress, the expression and secretion of UCHL5 in mouse VAT are up-regulated, and drive the increase of UCHL5 in circulation and myocardium; VAT removal significantly reduces myocardial collagen deposition, myofibroblast activation and impaired systolic function. Intravenous injection of exogenous UCHL5 recombinant protein can independently induce left ventricular systolic dysfunction (EF, FS decreased, LVIDd, LVIDs expanded) and interstitial collagen deposition (Masson, Sirius Red enhanced), and synchronous administration of UCHL inhibitor b-AP15 can reverse / reduce the above functional and structural damage, proving that UCHL5 is a drugable pro-fibrotic effector molecule. Clinically, the plasma UCHL5 of hypertensive patients is elevated, and positively correlated with Ang II and fibrosis markers (PICP, sST2). Therefore, the present application provides: (1) VAT-derived UCHL5 (tissue, plasma, exosome level and its dynamics) as a biomarker for identifying, stratifying and monitoring VAT-driven myocardial fibrosis / reconstruction risk; (2) inhibitors targeting the VAT-UCHL5 axis (expression, activity, release and myocardial effect) (such as neutralizing antibodies, ligand / receptor interface blockers, siRNA / ASO, small molecule inhibitors, b-AP15 or its improved substances, etc.) for use in the preparation of drugs for preventing or treating myocardial fibrosis and improving cardiac function; (3) a quantitative detection kit / composition for serum / plasma, VAT tissue or exosome UCHL5. This strategy realizes the dual precision of pathogenic tissue + target molecule, and improves the efficacy and safety.

[0119] The applications and extensions of the present application include but are not limited to: (1) source expansion or replacement (i) Visceral fat refers to fat in the perirenal, abdominal cavity, mesentery and other positions, including white adipocytes and beige adipocytes.

[0120] (ii) Visceral fat sources include visceral fat and its stromal vascular fraction (SVF) or extracellular vesicles secreted by it.

[0121] (2) Target level downshift / upshift / lateral regulation (i) Upstream regulation: inhibit the transcription or inflammatory pathways (such as NF-κB, PPAR-related co-regulatory factors) that promote adipocyte UCHL5 expression.

[0122] (ii) Expression and processing: intervene in the transcription, mRNA stability, translation, splicing or deubiquitination active site conformation of UCHL5.

[0123] (iii) Secretion / Loading: Block exosome / vesicle loading, vesicle release, and extracellular stability.

[0124] (iv) Receptor / Binding: Block UCHL5 interaction with potential membrane receptors, co-factors, or ECM anchoring structures (even if specific receptors are to be further defined).

[0125] (v) Downstream signaling: Inhibit latent TGF-b activation module, Smad cascade, and fibrosis-related transcriptional programs.

[0126] (3) “Functionally equivalent molecule groups” or combination replacement (i) Construct a weighted index or multi-marker panel of UCHL5 with other adipose-secreted or ECM pro-fibrotic molecules (e.g. THBS1 / THBS4, Periostin, CTGF, Gal-3, MMP-2 / 9, PICP, ICTP).

[0127] (ii) Establish a “Fibrosis-Drive Index” =∑(each marker x weight); UCHL5 can be either a mandatory item or a panel core or trigger in certain embodiments.

[0128] (iii) Use ratio / derived indices: UCHL5 / Ang II, UCHL5 / sST2, (UCHL5 x Ang II) / TGF-b1, UCHL5 / Albumin normalization, etc.

[0129] (iv) Multi-modal fusion: Blood UCHL5 combined with imaging (ultrasound strain, MRI ECV, T1 mapping, CT fat density) and clinical metabolic parameters to constitute a fusion score.

[0130] (4) Delivery / intervention platform diversification (i) Vector classes: Lipid nanoparticles (LNP), polymeric nanoparticles, nanogels, AAV / AAV derivatives, non-viral DNA / RNA delivery systems, engineered exosomes, microsphere sustained release, degradable patches.

[0131] (ii) Targeting strategies: Adipose-specific promoters (AdipoQ, aP2), lipotropins / aptamers, receptor ligands (for adipose vascular endothelium or transporters), surface modifications (hydrophobic chains, glycosylation), or local injection (epicardial fat pad / peritoneal localization).

[0132] (iii) Intervention forms: Small molecule inhibitors, nucleic acid drugs (siRNA, ASO, shRNA, CRISPRi), neutralizing antibodies / nanobodies, aptamer blockade, PROTAC / molecular glue / LYTAC degradation, blocking peptides, competitive substrate decoys.

[0133] (iv) Indirect strategy: Induce remodeling of adipose metabolic phenotype (e.g. beta3 receptor agonism, cold stimulation mimicry) to secondarily reduce UCHL5 secretion propensity.

[0134] (5) Treatment positioning and administration modality reshaping (i) Systemic vs. local: Intravenous, subcutaneous, intraperitoneal, trans-catheter epicardial adipose region injection, local sustained release implantation.

[0135] (ii) Delivery time strategy: Single bolus + maintenance, pulsed, responsive (triggered based on subsequent monitoring metrics).

[0136] (iii) Combination regimen: Combination with RAAS inhibition, ARNI, SGLT2 inhibitor, beta blocker, MRA, anti-inflammatory (IL-1 beta / NLRP3 inhibition), anti-fibrotic small molecule (pirfenidone, etc.), metabolic modulation (GLP-1 receptor agonist, PPAR modulator).

[0137] (6) Inhibition mechanism or regulatory hierarchy reshaping (i) Expression inhibition: Transcriptional repression (CRISPRi, transcription factor regulation), mRNA cleavage (siRNA, ASO, RNase H mechanism).

[0138] (ii) Protein level: Active site occupancy, allosteric inhibition, E3 recruitment degradation (PROTAC), molecular glue facilitated degradation, extracellular neutralizing antibody.

[0139] (iii) Secretion / exosome regulation: Inhibition of vesicle biogenesis or blockade of exosome uptake.

[0140] (iv) Receptor / chaperone blockade: Aptamer, bispecific antibody, or functional peptide segment blockade of binding.

[0141] (v) Functional fragment competition: Design of truncated fragment / pseudosubstrate to block natural substrate deubiquitination.

[0142] (7) Detection and platform replacement (i) Immunological: ELISA, chemiluminescence, double antibody sandwich digitization (Simoa), aptamer array.

[0143] (ii) Mass spectrometry: Targeted MRM / PRM quantification or top quantification combined with stable isotope internal standard.

[0144] (iii) Electrochemistry / microfluidics: Chip-integrated multi-marker rapid quantification.

[0145] (iv) Exosome level: Detection of UCHL5 enrichment or activity after isolation of specific particle size / label (CD63, CD81).

[0146] (v) Multimodality integration: detection platform outputs combined score directly or interfaces with imaging systems.

[0147] (8) Expanding the population and scenarios of application (i) Pre-screening of fibrosis risk in individuals with hypertension or early left ventricular hypertrophy tendencies.

[0148] (ii) Assessment of myocardial remodeling associated with obesity / metabolic syndrome.

[0149] (iii) Monitoring of cardiotoxicity risk associated with chemotherapy / radiation / targeted or immunotherapy.

[0150] (iv) Different phenotypes of heart failure (including heart failure with preserved ejection fraction HFpEF, early stage of mild diastolic dysfunction).

[0151] (v) Monitoring of atrial or ventricular fibrosis tendencies (e.g. structural-matrix remodeling background of atrial fibrillation).

[0152] (vi) Other RAAS or inflammation-driven myocardial remodeling states.

[0153] It should be understood that the application of the present application is not limited to the above embodiments. Those skilled in the art can improve or transform it according to the above description, and all these improvements and transformations shall belong to the protection scope of the appended claims of the present application.

Claims

1. Application of reagents for detecting UCHL5 expression levels in the preparation of diagnostic products for myocardial fibrosis.

2. Application of reagents that inhibit UCHL5 expression levels in the preparation of drugs for the prevention of myocardial fibrosis.

3. Application of reagents that inhibit UCHL5 expression levels in the preparation of drugs for treating myocardial fibrosis.

4. The application according to claim 2 or 3, characterized in that, The reagents used to inhibit UCHL5 expression levels are small molecules, nucleic acids, gene editing systems, proteins, or peptides.

5. The application according to claim 4, characterized in that, The reagent used to inhibit UCHL5 expression is b-AP15.

6. A reagent kit for diagnosing myocardial fibrosis, characterized in that, The kit includes: reagents for detecting UCHL5 expression levels.

7. The reagent kit according to claim 6, characterized in that, The kit is designed to detect UCHL5 in plasma, serum, visceral adipose tissue, or exosomes.

8. A drug for preventing myocardial fibrosis, characterized in that, The drug includes: a reagent that inhibits the expression level of UCHL5.

9. A drug for treating myocardial fibrosis, characterized in that, The drug includes: a reagent that inhibits the expression level of UCHL5.

10. The medicament according to claim 8 or 9, characterized in that, The drug also includes one or more of the following: angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor-neprilysin inhibitors, β-adrenergic receptor blockers, mineral corticosteroid receptor antagonists, and sodium-glucose cotransporter 2 inhibitors.

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