Molecular marker for diagnosis, prognosis evaluation and treatment of cardiac fibrosis

Abstract

The invention relates to a molecular marker for diagnosis, prognosis evaluation and treatment of cardiac fibrosis, and belongs to the technical field of biological medicines. The invention provides a molecular marker capable of diagnosing cardiac fibrosis, evaluating the severity of cardiac fibrosis, evaluating the prognosis of cardiovascular diseases accompanied with cardiac fibrosis or evaluating the prognosis of acute myocardial infarction. The molecular marker comprises a hepatocyte growth factor. Research finds that the HGF is remarkably increased in plasma of a patient with cardiac fibrosis, and the plasma HGF level of a patient with aortic valve stenosis is also remarkably increased before an operation, so that the HGF can be used for diagnosing cardiac fibrosis and evaluating the severity of cardiac fibrosis. Researches find that timely transient increase of the HGF in plasma of a patient with acute myocardial infarction is significantly related to good prognosis, and continuous low-level increase, continuous high-level increase, recurrence and delayed increase of the HGF are significantly related to poor prognosis, so that the HGF can be used for evaluating prognosis of acute myocardial infarction.

Description

Technical Field

[0001] This invention relates to molecular biomarkers for the diagnosis, prognostic assessment, and treatment of cardiac fibrosis, and belongs to the field of biomedical technology. Background Technology

[0002] Cardiac fibrosis (also known as myocardial fibrosis) is characterized by excessive proliferation of cardiac fibroblasts and excessive production of extracellular matrix in myocardial tissue. Normally, the extracellular matrix provides mechanical support to myocardial tissue. However, excessive or uncontrolled cardiac fibrosis can severely affect the heart's systolic and diastolic functions, as well as its electrophysiological function. Studies have shown that cardiac fibrosis is associated with almost all cardiovascular diseases. Therefore, clinically, the degree of cardiac fibrosis can be used to predict the prognosis of a range of major cardiovascular diseases, including hypertension, diastolic heart failure, cardiomyopathy, and sudden cardiac death. Other studies have confirmed that cardiac fibrosis repair after acute myocardial infarction is crucial for restoring cardiac function; therefore, the occurrence of cardiac fibrosis can also be used to predict the prognosis of acute myocardial infarction.

[0003] Cardiac fibrosis is extremely complex, primarily in two aspects: first, it can occur at any stage of cardiovascular disease; second, it involves multiple cell types. Although fibroblasts are the main executive cells in cardiac fibrosis, other cell types also play important roles in the process. Currently, fibrosis is mainly studied by isolating a specific cell type. However, myocardial tissue is rich in a dense extracellular matrix, and isolating cells through digestion can affect gene expression. Due to these complex factors, our understanding of cardiac fibrosis is currently limited. Therefore, effective treatments for cardiac fibrosis are still lacking, and there are also few effective interventions to restore cardiac function by controlling cardiac fibrosis after acute myocardial infarction.

[0004] Meanwhile, the current clinical diagnosis of cardiac fibrosis largely relies on invasive tissue biopsies or cardiac magnetic resonance imaging (MRI) with delayed gadolinium enhancement (LGE) and T1 mapping. Invasive tissue biopsies typically involve direct contact with tissues or organs, and patients may experience risks of pain, bleeding, infection, or other complications. MRI with LGE and T1 mapping is complex, requires a high level of operator expertise, and is expensive. Finding plasma biomarkers directly related to the occurrence and development of cardiac fibrosis would be extremely helpful for its clinical diagnosis. To date, several plasma biomarkers associated with fibrosis have been reported in the literature; however, these biomarkers are neither derived from the heart nor do they reflect specific stages of fibrosis. Therefore, at present, there is still a lack of plasma biomarkers that can be used for the clinical diagnosis of cardiac fibrosis. Summary of the Invention

[0005] To address the above problems, the present invention provides a molecular biomarker, wherein the molecular biomarker includes hepatocyte growth factor (HGF, Scatter Factor); the molecular biomarker has any of the following functions:

[0006] (a) Diagnosis of cardiac fibrosis (also known as myocardial fibrosis);

[0007] (b) Assess the severity of cardiac fibrosis;

[0008] (c) Assess the prognosis of acute myocardial infarction;

[0009] Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction.

[0010] In one embodiment of the invention, the cardiac fibrosis includes cardiac fibrosis caused by pressure stress and / or cardiac fibrosis caused by myocardial infarction.

[0011] In one embodiment of the invention, the pressure load includes aortic stenosis and / or hypertension.

[0012] In one embodiment of the present invention, the myocardial infarction includes acute myocardial infarction.

[0013] In one embodiment of the present invention, the other cardiovascular diseases accompanied by cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

[0014] In one embodiment of the present invention, the diagnosis of cardiac fibrosis includes: determining whether a patient has cardiac fibrosis based on the concentration of hepatocyte growth factor in plasma; if the concentration of hepatocyte growth factor in the patient's plasma is much higher than that in the normal population, the patient is determined to have cardiac fibrosis; if the concentration of hepatocyte growth factor in the patient's plasma is close to that in the normal population, the patient is determined not to have cardiac fibrosis.

[0015] In one embodiment of the present invention, the assessment of the prognosis of acute myocardial infarction includes: judging the prognosis of a patient after acute myocardial infarction based on the change in the concentration of hepatocyte growth factor in plasma. If the concentration of hepatocyte growth factor in the patient's plasma increases transiently, the patient's prognosis is good. If the concentration of hepatocyte growth factor in the patient's plasma increases at a low level, increases at a high level, recurs, or increases delayed, the patient's prognosis is poor.

[0016] This invention also provides the application of reagents for detecting the above-mentioned molecular markers in a test sample in the preparation of a product, said product having any of the following functions:

[0017] (a) Diagnosis of cardiac fibrosis;

[0018] (b) Assess the severity of cardiac fibrosis;

[0019] (c) Assess the prognosis of acute myocardial infarction;

[0020] Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction.

[0021] In one embodiment of the invention, the cardiac fibrosis includes cardiac fibrosis caused by pressure stress and / or cardiac fibrosis caused by myocardial infarction.

[0022] In one embodiment of the invention, the pressure load includes aortic stenosis and / or hypertension.

[0023] In one embodiment of the present invention, the myocardial infarction includes acute myocardial infarction.

[0024] In one embodiment of the present invention, the other cardiovascular diseases accompanied by cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

[0025] In one embodiment of the present invention, the diagnosis of cardiac fibrosis includes: determining whether a patient has cardiac fibrosis based on the concentration of hepatocyte growth factor in plasma; if the concentration of hepatocyte growth factor in the patient's plasma is much higher than that in the normal population, the patient is determined to have cardiac fibrosis; if the concentration of hepatocyte growth factor in the patient's plasma is close to that in the normal population, the patient is determined not to have cardiac fibrosis.

[0026] In one embodiment of the present invention, the assessment of the prognosis of acute myocardial infarction includes: judging the prognosis of a patient after acute myocardial infarction based on the change in the concentration of hepatocyte growth factor in plasma. If the concentration of hepatocyte growth factor in the patient's plasma increases transiently, the patient's prognosis is good. If the concentration of hepatocyte growth factor in the patient's plasma increases at a low level, increases at a high level, recurs, or increases delayed, the patient's prognosis is poor.

[0027] In one embodiment of the present invention, the product includes a test kit.

[0028] The present invention also provides a product comprising a reagent for detecting the above-mentioned molecular markers in a sample to be tested; the product having any of the following functions:

[0029] (a) Diagnosis of cardiac fibrosis;

[0030] (b) Assess the severity of cardiac fibrosis;

[0031] (c) Assess the prognosis of acute myocardial infarction;

[0032] Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction.

[0033] In one embodiment of the invention, the cardiac fibrosis includes cardiac fibrosis caused by pressure stress and / or cardiac fibrosis caused by myocardial infarction.

[0034] In one embodiment of the invention, the pressure load includes aortic stenosis and / or hypertension.

[0035] In one embodiment of the present invention, the myocardial infarction includes acute myocardial infarction.

[0036] In one embodiment of the present invention, the other cardiovascular diseases accompanied by cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

[0037] In one embodiment of the present invention, the diagnosis of cardiac fibrosis includes: determining whether a patient has cardiac fibrosis based on the concentration of hepatocyte growth factor in plasma; if the concentration of hepatocyte growth factor in the patient's plasma is much higher than that in the normal population, the patient is determined to have cardiac fibrosis; if the concentration of hepatocyte growth factor in the patient's plasma is close to that in the normal population, the patient is determined not to have cardiac fibrosis.

[0038] In one embodiment of the present invention, the assessment of the prognosis of acute myocardial infarction includes: judging the prognosis of a patient after acute myocardial infarction based on the change in the concentration of hepatocyte growth factor in plasma. If the concentration of hepatocyte growth factor in the patient's plasma increases transiently, the patient's prognosis is good. If the concentration of hepatocyte growth factor in the patient's plasma increases at a low level, increases at a high level, recurs, or increases delayed, the patient's prognosis is poor.

[0039] In one embodiment of the present invention, the product includes a test kit.

[0040] The present invention also provides a prediction model having any of the following functions:

[0041] (a) Diagnosis of cardiac fibrosis;

[0042] (b) Assess the severity of cardiac fibrosis;

[0043] (c) Assess the prognosis of acute myocardial infarction;

[0044] Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction;

[0045] The prediction model includes a detection module, a data analysis module, and a prediction module;

[0046] The detection module is used to detect the levels of the aforementioned molecular markers in the sample to be tested;

[0047] The data analysis module is used to analyze the detection results output by the detection module;

[0048] The prediction module is used to classify the sample to be tested based on the analysis results output by the data analysis module, determining whether it has a disease or a good or bad prognosis.

[0049] In one embodiment of the invention, the cardiac fibrosis includes cardiac fibrosis caused by pressure stress and / or cardiac fibrosis caused by myocardial infarction.

[0050] In one embodiment of the invention, the pressure load includes aortic stenosis and / or hypertension.

[0051] In one embodiment of the present invention, the myocardial infarction includes acute myocardial infarction.

[0052] In one embodiment of the present invention, the other cardiovascular diseases accompanied by cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

[0053] In one embodiment of the present invention, the diagnosis of cardiac fibrosis includes: determining whether a patient has cardiac fibrosis based on the concentration of hepatocyte growth factor in plasma; if the concentration of hepatocyte growth factor in the patient's plasma is much higher than that in the normal population, the patient is determined to have cardiac fibrosis; if the concentration of hepatocyte growth factor in the patient's plasma is close to that in the normal population, the patient is determined not to have cardiac fibrosis.

[0054] In one embodiment of the present invention, the assessment of the prognosis of acute myocardial infarction includes: judging the prognosis of a patient after acute myocardial infarction based on the change in the concentration of hepatocyte growth factor in plasma. If the concentration of hepatocyte growth factor in the patient's plasma increases transiently, the patient's prognosis is good. If the concentration of hepatocyte growth factor in the patient's plasma increases at a low level, increases at a high level, recurs, or increases delayed, the patient's prognosis is poor.

[0055] The present invention also provides a drug screening method, wherein the drugs include drugs for treating cardiac fibrosis, acute myocardial infarction and / or other cardiovascular diseases accompanied by cardiac fibrosis other than acute myocardial infarction;

[0056] The method includes: administering the drug to be screened to the experimental subjects; obtaining the results related to the above molecular markers after the administration of the drug to be screened to the experimental subjects; and determining whether the drug to be screened is effective or ineffective based on the obtained results.

[0057] This invention also provides the use of hepatocyte growth factor, hepatocyte growth factor signaling pathway blockers, and / or neutrophil infiltration inhibitors in the preparation of pharmaceuticals, wherein the pharmaceuticals have any of the following functions:

[0058] (a) Prevention and / or treatment of cardiac fibrosis;

[0059] (b) Prevention and / or treatment of acute myocardial infarction;

[0060] Alternatively, (c) prevent and / or treat other cardiovascular diseases associated with cardiac fibrosis, other than acute myocardial infarction.

[0061] In one embodiment of the invention, the cardiac fibrosis includes cardiac fibrosis caused by pressure stress and / or cardiac fibrosis caused by myocardial infarction.

[0062] In one embodiment of the invention, the pressure load includes aortic stenosis and / or hypertension.

[0063] In one embodiment of the present invention, the myocardial infarction includes acute myocardial infarction.

[0064] In one embodiment of the present invention, the other cardiovascular diseases accompanied by cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

[0065] In one embodiment of the present invention, when the drug has the function of preventing and / or treating cardiac fibrosis or cardiovascular diseases accompanied by cardiac fibrosis, the drug contains hepatocyte growth factor and / or hepatocyte growth factor signaling pathway blocker.

[0066] In one embodiment of the present invention, when the drug has the function of preventing and / or treating acute myocardial infarction, the drug contains hepatocyte growth factor, or the drug contains a hepatocyte growth factor signaling pathway blocker and / or a neutrophil infiltration inhibitor.

[0067] In one embodiment of the present invention, the treatment of acute myocardial infarction includes treating acute myocardial infarction by controlling fibroblast proliferation dominated by acute inflammatory response and / or controlling myofibroblast-mediated fibrosis repair.

[0068] In one embodiment of the present invention, the hepatocyte growth factor signaling pathway blocker includes hepatocyte growth factor inhibitors, AKT inhibitors, mTORC1 inhibitors and / or MET inhibitors.

[0069] In one embodiment of the present invention, the neutrophil infiltration inhibitor includes erythromycin.

[0070] The present invention also provides a medicament having any of the following functions:

[0071] (a) Prevention and / or treatment of cardiac fibrosis;

[0072] (b) Prevention and / or treatment of acute myocardial infarction;

[0073] Alternatively, (c) prevent and / or treat other cardiovascular diseases associated with cardiac fibrosis, other than acute myocardial infarction;

[0074] The drug contains hepatocyte growth factor, hepatocyte growth factor signaling pathway blockers, and / or neutrophil infiltration inhibitors.

[0075] In one embodiment of the invention, the cardiac fibrosis includes cardiac fibrosis caused by pressure stress and / or cardiac fibrosis caused by myocardial infarction.

[0076] In one embodiment of the invention, the pressure load includes aortic stenosis and / or hypertension.

[0077] In one embodiment of the present invention, the myocardial infarction includes acute myocardial infarction.

[0078] In one embodiment of the present invention, the other cardiovascular diseases accompanied by cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

[0079] In one embodiment of the present invention, when the drug has the function of preventing and / or treating cardiac fibrosis or cardiovascular diseases accompanied by cardiac fibrosis, the drug contains hepatocyte growth factor and / or hepatocyte growth factor signaling pathway blocker.

[0080] In one embodiment of the present invention, when the drug has the function of preventing and / or treating acute myocardial infarction, the drug contains hepatocyte growth factor, or the drug contains a hepatocyte growth factor signaling pathway blocker and / or a neutrophil infiltration inhibitor.

[0081] In one embodiment of the present invention, the treatment of acute myocardial infarction includes treating acute myocardial infarction by controlling fibroblast proliferation dominated by acute inflammatory response and / or controlling myofibroblast-mediated fibrosis repair.

[0082] In one embodiment of the present invention, the hepatocyte growth factor signaling pathway blocker includes hepatocyte growth factor inhibitors, AKT inhibitors, mTORC1 inhibitors and / or MET inhibitors.

[0083] In one embodiment of the present invention, the neutrophil infiltration inhibitor includes erythromycin.

[0084] The technical solution of this invention has the following advantages:

[0085] 1. The present invention provides a molecular marker comprising hepatocyte growth factor (HGF, Scatter Factor); the molecular marker having any of the following functions: (a) diagnosing cardiac fibrosis; (b) evaluating the severity of cardiac fibrosis; (c) assessing the prognosis of cardiovascular diseases associated with cardiac fibrosis; or, (d) assessing the prognosis of acute myocardial infarction.

[0086] Studies have found that HGF levels are significantly elevated in the plasma of patients with cardiac fibrosis, and preoperative plasma HGF levels are also significantly elevated in patients with aortic stenosis. Therefore, HGF can be used to diagnose cardiac fibrosis and assess its severity. Meanwhile, cardiovascular disease patients often have concurrent cardiac fibrosis, and excessive cardiac fibrosis in cardiovascular disease patients generally has a poor prognosis. Therefore, HGF can also be used to assess the prognosis of cardiovascular disease patients with concurrent cardiac fibrosis.

[0087] Cardiac repair following acute myocardial infarction (AMI) comprises two stages: acute inflammatory response and fibrotic repair. Acute inflammatory response generally occurs in the early stages of cardiac repair, while fibrotic repair typically occurs in the later stages or after the acute inflammatory response has ended. Studies have found that for acute inflammatory responses in the heart, plasma HGF levels in AMI patients are positively correlated with the activation status of neutrophils. The activation status of neutrophils can indicate whether timely and effective acute inflammation has occurred in the body of AMI patients. Regarding the overall cardiac repair process following AMI, which includes both acute inflammatory response and fibrotic repair, timely and transient increases in plasma HGF levels are significantly associated with a good prognosis, while persistently low, persistently high, recurrent, and delayed increases in HGF levels are significantly associated with a poor prognosis (where a good prognosis means that AMI patients can promptly and effectively repair cardiac damage and restore cardiac function after treatment, and a poor prognosis means that AMI patients cannot promptly and effectively repair cardiac damage and restore cardiac function after treatment). Therefore, HGF can be used to assess the prognosis of acute myocardial infarction.

[0088] 2. This invention provides the use of hepatocyte growth factor, hepatocyte growth factor signaling pathway blocker and / or neutrophil infiltration inhibitor in the preparation of a medicament having any of the following functions: (a) prevention and / or treatment of cardiac fibrosis; (b) prevention and / or treatment of cardiovascular diseases associated with cardiac fibrosis; or, (c) prevention and / or treatment of acute myocardial infarction.

[0089] Cardiac repair following acute myocardial infarction (AMI) involves two stages: acute inflammatory response and fibrotic repair. Acute inflammatory response generally occurs in the early stages of cardiac repair, while fibrotic repair typically occurs in the later stages or after the acute inflammatory response has ended. Studies have found that elevated plasma HGF levels after myocardial infarction originate from neutrophils, not cardiac fibroblasts; HGF-MET signaling does not affect the migration and infiltration of neutrophils into myocardial tissue after myocardial infarction; inhibiting neutrophil infiltration suppresses the inflammatory response in damaged tissue; and inhibiting the inflammatory response in damaged tissue is detrimental to cardiac repair after AMI. This indicates that the release of HGF from neutrophils participates in fibroblast proliferation dominated by acute inflammatory response after AMI, meaning the HGF signaling pathway is crucial for fibroblast proliferation dominated by acute inflammatory response after AMI. Research has shown that HGF can significantly promote fibroblast proliferation but inhibit collagen synthesis in myofibroblasts; inhibiting MET or mTORC1 signaling blocks the HGF-promoting effect on fibroblast proliferation but promotes fibrosis in myofibroblasts. It is evident that HGF-MET signaling can promote the proliferation of cardiac fibroblasts after myocardial infarction but inhibit the collagen synthesis function of myofibroblasts (i.e., inhibit fibrosis). This indicates that the HGF signaling pathway plays a crucial role in myofibroblast-mediated fibrosis repair after acute myocardial infarction. Therefore, in cardiac fibrosis and acute myocardial infarction, the different roles of the HGF signaling pathway in neutrophil-dominated fibroblast proliferation and myofibroblast-mediated extracellular matrix synthesis can be regulated based on plasma HGF levels and their dynamic changes. This can achieve therapeutic effects on cardiac fibrosis and control of damage repair after acute myocardial infarction. Compounds that can regulate the HGF signaling pathway and the release of HGF from neutrophils, such as HGF, HGF signaling blockers, and / or neutrophil infiltration inhibitors, can be used to prepare drugs for the treatment of cardiac fibrosis and acute myocardial infarction. Taking acute myocardial infarction as an example, if the HGF level in patients with acute myocardial infarction remains high or rises again after treatment, hepatocyte growth factor signaling pathway blockers and / or neutrophil infiltration inhibitors can be administered to improve patient prognosis; if the HGF level in patients with acute myocardial infarction is insufficient after treatment (e.g., persistently low HGF level), HGF supplementation can be used to improve patient prognosis; if the HGF level in patients with acute myocardial infarction rises late after treatment, the activation time of the HGF signaling pathway can be adjusted by administering hepatocyte growth factor signaling pathway blockers to improve patient prognosis. Attached Figure Description

[0090] Figure 1 WGCNA analysis flowchart. Based on LCA and TAC models, eight time points spanning the acute and chronic phases were selected.

[0091] Figure 2 The brown module is associated with cardiac mechanical stress and cardiomyocyte contraction. In the brown module, enrichment is calculated relative to a random expectation (relative to the mouse transcriptome). An enrichment factor of 1 indicates no enrichment. The p-value is calculated based on the hypergeometric distribution.

[0092] Figure 3 Turquoise, pink, and yellow modules are associated with neutrophils / proliferating fibroblasts, focal adhesions, and myofibroblasts, respectively.

[0093] Figure 4 The different dynamic characteristics of turquoise, pink, and yellow modules during cardiac fibrosis were investigated. Genes from different modules were compared with upregulated genes from cardiac fibroblasts at 3, 7, 2, and 4 weeks post-myocardial infarction, and enrichment was calculated.

[0094] Figure 5 Plasma HGF was significantly elevated in a mouse model of acute myocardial infarction. Plasma HGF peaked on day 3 post-infarction but declined on day 7. HGF levels were measured on day 3 after intraperitoneal injection of erythromycin daily following myocardial infarction.

[0095] Figure 6 Cardiac fibroblasts did not increase HGF secretion on day 3 after myocardial infarction. Cardiac fibroblasts were isolated from sham-operated and MI mouse models and cultured in 2 mL of culture medium for 4 hours. HGF levels in the culture medium were measured.

[0096] Figure 7 Neutrophils secrete HGF under pro-inflammatory stimulation. Neutrophils were isolated from peripheral blood, cultured in culture medium, and treated with serum amyloid a (10 μg / mL) for 5 hours, after which HGF levels were measured.

[0097] Figure 8 CEBPδ controls HGF production by neutrophils. Neutrophils were isolated from the peripheral blood of Cebpδ knockout mice, cultured in culture medium, treated with serum amyloid a (10 μg / mL) for 5 hours, and then HGF levels were measured.

[0098] Figure 9 HGF does not affect neutrophil infiltration in myocardial tissue after acute myocardial infarction. Leukocytes and neutrophils were isolated from the left ventricle of mice with acute myocardial infarction and those treated with JNJ, and the proportion of neutrophils to leukocytes was calculated. Leukocytes and neutrophils (CD45+CD11b+Ly6G+) were isolated using a BD FACSARIAIII flow cytometer.

[0099] Figure 10The TNF superfamily ligands and their receptors that induce non-classical NF-κB are enriched in the turquoise but not the yellow module.

[0100] Figure 11 TNFR2 interacting proteins are associated with the turquoise module and proliferating fibroblasts. These interacting proteins were derived from the GPS protein interaction database and compared with upregulated genes from cardiac fibroblasts at 3, 7, 2, and 4 weeks post-myocardial infarction.

[0101] Figure 12 Co-localization of TNFα and TNFR2 in myocardial tissue membrane on day 3 post-myocardial infarction. Red: TNFα signal, green: TNFR2 signal. Scale bar: 25 μm.

[0102] Figure 13 NF-κB2 (nuclear green signal) was significantly increased in cardiac fibroblasts on day 3 post-myocardial infarction, and ACTA2 (red) labeled cardiac fibroblasts. Scale bar: 25 μm.

[0103] Figure 14 TNFα from neutrophil membranes promotes cardiac fibroblast proliferation via NF-κB2. Electroporation may affect the co-culture of fibroblasts and neutrophils; therefore, the percentage of EdU-positive fibroblasts decreased in all three groups of cells that underwent electroporation.

[0104] Figure 15 : Neutrophil membrane TNFα activates non-canonical NF-κB2 in cardiac fibroblasts. Fibroblasts were cultured alone, or co-cultured with neutrophils, and treated with the TACE inhibitor TMI-1 or siRNA targeting NF-κB2. Scale bar: 50 μm.

[0105] Figure 16 Non-classical NF-κB2 activates MET expression in fibroblasts. Fibroblasts and neutrophils were co-cultured. Neutrophils were washed away, and the purified fibroblasts were used for Western blotting.

[0106] Figure 17 The HGF-MET signaling pathway is activated in myocardial tissue during acute myocardial infarction. Cardiac fibroblasts were isolated and cultured from mice with sham surgery and myocardial infarction models. Fibroblasts were treated with HGF and MET inhibitors. Western blot analysis of fibroblasts was performed using antibodies against MET and phospho-MET (Y1234 / Y1235).

[0107] Figure 18The HGF-MET signaling pathway was activated in myocardial tissue of a TAC mouse model. Cardiac fibroblasts were isolated from sham-operated and TAC mouse models. Western blot analysis of fibroblasts was performed using antibodies against MET and phosphate-MET (Y1234 / Y1235).

[0108] Figure 19 HGF promotes the proliferation of cardiac fibroblasts. Fibroblasts were isolated from myocardial tissue on day 5 after myocardial infarction (MI) and cultured in vitro. They were treated with HGF, a MET inhibitor, and rapamycin, and the number of viable cells was determined using a sensitive colorimetric assay to compare cell proliferation.

[0109] Figure 20 Differential gene expression analysis after HGF treatment. Genes related to the turquoise module and mTORC1 signaling were enriched in genes upregulated after HGF treatment. Genes downregulated after HGF treatment were enriched in the yellow module and the EPRS1 target.

[0110] Figure 21 The targets of EPRS1 were enriched in the extracellular matrix proteins of the yellow module and the core, but not in the turquoise module. This enrichment analysis result is consistent with the role of EPRS1 in ECM protein synthesis.

[0111] Figure 22 HGF activates the AKT-mTORC1 pathway in cardiac fibroblasts. Fibroblasts were isolated from the left ventricle of mice on day 3 post-myocardial infarction, cultured, and treated with HGF and rapamycin. Immunofluorescence staining was performed using antibodies targeting the activated forms of AKT and p70 S6 kinase. Scale bar: 25 μm.

[0112] Figure 23 In a mouse model of myocardial infarction, MET-mediated signaling activated the AKT-mTORC1 pathway in cardiac fibroblasts. IHC was performed using left ventricular tissue on day 3 post-myocardial infarction. Scale bar: 50 μm.

[0113] Figure 24 In the TAC mouse model, the AKT-mTORC1 pathway in cardiac fibroblasts is activated by a MET-mediated pathway. IHC was performed using left ventricular tissue on day 5 post-TAC surgery. Scale bar: 50 μm.

[0114] Figure 25 The MET-AKT-mTORC1 pathway promotes the proliferation of cardiac fibroblasts in a mouse model of myocardial infarction. IHC was performed using left ventricular tissue on day 3 post-myocardial infarction. Scale bar: 50 μm.

[0115] Figure 26The MET-AKT-mTORC1 pathway promotes the proliferation of cardiac fibroblasts in a TAC mouse model. IHC was performed using left ventricular tissue on day 5 post-TAC surgery. Scale bar: 50 μm.

[0116] Figure 27 The HGF-AKT-mTORC1-EPRS1 pathway inhibits TGFβ1-induced fibroblast differentiation. Cardiac fibroblasts were isolated from the left ventricle on day 3 post-myocardial infarction, cultured, and treated with TGFβ1, HGF, JNJ (MET inhibitor), RAP (rapamycin, mTORC1 inhibitor), and Halofuginone (EPRS1 inhibitor). Immunofluorescence staining with anti-ACTA2 antibody revealed the fibroblasts. Scale bar: 50 μm.

[0117] Figure 28 During TGFβ1-induced fibroblast differentiation, the HGF-AKT-mTORC1-EPRS1 pathway inhibits collagen synthesis. Cardiac fibroblasts were isolated from the left ventricle on day 3 post-myocardial infarction, cultured, and... Figure 28 The mixture was processed, and the hydroxyproline content was determined. Halofuginone, HAL.

[0118] Figure 29 Activated mTORC1 promotes EPRS1 phosphorylation. Cardiac fibroblasts were isolated from the left ventricle on day 7 post-myocardial infarction, cultured for 4 hours, and treated with HGF and rapamycin. Western blot analysis was performed using antibodies against EPRS1 and its phosphorylation.

[0119] Figure 30 The MET-AKT-mTORC1 pathway inhibits collagen synthesis in the heart of a mouse model of myocardial infarction (MI). Patients were treated with MET inhibitors and rapamycin from day 3 to day 10 post-MI, and HGF (250 μg / kg) was injected from day 4 to day 7 post-MI. Hydroxyproline levels in the left ventricle were measured.

[0120] Figure 31 The MET-AKT-mTORC1 pathway inhibited collagen synthesis in the heart of a TAC mouse model. Left ventricular hydroxyproline levels were measured after treatment with MET inhibitors and rapamycin from day 7 to day 15 post-TAC.

[0121] Figure 32 Patients with myocardial fibrosis and aortic stenosis have elevated plasma HGF levels before surgery.

[0122] Figure 33Dynamic changes in troponin T and HGF after acute myocardial infarction. Within two days after an acute myocardial infarction, serum troponin T levels rise sharply. This study monitored the dynamic changes in HGF levels in 46 hospitalized patients with acute myocardial infarction (including ST-segment elevation and non-ST-segment elevation myocardial infarction). The results showed that the dynamic changes in HGF could be categorized into five types: timely transient increase, sustained low-level increase, sustained high-level increase, recurrence, and delayed increase.

[0123] Figure 34 The dynamic changes in HGF after acute myocardial infarction were significantly correlated with the prognosis of patients during hospitalization. Among 46 patients with acute myocardial infarction, 15 out of 17 patients in the timely transient elevation group had a good prognosis, and only 2 had a poor prognosis; conversely, in the persistent elevation, recurrence, and delayed repair groups, all 18 patients had a poor prognosis (chi-square test, p-value 4.066e-07).

[0124] Figure 35 The correlation between HGF and clinical biochemical markers (i.e., NLR, BNP, creatine kinase myocardial type (CK-MB) levels and activity, and hs-cTnT) detected after acute myocardial infarction. The correlations between the markers were calculated using the Spearman method, and the p-values ​​are shown in parentheses below the correlation coefficients.

[0125] Figure 36 Inflammation and HGF signal intensity determine the prognosis of acute myocardial infarction (AMI). In an AMI model induced by permanent left coronary artery ligation (LCA), the survival rate was approximately 78% (N=3 groups, total 28). After AMI, administration of the MET inhibitor JNJ resulted in a survival rate of approximately 32% (N=3 groups, total 28). After AMI, administration of erythromycin resulted in a survival rate of approximately 35% (N=3 groups, total 28). After AMI, administration of HGF resulted in a survival rate of approximately 46% (N=3 groups, total 25). Detailed Implementation

[0126] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0127] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0128] Experimental Example 1

[0129] The experimental procedure is as follows:

[0130] 1. Using WGCNA analysis, three gene clusters associated with cardiac fibrosis were identified.

[0131] Myocardial infarction (MI) and pressure overload are likely two of the most common factors leading to cardiac fibrosis. Myocardial infarction is one of the most common causes of heart failure and can trigger reparative fibrosis. Hypertension or valvular disease can cause pressure overload, inducing reactive or diffuse myocardial fibrosis. This study used a myocardial infarction model with permanent left coronary artery ligation (LCA) and a aortic arch coarctation (TAC) model as two representative and related models to investigate cardiac fibrosis.

[0132] Eighty-eight C57BL / 6J mice (purchased from Beijing Huafukang Experimental Animal Company, approximately 8 weeks old) were used to establish a myocardial infarction mouse model (MI mouse model) by permanent ligation of the left coronary artery (LCA) according to the literature “Curaj A, et al. J Vis Exp. 2015 May 4:(99):e52197.” and “Zaw AM, et al. J Vis Exp. 2017 Mar 14:(121):55293.”. A aortic arch constriction mouse model (TAC mouse model) was also established by aortic arch constriction (TAC mouse model) for subsequent studies.

[0133] This study selected eight time points to investigate the dynamic process of cardiac fibrosis, covering both the acute and chronic phases. The acute phase included preoperative time (day 0), acute myocardial infarction, and day 1 and week after TAC surgery. The chronic phase included acute myocardial infarction and 1, 2, 4, 5, and 6 months after TAC surgery. To investigate cardiac fibrosis, this study employed weighted gene co-expression network analysis (WGCNA) to reveal common mechanisms of cardiac fibrosis. WGCNA divided 22,709 genes in the mouse transcriptome into 29 gene groups, with an average of 757 genes per gene group. Figure 1 ).

[0134] First, this study assessed whether the classified gene clusters reflected true cardiac biology. Brain natriuretic peptide (BNP), encoded by NPPB, is specifically expressed in cardiomyocytes. BNP is a recognized biomarker of cardiac mechanical stress, and elevated BNP levels are commonly used to diagnose heart failure. NPPB was clustered into the Brown module (or gene cluster). This study compared the cardiomyocyte-enriched cluster with the Brown module using a published cardiomyocyte-enriched gene set (see "Uhlén M etal. Science. 2015 Jan 23; 347(6220):1260419."). The cardiomyocyte-enriched gene cluster was highly enriched in the Brown module, indicating that the Brown module is primarily composed of genes derived from cardiomyocytes. Figure 2 Genes related to cardiomyocyte contraction are highly enriched in the Brown module. Intercalated discs (IDs) in myocardial tissue connect adjacent cardiomyocytes and provide mechanosensing. Genes constituting IDs are also highly enriched in the Brown module. Figure 2 These analyses strongly suggest that the Brown module is genetically derived from cardiomyocytes and senses mechanical stretch. The transcription factors Mef2a and Mef2d, which directly activate BNP expression, are both clustered in the Brown module. Figure 2 These data demonstrate that WGCNA correctly clusters functionally related genes into the same module or gene group.

[0135] Next, this study identified which modules were associated with cardiac fibroblasts. On day 3 post-myocardial infarction, cardiac fibroblasts were predominantly proliferative, while cardiac tissue two weeks post-infarction was rich in myofibroblasts. This study used upregulated genes from fibroblasts on day 3 or two weeks post-infarction for enrichment analysis. The turquoise module (5415) was similar to fibroblasts on day 3 post-infarction, while the pink (435) and yellow (1319) modules were associated with myofibroblasts. Figure 3 Following myocardial infarction, neutrophils infiltrate myocardial tissue, peaking around day 3. Neutrophil-specific genes and cell cycle-related genes are enriched in the turquoise module. These analyses suggest that the turquoise module is associated with the proliferation of both neutrophils and fibroblasts. Compared to proliferating fibroblasts, myofibroblasts secrete higher levels of extracellular matrix, including collagen. Consistent with genes upregulated 2 weeks post-myocardial infarction, genes associated with core extracellular matrix or extracellular matrix assembly are highly enriched in the pink and yellow modules. Figure 3These analyses strongly suggest that the pink and yellow modules are associated with myofibroblasts. This study assessed the dynamic changes of these modules during the progression of cardiac fibrosis by comparing upregulated genes in cardiac fibroblasts at days 3, 7, 2, and 4 weeks post-myocardial infarction. Figure 4 The turquoise module peaked on day 3 after myocardial infarction and gradually declined. The pink module peaked 2 weeks after myocardial infarction and then declined. The yellow module remained active even 4 weeks after myocardial infarction.

[0136] Based on these analyses, this study concludes that the turquoise module is composed of genes associated with acute inflammation and fibroblast proliferation, while the yellow module is composed of genes associated with myofibroblasts.

[0137] 2. The necessity of HGF growth factor release from neutrophils for cardiac repair after acute myocardial infarction

[0138] Several growth factor families, such as PDGF, FGF, EGF, and HGF (hepatocyte growth factor), are grouped in the turquoise module. The roles of PDGF and FGF in regulating fibroblast proliferation are well-studied. Among these growth factors, HGF and its receptor MET are also in the turquoise module, but the HGF-MET signaling pathway has not been investigated in cardiac fibrosis. This study used a left coronary artery ligation (LCA) myocardial infarction model and a aortic arch constriction (TAC) mouse model to investigate the role of the HGF-MET signaling pathway in cardiac fibrosis.

[0139] This study first determined the dynamic changes of HGF after acute myocardial infarction. Twelve mice with myocardial infarction induced by permanent left coronary artery ligation (LCA) were used as the model group, and three mice with myocardial infarction induced by LCA were used as the experimental group. Following myocardial infarction, the experimental group mice were intraperitoneally injected daily with erythromycin (purchased from MedChemExpress, catalog number HY-B0220, using 0.1 mL PBS buffer as the solvent) at a dose of 100 mg / kg body weight from day 1 to day 7 post-infarction. During the intervention period, sham-operated mice served as controls. Plasma HGF levels in the mice were measured daily by ELISA. The results showed that plasma HGF levels in the model group mice significantly increased 3 days after myocardial infarction but decreased 7 days after myocardial infarction. Figure 5 Furthermore, compared with the model group mice, erythromycin intervention significantly reduced the increase in plasma HGF in the experimental group mice 3 days after myocardial infarction. Figure 5 ).

[0140] This study isolated cardiac fibroblasts from mice on day 3 post-myocardial infarction and measured their secreted HGF levels. Six mice with myocardial infarction models obtained through permanent left coronary artery ligation (LCA) were used, with sham-operated mice as controls. Cardiac fibroblasts were isolated from these mice on day 3 post-myocardial infarction (i.e., day 3 after myocardial infarction surgery) using the isolation method described in the literature "Pramod Sahadevan, Bruce GAllen, Methods. 2022 Jul; 203:187-195."). Fibroblasts were then processed at a concentration of 2 × 10⁻⁶ cells / mL. 5 Each cell was inoculated into 2 mL of DMEM / F-12 serum-free medium (Gibco) supplemented with 10% (v / v) fetal bovine serum (Gibco) and cultured at 37°C in a sterile cell culture incubator with 5% (v / v) CO2 for 4 hours. After culture, the culture supernatant was collected, and the HGF level secreted by cardiac fibroblasts was detected by ELISA. Measurements showed no difference in HGF levels between the myocardial infarction group and the sham-operated group, indicating that cardiac fibroblasts are not the source cells of plasma HGF. Figure 6 ).

[0141] Neutrophils are the most abundant white blood cells in the blood and play a crucial role in tissue wound healing. According to literature reports, SAA can activate neutrophils and induce degranulation. Since the turquoise module is associated with neutrophils, and neutrophils infiltrate myocardial tissue after myocardial infarction, this study aimed to confirm whether HGF originates from neutrophils. In this study, neutrophils were isolated from peripheral blood of mice and stimulated with serum amyloid A (SAA). Neutrophils were isolated from peripheral blood of C57BL / 6J mice (isolation method according to the literature "Ubags NDJ, Suratt BT. Methods Mol Biol. 2018; 1809:45-57."), and neutrophils were harvested at a concentration of 6 × 10⁶ cells / mL. 4 Two cells were inoculated into 2 mL of DMEM / F-12 serum-free medium supplemented with 10% (v / v) fetal bovine serum. Serum amyloid A (SAA) was added to the medium to stimulate neutrophils. After inoculation, the cells were incubated at 37°C and 5% (v / v) CO2 for 5 hours. After incubation, the culture supernatant was collected, and the level of HGF secreted by neutrophils was detected by ELISA. Measurements showed that HGF levels in the neutrophil culture supernatant increased significantly after stimulation. Figure 7 These results confirm that the elevated plasma HGF levels after myocardial infarction originate from neutrophils.

[0142] CEBP family transcription factors regulate the production of inflammatory cytokines in neutrophils. Within the CEBP family, CEBPδ is expressed in mature neutrophils. In this study, neutrophils were isolated from peripheral blood of Cebpδ knockout mice (purchased from Cyagen, catalog number KOCMP-12609-Cebpd-B6J-VA) and stimulated to replicate the above experimental procedure. Measurements revealed an almost 500-fold reduction in HGF levels, and no change in HGF release after SAA treatment. Figure 8 These data indicate that Cebpδ regulates the production of HGF by neutrophils.

[0143] HGF-MET signaling has been reported to be involved in the recruitment of anti-tumor neutrophils. This study blocked HGF-MET signaling and detected neutrophil infiltration in myocardial tissue after myocardial infarction. Six mice with myocardial infarction, obtained through permanent left coronary artery ligation (LCA), were randomly divided into two groups: a control group (n=3) and a treatment group (n=3). After grouping, the treatment group mice were administered the MET inhibitor JNJ-38877605 (purchased from MedChemExpress, catalog number HY-50683, using 0.1 mL PBS buffer as a solvent) by gavage at a dose of 50 mg / kg body weight. The control group mice were administered the same amount of the solvent by gavage as a control. After gavage, leukocytes (CD45+) and neutrophils (CD45+CD11b+Ly6G+) in the myocardial tissue of the mouse models on day 1 post-myocardial infarction (i.e., post-operative day 1) using BD FACSARIAIII flow cytometry were separated by fluorescence-activated cell sorting (FACS), and the proportion of neutrophils to leukocytes was calculated. Measurements showed no change in the total number of neutrophils or the neutrophil-to-leukocyte ratio. These data indicate that HGF-MET signaling does not affect the migration and infiltration of neutrophils into myocardial tissue after myocardial infarction. Figure 9 ).

[0144] These data confirm that the release of HGF by neutrophils is crucial for cardiac repair after myocardial infarction.

[0145] 3. Following acute myocardial infarction, the neutrophil-mediated membrane TNFα signaling pathway activates non-canonical NF-κB, thereby inducing the expression of HGF receptor MET protein in fibroblasts.

[0146] The MET receptor was classified in the turquoise module, indicating that its expression is also related to HGF signaling or neutrophil-fibroblast interactions. Membrane TNFα signaling induces the non-canonical NF-κB pathway, promoting cell proliferation. TNF superfamily ligands and their receptors that induce non-canonical NF-κB signaling are highly enriched in the turquoise module but not in the yellow module. Figure 10Consistent with this observation, proteins that interact with TNFR2 are associated with the turquoise module and proliferating fibroblasts. Figure 11 These analyses suggest that membrane TNFα expressed by neutrophils binds to TNFR2 in fibroblasts, inducing non-classical NF-κB signaling, thereby promoting MET expression in fibroblasts. This lays the foundation for HGF-MET signaling.

[0147] In this study, myocardial tissue was collected from mice on day 3 after acute myocardial infarction (i.e., day 3 post-myocardial infarction surgery) for staining analysis. The results showed that on day 3 post-myocardial infarction, the co-localization of TNFα and TNFR2 was significantly increased. Figure 12 Membrane TNFα induces the processing of NF-κB2 precursor protein p100 into p52, and NF-κB2 / p52 and RELB mediate non-canonical NF-κB signaling. On day 3 post-myocardial infarction, NF-κB2 levels in fibroblasts are indeed significantly increased. Figure 13 ).

[0148] This study investigated the non-canonical NF-κB pathway induced by membrane TNFα in a co-culture system of fibroblasts and neutrophils. An acute myocardial infarction mouse model was established, and cardiac fibroblasts were isolated from mice on day 3 post-myocardial infarction (i.e., day 3 post-infarction surgery). Neutrophils were isolated from peripheral blood of C57BL / 6J mice. Cardiac fibroblasts and neutrophils were co-cultured in DMEM / F-12 serum-free medium supplemented with 10% (v / v) fetal bovine serum to observe the interaction between fibroblasts and neutrophils. The results showed that co-culturing fibroblasts and neutrophils significantly increased the proliferation of fibroblasts compared to fibroblasts alone. Figure 14 This proliferative effect may be partly attributed to membrane TNFα signaling and the release of growth factors such as HGF from neutrophils. If membrane TNFα signaling does indeed play a role, then enhancing this signal should increase the proliferation of fibroblasts. Tumor necrosis factor-α converting enzyme (TACE, also known as ADAM-17) is a membrane-bound metalloproteinase that cleaves membrane TNFα, releasing soluble TNFα. Inhibition of TACE does significantly increase the percentage of proliferating fibroblasts. Figure 14 ).

[0149] This study further investigated whether NF-κB2 / p52 is crucial for fibroblast proliferation. An acute myocardial infarction mouse model was established. Cardiac fibroblasts were isolated from mice on day 3 post-myocardial infarction (i.e., day 3 after surgery). NF-κB2 knockdown siRNA was delivered to the fibroblasts (the NF-κB2 knockdown siRNA sequence can be found in the literature "Schumm K, et al. EMBO J. 2006 Oct 18; 25(20):4820-32."). The NF-κB2 knockdown fibroblasts were cultured individually or co-cultured with neutrophils in DMEM / F-12 serum-free medium supplemented with 10% (v / v) fetal bovine serum to observe the proliferation of NF-κB2 knockdown fibroblasts and their MET expression. Since primary fibroblasts isolated from mouse hearts are difficult to transfect with liposomes, this study used electroporation to deliver NF-κB2 knockdown siRNA into fibroblasts (electroplated fibroblasts were suspended in OptiMEM-GlutaMAX solution at a cell density of 1×10⁻⁶ cells). 5 The electroporation conditions of the Gene Pulser MXcell electroporation system (Bio-Rad) were: 300V, 500μF, 1000-ohm pulse, exponential decay program. This study achieved an electroporation efficiency exceeding 60% and confirmed that NF-κB2 in fibroblasts was significantly knocked down. Figure 15 Knockdown of NF-κB2 significantly reduced the percentage of proliferating fibroblasts. Figure 14 Knockdown of NF-κB2 significantly reduced the expression of the MET receptor in fibroblasts. Figure 16 ).

[0150] These data confirm that membrane TNFα signaling induces the non-classical NF-κB pathway, which mediates proliferation.

[0151] 4. HGF promotes the proliferation of cardiac fibroblasts after acute myocardial infarction.

[0152] HGF-MET is involved in the development of many tumors and in liver regeneration. HGF binds to the MET receptor, inducing MET dimerization and trans-autophosphorylation, which activates the Y1234 and Y1235 tyrosine residues of the phosphorylation kinase ring. Therefore, phosphorylation of Y1234 and Y2235 can be used as a marker of MET activation. To confirm whether HGF can promote the proliferation of cardiac fibroblasts after acute myocardial infarction, six mice with myocardial infarction model obtained by permanent ligation of the left coronary artery (LCA) were divided into two groups: a control group (n=6) and a treatment group (n=6). After the grouping, the treatment group mice were administered the MET inhibitor JNJ-38877605 (purchased from MedChemExpress, catalog number HY-50683, using 0.1 mL PBS buffer as solvent) by gavage at a dose of 50 mg / kg mouse body weight. The control group mice were administered the same amount of solvent by gavage as a control. The administration was carried out once a day from the day of surgery until 3 days after myocardial infarction. After the gavage, on the 3rd day after myocardial infarction, Western blot analysis of fibroblasts was performed using antibodies against MET and phosphorylated MET (Y1234 / Y1235) to detect the levels of MET and phosphorylated MET (Y1234 / Y1235) in mouse cardiac fibroblasts. Studies have found that on the third day after myocardial infarction, MET and phosphorylated MET (Y1234 / Y1235) increase. MET inhibitors effectively block HGF-MET signaling. Figure 17 Repeating the above experimental procedure in the TAC model yielded similar results. Figure 18 These data indicate that HGF activates MET in fibroblasts in both the left coronary artery permanent ligation (LCA) myocardial infarction (MI) model and the aortic arch constriction (TAC) model.

[0153] This study then examined whether HGF directly promotes the proliferation of cardiac fibroblasts. Six mice with myocardial infarction (MI) models obtained through permanent left coronary artery ligation (LCA) were used. Cardiac fibroblasts were isolated from these mice on day 3 post-myocardial infarction (i.e., day 3 after surgery) using the isolation method described in the literature "Pramod Sahadevan, Bruce G Allen, Methods. 2022 Jul; 203:187-195."). Fibroblasts were then used at a concentration of 2 × 10⁻⁶ cells / mL. 5The inoculum was inoculated into 2 mL of DMEM / F-12 serum-free medium supplemented with 10% (v / v) fetal bovine serum and cultured in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for 48 hours. After culture, the fibroblasts were transferred to serum-free DMEM / F-12 medium and cultured in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for another 24 hours. After culture, the cardiac fibroblasts were treated with 60 ng / mL HGF, 0.1 μM MET inhibitor JNJ-38877605, and 0.05 μM rapamycin, respectively. After treatment, the cells were incubated in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for 72 hours. After incubation, the cells were analyzed by a sensitive colorimetric assay (using a Cell Counting Kit). 8. The number of viable cells was detected using the methods AC11L054, LFF23063, and Life-iLab to compare cell proliferation. The study found that HGF significantly promoted fibroblast proliferation, while inhibiting MET or mTORC1 signaling completely blocked the HGF-promoted fibroblast proliferation effect. Figure 19 ).

[0154] These data show that HGF-MET signaling promotes the proliferation of cardiac fibroblasts after myocardial infarction.

[0155] 5. RNA sequencing revealed that HGF may promote fibroblast proliferation by activating the AKT-mTORC1 pathway, but inhibit collagen synthesis in myofibroblasts.

[0156] To investigate how HGF regulates fibrosis, this study used cardiac fibroblasts treated with 60 ng / mL HGF in the previous experiments and performed RNA-seq analysis using the Illumina sequencing platform. This study identified 1688 upregulated genes and 1696 downregulated genes. Compared to downregulated genes, upregulated genes were more enriched in the turquoise module, indicating that HGF promotes the regulation of fibrosis in the turquoise module (…). Figure 20 AKT-mTORC1 is an important downstream pathway promoting cell proliferation. The enrichment of upregulated genes related to the mTORC1 pathway indicates that HGF directly activates AKT-mTORC1 in fibroblasts. Figure 20 Conversely, downregulated genes were highly enriched in the yellow module, indicating that HGF inhibits the function of the yellow module. In fact, genes related to the core extracellular matrix were highly enriched in the downregulated genes. Figure 20 ).

[0157] ECM proteins, especially collagen, are rich in proline. Proline and hydroxyproline account for approximately 25% of the amino acids in collagen. The translation of these ECM proteins is controlled by EPRS1 (glutamyl-prolyl tRNA synthetase). EPRS1 catalyzes the attachment of proline (Pro) to its corresponding tRNA to synthesize Pro-containing proteins, playing a crucial role in cardiac fibrosis by promoting the synthesis of Pro-rich fibrotic proteins. Eighty-three genes containing the Pro-Pro motif have been identified as preferential regulatory targets of EPRS1. These regulatory targets of EPRS1 are enriched in genes downregulated after HGF treatment. These regulatory targets of EPRS1 are highly enriched in the core extracellular matrix and the yellow module. Figure 21 ).

[0158] These data and analyses indicate that HGF activates the AKT-mTORC1 pathway to promote fibroblast proliferation, but inhibits myofibroblast function through EPRS1.

[0159] 6. HGF activates the AKT-mTORC1 pathway to promote fibroblast proliferation.

[0160] Growth factors bind to the extracellular regions of their receptor tyrosine kinases (RTKs), activating them. A major downstream effector pathway of RTKs is the PI3K-AKT-mTORC1 pathway. Phosphorylation of AKT Ser473 marks the activation of this kinase. p70 S6 kinase is a substrate of mTORC1, and phosphorylation of p70 S6 kinase (Thr389) can be used as a marker of mTORC1 activation. To verify the mechanism by which HGF regulates fibrosis, this study used cardiac fibroblasts treated with 60 ng / mL HGF and 0.05 μM rapamycin in the previous experiments. Immunofluorescence staining was performed using antibodies against activated forms of AKT and p70 S6 kinase to assess AKT and mTORC1 activation by phosphorylation of AKT Ser473 and p70 S6 kinase Thr389. The results showed that HGF treatment activated both kinases in fibroblasts. Rapamycin is an inhibitor of mTORC1, which inhibits HGF-induced phosphorylation of p70 S6 kinase. Figure 22 ).

[0161] This study further validated the HGF-induced AKT-mTORC1 pathway in a myocardial infarction (MI) model with permanent left coronary artery ligation (LCA) and aortic arch coarctation (TAC) model. Left ventricular tissue was collected from the mouse models on day 3 post-MI (i.e., day 3 after myocardial infarction surgery) and day 5 after TAC surgery for IHC analysis. The results showed that the MET inhibitor JNJ inhibited HGF signaling and completely blocked the activation of AKT and mTORC1. Rapamycin also blocked the phosphorylation of p70 S6 kinase (…). Figure 23 , Figure 24 This study further investigated the role of the HGF-MET-AKT-mTORC1 pathway in fibroblast proliferation. In this study, a novel thymidine analog, EdU, was used to label proliferating cells in myocardial tissue (labeling method described in the literature "Fu X, et al. J Clin Invest. 2018 May 1; 128(5):2127-2143"). On the 3rd day after myocardial infarction, treatment with MET inhibitors or rapamycin significantly inhibited fibroblast proliferation. Figure 25 Treatment with MET inhibitors or rapamycin also significantly inhibited cardiac fibroblast proliferation in the TAC model. Figure 26 ).

[0162] 7. HGF inhibits collagen synthesis in myofibroblasts through mTORC1-S6K1-mediated EPRS1 phosphorylation.

[0163] TGFβ1 promotes the differentiation of fibroblasts into contractile myofibroblasts. To determine the role of HGF in regulating myofibroblasts, this study used a TGFβ1-induced fibroblast differentiation model. Six mice with myocardial infarction (LCA) were used as models. Cardiac fibroblasts were isolated from these mice on day 3 post-myocardial infarction (i.e., day 3 after surgery) using the isolation method described in the literature "Pramod Sahadevan, Bruce GAllen, Methods. 2022 Jul; 203:187-195."). Fibroblasts were then harvested at a concentration of 2 × 10⁻⁶ cells / mL. 5Fibroblasts were inoculated into 2 mL of DMEM / F-12 serum-free medium supplemented with 10% (v / v) fetal bovine serum and cultured at 37°C and 5% (v / v) CO2 for 48 hours. After culture, the fibroblasts were transferred to serum-free DMEM / F-12 medium and cultured at 37°C and 5% (v / v) CO2 for another 24 hours. After culture, 600 pmol / L TGFβ1 (purchased from MedChemExpress, HY-P70648), 60 ng / mL HGF, and 0.1 μM MET inhibitor were added to the culture system. Cardiac fibroblasts were treated with JNJ-38877605, 0.05 μM RAP (rapamycin, an mTORC1 inhibitor), and 50 ng / mL Halofuginone (EPRS1 inhibitor, purchased from Shanghai Bied Pharmaceutical Technology Co., Ltd., catalog number BD132622). After addition, the cells were incubated at 37°C and 5% (v / v) CO2 in a sterile cell culture incubator for 72 hours. After incubation, the fibroblasts were treated with an antibody against ACTA2 and immunofluorescence staining was performed to visualize the fibroblasts. The supernatant was collected and the hydroxyproline content was determined using a hydroxyproline assay kit (BC0250, Solarbio). The results showed that HGF treatment significantly inhibited the function of myofibroblasts, and MET inhibitors or rapamycin could block the effect of HGF. Figure 27 Collagen is composed of a repeating sequence of Gly-Pro-X (where X is any amino acid) or Gly-X-Hyp (hydroxyproline), with hydroxyproline accounting for approximately 14% of the dry weight of collagen. Due to its abundant content, measuring hydroxyproline can provide a quantitative comparison of collagen content. HGF treatment significantly inhibits collagen synthesis. Figure 28 ).

[0164] Six mice with myocardial infarction (MI) models obtained through permanent left coronary artery ligation (LCA) were used. Cardiac fibroblasts were isolated from these mice on day 7 post-MI (i.e., day 7 after myocardial infarction surgery) using the isolation method described in the literature "Pramod Sahadevan, Bruce G Allen, Methods. 2022 Jul; 203:187-195."). Fibroblasts were then collected at a concentration of 2 × 10⁻⁶ cells / mL. 5Inoculation was performed on 2 mL of DMEM / F-12 serum-free medium supplemented with 10% (v / v) fetal bovine serum. The cells were cultured at 37°C and 5% (v / v) CO2 for 4 hours. After incubation, cardiac fibroblasts were treated with 60 ng / mL HGF, 0.1 μM MET inhibitor JNJ-38877605, and 0.05 μM rapamycin, respectively. The cells were then incubated at 37°C and 5% (v / v) CO2 for 72 hours. Following incubation, Western blot analysis was performed using antibodies against EPRS1 and its phosphorylation. Data analysis showed that HGF treatment inhibited downstream regulatory targets of EPRS1. Recent studies have found that EPRS1 is a direct substrate of p70 S6 kinase. EPRS1 encodes a bifunctional glutamyl-prolyl-tRNA synthetase. Upon activation of p70 S6 kinase, phosphorylation of Ser999 of EPRS1 induces its dissociation from the aminoacyl-tRNA synthetase multienzyme complex, thereby reducing the translation of proline-rich mRNA. Activation of the HGF-MET pathway enhances EPRS1 (Ser999) phosphorylation, an effect that can be blocked by rapamycin. Figure 29 ).

[0165] This study further validated in vivo whether HGF signaling inhibits myofibroblast function. Nineteen mice with myocardial infarction, obtained through permanent left coronary artery ligation (LCA), were divided into three groups: a control group (n=7), a MET inhibitor intervention group (n=5), and a rapamycin intervention group (n=7). After grouping, mice in the MET inhibitor intervention group were administered the MET inhibitor JNJ-38877605 (purchased from MedChemExpress, catalog number HY-50683, using 0.1 mL PBS buffer as a solvent) at a dose of 50 mg / kg body weight via gavage. Mice in the rapamycin intervention group were administered rapamycin (purchased from MedChemExpress, catalog number HY-10219, using 0.1 mL PBS buffer as a solvent) intraperitoneally at a dose of 10 mg / kg body weight via injection. Using PBS buffer as a solvent, mice were given an equal amount of the solvent as a control. Intervention was administered daily from day 3 to day 10 post-myocardial infarction. After the intervention, on day 10 post-myocardial infarction (i.e., day 10 post-myocardial infarction), the hydroxyproline content in the left ventricle of mice was measured using a hydroxyproline assay kit (BC0250, Solarbio). The results showed that in an acute myocardial infarction model, blocking the HGF signaling pathway with MET inhibitors or rapamycin significantly increased hydroxyproline levels. Figure 30The above experimental procedure was repeated in the TAC model. During the experiment, intervention with MET inhibitors and rapamycin was performed from day 7 to day 15 after TAC. It was found that inhibition of the HGF signaling pathway also led to a significant increase in hydroxyproline levels. Figure 31 ).

[0166] 8. HGF released by neutrophils controls myocardial fibrosis and myocardial injury repair after acute myocardial infarction.

[0167] Diagnosis of cardiac fibrosis largely relies on invasive tissue biopsy or detection via delayed gadolinium enhancement (LGE) and T1 mapping on cardiac magnetic resonance imaging. Because fibrosis involves multiple cell types, identifying plasma biomarkers for cardiac fibrosis has significant clinical value. To date, several molecular biomarkers for fibrosis have been reported in the literature, but these biomarkers are considered neither cardiac-derived nor known to reflect specific stages of fibrosis. HGF is classified into the turquoise module, which represents proliferating cardiac fibroblasts. This study selected 100 patients clinically diagnosed with myocardial fibrosis via delayed gadolinium enhancement on cardiac magnetic resonance imaging (collected from Fuwai Central China Cardiovascular Hospital) and found that HGF was significantly elevated in the plasma of patients with cardiac fibrosis. Figure 32 Patients with aortic stenosis experience increased cardiac afterload, leading to cardiac remodeling and myocardial fibrosis. This study included 15 patients with aortic stenosis (collected from Fuwai Central China Cardiovascular Hospital) and found that preoperative plasma HGF levels were significantly elevated in these patients. Figure 32 ).

[0168] Following acute myocardial infarction (AMI), cardiac repair is crucial for restoring cardiac function. Within two days of AMI, necrotic cardiomyocytes release large amounts of troponin, and a sharp increase in troponin T or troponin I in the blood is a clinical diagnostic criterion for AMI. This study monitored the dynamic changes in HGF levels in 46 hospitalized patients with acute myocardial infarction (ST-elevation and non-ST-elevation) (data collected from Fuwai Central China Cardiovascular Hospital). The study found that the dynamic changes in HGF could be broadly categorized into five types: timely transient elevation (significantly elevated within 3 days after infarction, followed by a decrease), persistent low-level elevation, persistent high-level elevation (more than 3 consecutive days, exceeding 10 times the normal level), recurrent and delayed elevation (…). Figure 33 The results of this study have also demonstrated that a timely and transient increase in plasma HGF represents a normal inflammatory response. Figure 5 Persistent low-level elevation and other types should be considered abnormal inflammatory responses.

[0169] To explore whether dynamic changes in hemoglobin (HGF) can predict the prognosis of hospitalized patients with acute myocardial infarction (AMI), this study categorized dynamic HGF changes into three groups: timely transient elevation, sustained low-level elevation, and other types (sustained high-level elevation, recurrent elevation, and delayed elevation). To assess the prognosis of hospitalized AMI patients, this study considered the following four conditions as post-AMI heart failure events: 1) new-onset heart failure; 2) worsening heart failure (progression from Killip classification II to III / IV, or from III to IV); 3) cardiogenic shock; and 4) death due to cardiogenic shock or heart failure. A good prognosis was considered to be achieved when patients met either of the following two criteria: 1) left ventricular ejection fraction (EF) > 50% at discharge and no heart failure events occurred; or 2) EF between 40% and 50% at discharge but improved during hospitalization and no heart failure events occurred during hospitalization. This study considered a poor prognosis for patients meeting any of the following criteria: 1) EF < 40% at discharge; 2) EF between 40% and 50% at discharge and decreasing during hospitalization; 3) Heart failure events during hospitalization. This study analyzed the correlation between dynamic changes in HGF after acute myocardial infarction and the prognosis of acute myocardial infarction patients during hospitalization. The analysis revealed that among the 46 hospitalized patients with acute myocardial infarction monitored, a timely and transient increase was significantly associated with a good prognosis, while other types of HGF were significantly associated with a poor prognosis. Figure 34 ).

[0170] The neutrophil-to-leukocyte ratio (NLR) can reflect the body's inflammatory state to some extent. Brain natriuretic peptide (BNP) is a recognized marker of heart failure, while the mass and activity of creatine kinase myocardial type (CK-MB) and high-sensitivity troponin (hs-cTnT) reflect cardiomyocyte damage. These clinical indicators can reflect the condition of patients with myocardial infarction to some extent. This study analyzed the correlation between HGF and clinical biochemical indicators (i.e., NLR, BNP, creatine kinase myocardial type (CK-MB) mass and activity, and hs-cTnT) detected after acute myocardial infarction. The analysis found that HGF was moderately correlated with NLR (Spearman correlation coefficient 0.5). This further confirms that HGF levels can reflect the activation state of neutrophils, thus accurately reflecting the inflammatory repair in patients with myocardial infarction. However, the correlation between HGF and cardiomyocyte indicators was low. Figure 35 ).

[0171] To investigate the importance of the HGF signaling pathway in myocardial repair after acute myocardial infarction, this study injected a MET inhibitor into a mouse model of myocardial infarction to block HGF signaling. Fifty-three mice with myocardial infarction, obtained through permanent left coronary artery ligation (LCA), were randomly divided into six groups: three control groups (n=28 each) and three treatment groups (n=25 each). After grouping, the treatment groups were administered a MET inhibitor (purchased from MedChemExpress (HY-50683), using 0.1 mL PBS buffer as the solvent) at a dose of 50 mg / kg body weight via gavage. The control groups were administered the same amount of the inhibitor via gavage as a control. Gavage was administered once daily for seven consecutive days, starting from day 1 post-myocardial infarction. The survival details of the mice were then observed. The study confirmed that adequate HGF signaling is crucial for cardiac repair after myocardial infarction. In an acute myocardial infarction mouse model, the survival rate of mice with myocardial infarction in the control group was 78%, while the survival rate of mice with myocardial infarction in the treatment group was approximately 32%. This demonstrates that blocking the HGF signaling pathway with MET inhibitors significantly reduced mouse survival. Figure 36 ).

[0172] The results of this study have also demonstrated that erythromycin can inhibit neutrophil infiltration and thus suppress the inflammatory response in damaged tissues, but erythromycin does not significantly affect neutrophil degranulation. Figure 5 To further investigate whether inhibiting neutrophil infiltration affects the survival of mice with myocardial infarction, this study injected erythromycin into a mouse model of myocardial infarction. Fifty-six mice with myocardial infarction, obtained through permanent left coronary artery ligation (LCA), were divided into six groups: a control group and three treatment groups. After grouping, the treatment groups received intraperitoneal injections of erythromycin (purchased from MedChemExpress, catalog number HY-B0220, using 0.1 mL PBS buffer as a solvent) at a dose of 100 mg / kg body weight. The control group received an equal volume of the solvent intraperitoneally as a control. Intervention was administered once daily from day 1 to day 6 post-myocardial infarction. Survival details were observed after the intervention. The results showed that erythromycin significantly reduced the survival rate of mice, further demonstrating that inhibiting inflammation is detrimental to cardiac repair. Figure 36 ).

[0173] To investigate how excessive HGF signaling affects repair after acute myocardial infarction, this study administered HGF on days 4–7 post-infarction and found that HGF strongly inhibited myocardial fibrosis. Figure 30To further confirm this conclusion, 53 mice with myocardial infarction model obtained through permanent left coronary artery ligation (LCA) were divided into 6 groups, with 3 groups in each control and treatment group. After grouping, the mice in the three treatment groups were injected intravenously with HGF (purchased from R&D, catalog number 2207-HG-025, using 0.1 mL PBS buffer as solvent) at a dose of 250 μg / kg mouse body weight. The control group received an equal amount of solvent via intravenous injection. The intervention was performed once daily from day 1 to day 3 after myocardial infarction. After the intervention, the survival details of the mice were observed. The results showed that due to its inhibition of cardiac repair, HGF injection on days 4 to 6 after myocardial infarction significantly increased the mortality rate of mice. Figure 36 These results indicate that persistently elevated HGF levels are also detrimental to cardiac repair. Autopsy and pathological examination of all treated myocardial infarction model mice confirmed that these acute myocardial infarction model mice died from cardiac rupture.

[0174] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A molecular marker, characterized in that, The molecular marker includes hepatocyte growth factor; the molecular marker has any of the following functions: (a) Diagnosis of cardiac fibrosis; (b) Assess the severity of cardiac fibrosis; (c) Assess the prognosis of acute myocardial infarction; Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction.

2. The molecular marker as described in claim 1, characterized in that, The cardiac fibrosis includes cardiac fibrosis caused by pressure overload and / or cardiac fibrosis caused by myocardial infarction; Other cardiovascular diseases associated with cardiac fibrosis besides acute myocardial infarction include hypertension, diastolic heart failure, cardiomyopathy, and / or sudden cardiac death.

3. The application of the reagent for detecting the molecular marker of claim 1 or 2 in a test sample in the preparation of a product, characterized in that, The product has any of the following functions: (a) Diagnosis of cardiac fibrosis; (b) Assess the severity of cardiac fibrosis; (c) Assess the prognosis of acute myocardial infarction; Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction.

4. A product characterized in that, The product includes a reagent for detecting the molecular markers of claim 1 or 2 in a sample to be tested; the product has any of the following functions: (a) Diagnosis of cardiac fibrosis; (b) Assess the severity of cardiac fibrosis; (c) Assess the prognosis of acute myocardial infarction; Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction.

5. A prediction model, characterized in that, The prediction model has any of the following functions: (a) Diagnosis of cardiac fibrosis; (b) Assess the severity of cardiac fibrosis; (c) Assess the prognosis of acute myocardial infarction; Alternatively, (d) assess the prognosis of other cardiovascular diseases with cardiac fibrosis besides acute myocardial infarction; The prediction model includes a detection module, a data analysis module, and a prediction module; The detection module is used to detect the level of the molecular markers described in claim 1 or 2 in the sample to be tested; The data analysis module is used to analyze the detection results output by the detection module; The prediction module is used to classify the sample to be tested based on the analysis results output by the data analysis module, determining whether it has a disease or a good or bad prognosis.

6. A drug screening method, characterized in that, The drugs include those for treating cardiac fibrosis, acute myocardial infarction, and / or other cardiovascular diseases with cardiac fibrosis other than acute myocardial infarction; The method includes: administering the drug to be screened to the experimental subjects; obtaining results related to the molecular markers described in claim 1 or 2 after the administration of the drug to be screened to the experimental subjects; and determining whether the drug to be screened is effective or ineffective based on the obtained results.

7. The use of hepatocyte growth factor, hepatocyte growth factor signaling pathway blockers, and / or neutrophil infiltration inhibitors in the preparation of pharmaceuticals, characterized in that, The drug has any of the following functions: (a) Prevention and / or treatment of cardiac fibrosis; (b) Prevention and / or treatment of acute myocardial infarction; Alternatively, (c) prevent and / or treat other cardiovascular diseases associated with cardiac fibrosis, other than acute myocardial infarction.

8. The application as described in claim 7, characterized in that, When the drug has the function of preventing and / or treating cardiac fibrosis or other cardiovascular diseases accompanied by cardiac fibrosis other than acute myocardial infarction, the drug contains hepatocyte growth factor and / or hepatocyte growth factor signaling pathway blocker. When the drug has the function of preventing and / or treating acute myocardial infarction, the drug contains hepatocyte growth factor, or the drug contains a hepatocyte growth factor signaling pathway blocker and / or a neutrophil infiltration inhibitor.

9. The application as described in claim 7 or 8, characterized in that, The treatment of acute myocardial infarction includes treating acute myocardial infarction by controlling fibroblast proliferation dominated by acute inflammatory response and / or controlling myofibroblast-mediated fibrosis repair.

10. A drug, characterized in that, The drug has any of the following functions: (a) Prevention and / or treatment of cardiac fibrosis; (b) Prevention and / or treatment of acute myocardial infarction; Alternatively, (c) prevent and / or treat other cardiovascular diseases associated with cardiac fibrosis, other than acute myocardial infarction; The drug contains hepatocyte growth factor, hepatocyte growth factor signaling pathway blockers, and / or neutrophil infiltration inhibitors.

Images