Chronic heart failure blood stasis syndrome biomarker and application thereof

Abstract

The invention discloses a chronic heart failure blood stasis syndrome biomarker and application thereof. The biomarker is selected from at least one of AFM, PCSK9 and APOC1. The chronic heart failure blood stasis syndrome biomarker provided by the invention has high sensitivity and specificity when being applied to clinical diagnosis or auxiliary diagnosis of chronic heart failure blood stasis syndrome patients, and can effectively distinguish the chronic heart failure blood stasis syndrome patients from healthy people and chronic heart failure non-blood stasis syndrome patients; reliable basis and practical guidance can be provided for clinical diagnosis and drug treatment of the blood stasis syndrome of the chronic heart failure, the accuracy of clinical diagnosis of the heart failure is improved, and the accuracy and standardization degree of traditional Chinese medicine syndrome differentiation diagnosis are improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a biomarker for blood stasis syndrome in chronic heart failure and its application in the preparation of products for the diagnosis or auxiliary diagnosis of blood stasis syndrome in chronic heart failure. Background Technology

[0002] Heart failure (HF) is a complex clinical syndrome caused by various factors leading to abnormal changes in the heart's structure or its diastolic and systolic functions. Its main clinical manifestations include chest tightness, shortness of breath, fatigue, dyspnea, and fluid retention. HF is not an independent disease but rather the end stage of the progression of heart disease. Based on the urgency of the condition, it is clinically divided into acute heart failure (AF) and chronic heart failure (CHF). Although treatment strategies for chronic heart failure are continuously being optimized, and drug and device therapies have made significant progress thanks to technological advancements, improving patient prognosis and quality of life, readmission and mortality rates remain high.

[0003] From the perspective of Traditional Chinese Medicine (TCM), the symptoms and clinical manifestations of chronic heart failure are similar to those of diseases such as palpitations, phlegm retention, and edema. Blood stasis syndrome is closely related to the occurrence and development of chronic heart failure. (From *Suwen*) The *Bi Lun* (Treatise on Bi Syndrome) states, "When the pulse is obstructed and further affected by pathogenic factors, it lodges internally in the heart… Heart obstruction is characterized by blocked pulses," vividly describing the pathological state of blood vessel obstruction and laying the theoretical foundation for the connection between blood stasis and heart failure. Modern research has found that the blood vessel obstruction emphasized in traditional Chinese medicine's theory of blood stasis has a potential link with the pathological mechanisms of circulatory disorders and hemodynamic abnormalities observed in patients with chronic heart failure in modern medicine. The 2016 *Expert Consensus on the Diagnosis and Treatment of Chronic Heart Failure Using Integrated Traditional Chinese and Western Medicine* points out that the location of chronic heart failure is in the heart, and its pathogenesis is characterized by "stasis," "deficiency," and "water retention." "Stasis" permeates the entire pathological process of chronic heart failure, clarifying the important role of blood stasis in this process. Blood stasis syndrome is one of the important pathological links in heart failure, having a profound impact on its occurrence, development, and prognosis. The *Huangdi Neijing* (Yellow Emperor's Inner Classic) records: "Heart obstruction is characterized by blocked pulses." The *Jinkui Yaolue* (Essential Prescriptions of the Golden Chamber) already stated: "When blood is not flowing smoothly, it becomes water retention." *Yilin Gaicuo* (Corrections of Errors in Medical Classics) states: "When the vital energy is deficient, it cannot reach the blood vessels; when the blood vessels lack energy, they will stagnate and become stagnant." Deficiency of heart qi is the underlying cause of heart failure. Qi deficiency weakens the body's ability to propel blood circulation, leading to blood stasis and ultimately blood stasis syndrome. Qi deficiency combined with impaired circulation is the key mechanism for blood stasis formation. Furthermore, heart failure patients often experience further damage to heart qi and exacerbate blood stasis due to emotional distress or external exposure to wind and cold. Qi flow is the foundation of blood flow. Impaired qi and blood circulation leads to blood stasis, congesting the heart and lungs – this is the central pathological link in heart failure. Modern medical research has found a high degree of consistency between blood stasis syndrome and the pathological mechanism of heart failure: heart failure patients often exhibit hemodynamic abnormalities, vascular endothelial dysfunction, hypercoagulable states, and microcirculatory disturbances. The pathogenesis of chronic heart failure follows a pattern of progression from qi to blood, from blood to fluids, from superficial to deep, and from mild to severe. In this process, qi, blood, and fluids interact, are mutually causal, and intertwine, collectively constituting a complex pathological state. Qi deficiency is the root cause of its onset, while blood stasis syndrome is the primary pathological manifestation. Therefore, in-depth exploration of the connotations of blood stasis syndrome in chronic heart failure is of profound significance.

[0004] Syndrome differentiation is the premise and foundation of TCM syndrome differentiation and treatment. Traditional TCM theory has formed a relatively systematic understanding of heart failure with blood stasis syndrome, the core pathogenesis of which lies in qi deficiency and blood stasis, and obstruction of the meridians. This understanding guides clinical syndrome differentiation and treatment, accumulating rich experience. However, traditional syndrome differentiation methods mainly rely on the physician's subjective observation, auscultation, inquiry, and palpation. The criteria for judgment have a certain degree of subjectivity and reliance on experience, lacking a quantifiable and repeatable objective evaluation system widely recognized by modern medicine. This subjectivity limits the standardization and normalization of TCM syndrome diagnosis and hinders the integration of TCM efficacy evaluation with international standards.

[0005] Biomarkers are increasingly used in the diagnosis and treatment of chronic heart failure (CHF). Currently, commonly used clinical biomarkers include natriuretic peptides (such as BNP and NT-proBNP), cardiac troponin (cTnI and cTnT), and inflammatory markers (such as IL-6 and CRP). These biomarkers play important roles in the prediction, diagnosis, and prognostic assessment of heart failure. However, a biomarker that can specifically identify blood stasis syndrome in chronic heart failure is still lacking. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker for chronic heart failure with blood stasis syndrome that can specifically identify chronic heart failure with blood stasis syndrome.

[0007] According to one aspect of the present invention, a biomarker for blood stasis syndrome in chronic heart failure is provided, which is selected from at least one of proprotein convertase subtilisin / kexin type 9 (PCSK9), AFM (Afamin), and apolipoprotein C1 (APOC1).

[0008] To address the lack of objective biological indicators for diagnosing blood stasis syndrome in chronic heart failure using Traditional Chinese Medicine (TCM), this invention collects serum samples from healthy individuals and those with and without blood stasis syndrome in chronic heart failure patients. Using proteomics technology, various differentially expressed proteins are screened, including UBB, PAM, AFM, PCSK9, B3GNT2, APOC1, F13A1, and IGLV7-43. Finally, by expanding the sample size for validation, three biomarkers for chronic heart failure with blood stasis syndrome—PCSK9, AFM, and APOC1—were obtained that can specifically identify this syndrome.

[0009] The biomarkers PCSK9, AFM, and APOC1 provided by this invention for chronic heart failure with blood stasis syndrome, when used alone or in any combination, have high sensitivity and specificity for the diagnosis or auxiliary diagnosis of chronic heart failure with blood stasis syndrome. They can effectively distinguish patients with chronic heart failure with blood stasis syndrome from healthy individuals and patients with chronic heart failure without blood stasis syndrome. They can serve as specific biomarkers for the diagnosis or auxiliary diagnosis of chronic heart failure with blood stasis syndrome, accurately identifying patients with chronic heart failure with blood stasis syndrome, and achieving precise differentiation and diagnosis of patients with chronic heart failure with blood stasis syndrome, thereby meeting the needs of refined clinical diagnosis and treatment and the development of personalized treatment plans.

[0010] The biomarkers for blood stasis syndrome in chronic heart failure provided by this invention can provide a reliable basis and practical guidance for the clinical diagnosis and drug treatment of blood stasis syndrome in chronic heart failure, improve the accuracy of clinical diagnosis of heart failure, and also help improve the accuracy and standardization of TCM syndrome differentiation and diagnosis, and help to promote the standardized application of TCM syndrome differentiation and treatment for chronic heart failure.

[0011] In this invention, the three proteins PCSK9, AFM, and APOC1 can be used individually as biomarkers for diagnosing or assisting in the diagnosis of whether a patient has chronic heart failure with blood stasis syndrome, and can be applied to the preparation of products capable of diagnosing or assisting in the diagnosis of chronic heart failure with blood stasis syndrome. Alternatively, two or three proteins can be used in combination as biomarkers for diagnosing or assisting in the diagnosis of chronic heart failure with blood stasis syndrome, and can be applied to the preparation of products capable of diagnosing or assisting in the diagnosis of chronic heart failure with blood stasis syndrome.

[0012] In this invention, products used for diagnosing or assisting in the diagnosis of blood stasis syndrome in chronic heart failure include, but are not limited to, reagents, kits, chips, or instruments used for diagnosing or assisting in the diagnosis of whether an examinee is a patient with blood stasis syndrome in chronic heart failure.

[0013] According to another aspect of the present invention, the use of a product for detecting the content of a biomarker for chronic heart failure with blood stasis syndrome in a sample in the preparation of a product for diagnosing or assisting in the diagnosis of chronic heart failure with blood stasis syndrome is provided, wherein the biomarker for chronic heart failure with blood stasis syndrome is selected from at least one of PCSK9, AFM, and APOC1.

[0014] In this invention, the product for detecting the content of biomarkers for blood stasis syndrome in chronic heart failure can be any product that uses any known technology in the art to detect the content of PCSK9, AFM, or APOC1 protein in a sample, including but not limited to reagents, kits, chips, and / or instruments suitable for detecting the content of related proteins by at least one of the following methods: Western blotting, immunoturbidimetry, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, mass spectrometry, protein chips, or proteomics.

[0015] Specifically, products for detecting the content of relevant proteins in samples can be mass spectrometers, ELISA protein detection reagents or kits, Western blotting reagents or kits, etc.

[0016] In some implementations, the sample is peripheral blood serum.

[0017] In some implementations, the product for detecting the content of biomarkers for blood stasis syndrome in peripheral blood serum of chronic heart failure can be a reagent, kit, chip, and / or instrument suitable for detecting the content of biomarkers for blood stasis syndrome in peripheral blood serum of chronic heart failure by enzyme-linked immunosorbent assay (ELISA).

[0018] In some implementations, the product for detecting the levels of biomarkers for blood stasis syndrome in peripheral blood serum of chronic heart failure can be a kit suitable for detecting the levels of biomarkers for blood stasis syndrome in peripheral blood serum of chronic heart failure by enzyme-linked immunosorbent assay (ELISA).

[0019] In some embodiments, the kit may be an ELISA protein detection kit. In addition to antibodies against the corresponding proteins, the kit may also include other components such as buffers, washing solutions, diluents, blocking solutions, stop solutions, and chromogenic solutions. Those skilled in the art can select appropriate components and amounts according to actual needs, and this invention does not impose specific limitations in this regard. Attached Figure Description

[0020] Figures 1-3 PCA diagrams of histones from the HF-Y group and the CON group, the HF-Y group and the HF group, and the HF group and the CON group, respectively. Figures 4-6 Volcano diagrams of differentially expressed proteins between the HF-Y group and the CON group, the HF-Y group and the HF group, and the HF group and the CON group, respectively. Figures 7-9 The top 30 GO enrichment items related to biological processes, cellular components, and molecular functions of differentially expressed proteins between the HF-Y group and the CON group, the HF-Y group and the HF group and the HF group and the CON group, respectively. Figures 10-12 Bubble diagrams of the first 20 KEGG pathways in the HF-Y group and CON group, the HF-Y group and HF group, and the HF group and CON group, respectively. Figure 13 The image shows the analysis results for Venn. Figure 14 The results of ELISA validation of the differential expression of B3GNT2, AFM, APOC1, UBB, F13A1, PAM and PCSK9 in healthy individuals (CON), patients with heart failure without blood stasis syndrome (HF), and patients with heart failure with blood stasis syndrome (HF-Y); Figure 15 ROC curves for AFM, PCSK9, and APOC1 used individually to distinguish between HF-Y and HF; Figures 16-19 ROC curves for identifying HF-Y and HF can be obtained by combining any two or three of the biomarkers from AFM, PCSK9, and APOC1. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. Unless otherwise specified, the reagents used in the embodiments are commercially available conventional products, and the experimental methods not specifically specified in the embodiments are conventional methods and conditions well known in the art, or according to the conditions recommended by the reagent kit and instrument manufacturer.

[0022] Example 1 1. Source of cases Cases were selected from healthy individuals in the community and outpatients or inpatients of Guangdong Provincial Hospital of Traditional Chinese Medicine. Peripheral venous blood was collected according to standard operating procedures, and serum samples were collected. Among them, there were 5 healthy individuals (CON), 5 patients with chronic heart failure without blood stasis syndrome (HF), and 5 patients with chronic heart failure with blood stasis syndrome (HF-Y).

[0023] 2. Disease Diagnostic Criteria (1) Diagnostic criteria for diseases in Western medicine The diagnostic criteria for chronic heart failure are based on the standards outlined in the "Guidelines for the Diagnosis and Treatment of Heart Failure in China 2024" published by the Chinese Society of Cardiology. The diagnostic process for chronic heart failure is followed.

[0024] a. Diagnostic criteria for heart failure: The diagnosis of heart failure is based on the "Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2024", the AHA / ACC "2013 Guidelines for the Management of Heart Failure", and the ESC "2016 Guidelines for the Diagnosis and Treatment of Acute and Chronic Heart Failure". The diagnosis is made by comprehensively analyzing the patient's clinical symptoms, signs, echocardiography, and NT-proBNP levels to determine whether the patient is in the blood stasis syndrome group or the non-blood stasis syndrome group.

[0025] b. Criteria for Determining Cardiac Function Classification According to the relevant guidelines of the New York Heart Association, the degree of impairment of cardiac function is classified into four grades based on the degree of activity that induces heart failure symptoms, as shown in Table 1.

[0026] Table 1 New York Heart Association Functional Classification

[0027] (2) Diagnostic criteria for diseases in Traditional Chinese Medicine: The diagnostic criteria for blood stasis syndrome refer to the "Practical Diagnostic Criteria for Blood Stasis Syndrome" issued by the Professional Committee on Promoting Blood Activation and Removing Blood Stasis of the Chinese Association of Integrative Medicine in 2016.

[0028] 1) Disease diagnosis: Main symptoms: chest tightness, shortness of breath, fatigue, and palpitations.

[0029] Secondary symptoms: In the early stages of the disease, patients mainly experience fatigue after activity, palpitations accompanied by shortness of breath, spontaneous sweating, and sudden onset of wheezing and coughing at night. Symptoms are relieved when sitting upright. They often experience lethargy or a weak voice, and a pale, lackluster complexion. As the disease progresses, palpitations become more frequent, and patients experience worsening shortness of breath after activity, inability to lie flat, and generalized edema of the limbs and eyelids. A history of chest pain, coronary heart disease, heart failure, or myocardial infarction is common.

[0030] 2) Syndrome diagnosis: Main criteria: (1) Dark purple tongue or ecchymosis or petechiae; (2) Bluish or dark purple face, lips, gums, eye area and finger (toe) tips; (3) Varicose veins or abnormal dilation of capillaries in different parts; (4) Extramenstrual blood (blood stasis or accumulation in organs, tissues, subcutaneous or serous cavities caused by bleeding); (5) Intermittent claudication; (6) Abdominal tenderness and resistance; (7) Amenorrhea or dark menstrual blood with clots; (8) Imaging showing objective evidence of vascular occlusion or moderate to severe stenosis (≥50%), thrombosis, infarction or embolism, or organ ischemia.

[0031] Secondary criteria: (1) Fixed pain, or stabbing pain, colic, or pain that is worse at night; (2) Numbness or hemiplegia of limbs; (3) Dysmenorrhea; (4) Skin roughness (rough, thickened skin, increased scales); (5) Mania or forgetfulness; (6) Choppy or intermittent pulse, or no pulse; (7) Enlarged organs, neoplasms, inflammatory or non-inflammatory masses, tissue hyperplasia; (8) Imaging examinations showing vascular stenosis (<50%); (9) Abnormal physical and chemical tests such as blood rheology, coagulation, fibrinolysis, and microcirculation, indicating blood stasis; (10) Trauma, surgery, or induced abortion within the past month.

[0032] A diagnosis of blood stasis syndrome can be made if one primary criterion or two secondary criteria are met. Each primary criterion is worth 2 points, and each secondary criterion is worth 1 point; this can be used as a quantitative diagnostic standard for blood stasis syndrome.

[0033] 3. Inclusion criteria (1) Patients with heart failure 1) Meets the diagnostic criteria for diseases in both traditional Chinese and Western medicine; 2) All enrolled patients signed informed consent forms; 3) The age range for enrollment is 18-80 years old; 4) Patients who have undergone rigorous diagnosis by two physicians with the title of associate chief physician or above, and who meet the clinical symptoms of chronic heart failure with blood stasis syndrome, can be included in the heart failure with blood stasis syndrome group. 5) Patients admitted to the TCM syndrome group must undergo careful diagnosis by two physicians with the title of associate chief physician or above.

[0034] (2) Healthy people 1) Age 18-80 years old; 2) A written informed consent form must be signed; 3) Previous physical examinations have not diagnosed the following systemic diseases: including respiratory system, digestive system, circulatory system, endocrine system, nervous system, etc.

[0035] 4. Exclusion criteria (1) Patients with heart failure 1) Patients who meet the relevant diagnostic criteria but cannot provide specific information, or patients who refuse to participate in this study; 2) Patients with impaired consciousness (including drowsiness, coma, or waking coma) or mental illness (such as schizophrenia, Alzheimer's disease, major depressive disorder, etc.) are not included in the study because they cannot fully understand the content of this research project or have difficulty cooperating with the TCM diagnosis process. 3) Complications such as cardiogenic shock or severe arrhythmias (ventricular rate > 160 beats / min, atrial flutter, rapid atrial fibrillation) that affect the diagnosis and data collection of this disease; 4) Comorbid severe liver and kidney dysfunction (ALT and AST levels are more than twice the normal range, or serum creatinine is >265 μmol / L). 5) Pregnant or planning-to-be-pregnant women, and breastfeeding women; 6) Comorbid active bleeding or severe hematopoietic system disease, malignant tumor, or patient life expectancy of less than 3 years; 7) Those with a long history of chronic gastroenteritis (such as Crohn's disease, multiple intestinal polyps, ulcerative colitis, etc.), long-term chronic diarrhea or constipation, or a history of hereditary intestinal diseases; 9) Patients who are unable to attend follow-up examinations on time and are expected to have poor compliance.

[0036] (2) Healthy people 1) Those who have taken gastrointestinal motility modifiers, anti-inflammatory drugs, or traditional Chinese medicine in the past two weeks; 2) Abnormally elevated liver and kidney function (ALT or AST levels exceeding the upper limit by 2 times, or serum creatinine > 265 μmol / L); 3) Patients who are expected to have poor compliance and fail to cooperate with subsequent follow-up examinations.

[0037] 5. Initial screening of DIA proteomics 5.1 Sample Preparation Grouping criteria: Healthy control group (CON): meets the modern medical standards for healthy individuals, but does not meet the diagnostic criteria for heart failure and blood stasis syndrome; Heart failure without blood stasis syndrome (HF): meets the diagnostic criteria for heart failure but does not meet the diagnostic criteria for blood stasis syndrome; Heart failure and blood stasis syndrome group (HF-Y): meets the diagnosis of heart failure and blood stasis syndrome.

[0038] The initial screening of serum samples was conducted with 5 cases per group.

[0039] 5 mL of venous blood was collected from each patient, allowed to stand at room temperature for 2 hours, and then incubated at 4°C and 1500× g Centrifuge for 10 minutes, collect the supernatant, aliquot it, and store it in a -80℃ refrigerator for later use.

[0040] 5.2 DIA Proteomics Detection Serum samples were retrieved from the Guangdong Provincial Hospital of Traditional Chinese Medicine's Biological Resources Center and tested by Shanghai Lumin Biotechnology Co., Ltd. The process included the following steps: (1) Sample processing First, transfer the frozen sample to a low protein binding tube (1.5 mL Eppendorf tube). Add 300 µL of lysis buffer (containing 1 mM PMSF). Sonicate using 1 second / 1 second intervals for 3 minutes at 80 W. Incubate at 15000× at 4°C. g Centrifuge for 15 minutes to remove insoluble particles, repeat once to further remove precipitate. Determine protein concentration by BCA method, aliquot and store at -80°C.

[0041] (2) Protein extraction The Plasma High Abundance Protein Removal Kit (small column) (ThermoScientific, 85165) and the Plasma High Abundance Protein Removal Kit (large column) (Millipore, 122642) were used. Specific steps included: Equilibrate the high-abundance protein column to room temperature. Remove the screw cap from the column and add 10 µL of sample directly to the resin in the column. Replace the column cap and invert it several times to ensure complete resin suspension. Gently mix at room temperature for 60 minutes, ensuring thorough mixing of the sample and resin. Unscrew the bottom seal, loosen the cap, and place the column into a 2 mL collection tube. Incubate at 4°C for 1000× [temperature missing]. g Centrifuge for 2 minutes. Discard the column containing resin; the collected filtrate is the sample from which high-abundance proteins have been removed and can be used for subsequent processing or storage.

[0042] (3) Proteolytic enzyme digestion SDS-polyacrylamide gel electrophoresis: Separation was performed using 12% SDS-PAGE, followed by staining, fixation for 2 hours, and staining for 12 hours. The gel was rinsed with water until the bands were clearly visible. The stained gel was scanned using an automated digital gel imaging system.

[0043] The sample was thoroughly mixed with pre-cooled acetone solution (5 times its volume), and the mixture was allowed to stand overnight at -20°C to allow precipitation to complete. The next day, the sample was transferred to a centrifuge and centrifuged at 12000 × 10⁻⁶ at 4°C. gCentrifuge for 10 minutes and carefully collect the precipitate. Then, allow the precipitate to air dry at room temperature for 3 minutes, and dissolve it in SDS lysis buffer for 2 hours. After complete dissolution, centrifuge again to remove undissolved impurities. Determine the solution concentration using the BCA protein concentration assay, and finally aliquot and store according to experimental requirements.

[0044] (4) Peptide labeling Resuspend the lyophilized sample in 100 µL of 200 mM TEAB and transfer 40 µL to a new tube for labeling. Add 200 µL of isopropanol to the iTRAQ reagent bottle, dissolve at room temperature for 5 minutes, and centrifuge. Add an equal volume of 100 µL of iTRAQ labeling reagent to each sample and incubate at room temperature for 2 hours. Add 200 µL of HPLC water and incubate for 30 minutes to terminate the reaction. Lyophilize the labeled peptide solution and store at -80°C.

[0045] (5) Proteomics library construction based on data-dependent acquisition (DDA) Sample pretreatment procedure: Peptide samples dried by vacuum centrifugation were reconstituted using mobile phase A containing 2% acetonitrile and 0.1% formic acid. Subsequently, the samples were centrifuged at 20000 × 10⁻⁶ ppm. g Centrifuge for 10 minutes and collect the supernatant for subsequent analysis.

[0046] Chromatographic separation system: A UltiMate 3000 UHPLC ultra-high performance liquid chromatograph (equipped with a nano-scale chromatography system) was used for sample separation. The specific operation steps were as follows: Sample loading stage: The pretreated sample solution was injected into an AcclaimPepMap 100 pre-column (C18, 100 µm × 2 cm, 5 µm) for online enrichment and desalting.

[0047] (6) Data Dependency Acquisition (DDA) Detection Full-scan MS was performed in the range of 350–1650 m / z with a mass resolution of 120,000. The 20 most intense peaks in the MS were fragmented using high-energy collisional dissociation (HCD). For DDA mode data acquisition using a Q-Exactive HF mass spectrometer, efficient peptide fragmentation and signal capture were achieved by setting the following parameters: maximum ion implantation time 30 ms; AGC target value 3 × 10⁻⁶. 6 Secondary mass spectrometry parameters: collision-induced dissociation energy set to NCE 28; fragment ion scan resolution 15,000 (@m / z 200); AGC target value 1×10⁻⁶. 5 Maximum ion implantation time: 54 ms; Dynamic exclusion window: 28 seconds; Precursor ion screening criteria: charge state range 2+ to 6+; Top 20 high-intensity precursor ions (threshold ≥ 5 × 10⁻⁶). 4Positive ion mode detection: Peptides separated by chromatography are introduced into the mass spectrometer via a nanoESI ion source (voltage 2.0 kV), and a gradient elution strategy is used to achieve efficient ionization. This parameter combination optimizes ion transport efficiency and fragmentation energy, ensuring high-reliability tandem mass spectrometry data in complex samples.

[0048] (7) Mass spectrometry detection based on data-independent acquisition (DIA) The peptides separated by liquid phase were ionized using a nanoESI ion source (spray voltage set to 1.9 kV), followed by data acquisition-independent analysis (DIA) using a Q-Exactive HF mass spectrometer. Fragmentation mode: high-energy collision-induced dissociation (HCD); collision energy: NCE28; fragmentation resolution: 30,000 (@m / z 200); AGC target value: 1×10⁻⁶. 6 Maximum ion implantation time: Automatic mode (≤100 ms); Dynamic exclusion time: 28 seconds. This detection strategy optimizes ionization efficiency and fragmentation energy parameters, combined with a wide-range mass window scanning technique, to ensure unbiased acquisition of full-spectrum peptide signals in complex sample backgrounds. The dynamic range of ion intensity is monitored in real time during data acquisition, and automatic gain control (AGC) technology is used to maintain the stability of detection sensitivity.

[0049] (8) DDA data processing flow DDA Data Validation: MaxQuant software (version 1.6.14) was used to supplement the analysis of the raw data acquired in DDA mode. Peptide identification was performed based on the UniProt protein database, with the following search parameters set: Enzyme digestion specificity: Trypsin / P (maximum of 2 miscleavages allowed); Fixed modification: Carbamidomethyl (C); Variable modification: Oxidation (M), Acetyl (Protein N-term); Mass tolerance: Level 1 mass spectrometry ±4.5 ppm, Level 2 mass spectrometry ±20 mmu. A target decoy search strategy was used to control the false detection rate (FDR) to ≤1%, ensuring the reliability of the identification results. This analytical workflow integrates multiple software platforms to achieve complete resolution from raw mass spectrometry signals to quantitative proteomics data, providing fundamental data support for subsequent differential expression analysis and functional annotation. A fold change (FC) ≥1.2 or <0.83 was used as the threshold for peptide identification. P A value <0.05 is used as a scoring criterion for significant protein expression.

[0050] (9) Bioinformatics analysis The bioinformatics analysis workflow for quantitative proteomics based on DIA technology is as follows: Data processing and quality control stage: First, a reliable protein set is screened using protein confidence filtering (1% FDR threshold) and missing value imputation strategies. Then, rigorous statistical tests (such as Student's t-test or SAM algorithm) are used to identify differentially expressed proteins, with |log2(fold change)| > 1.2 or < 0.83 and p-value < 0.05 as the screening criteria.

[0051] Functional annotation and pathway enrichment analysis: Multi-dimensional functional analysis of differentially expressed proteins; Molecular functional annotation: Enrichment analysis of biological processes, cellular components, and molecular functions was conducted using the Gene Ontology (GO) database (FDR≤0.05); Pathway mapping: Signal transduction network analysis was performed based on pathway databases such as KEGG and BioCarta, and significantly enriched biological pathways were identified using hypergeometric tests (p<0.05); Protein-protein interaction network construction: Protein-protein interaction (PPI) networks were constructed using the STRING database (v11.5), and module analysis was performed using Cytoscape software; Data visualization and candidate selection: Dimensionality reduction and feature extraction were achieved using the following methods: Expression pattern clustering: A hierarchical clustering algorithm (Euclidean distance, Ward method) was used to generate heatmaps to visualize differential patterns; Correlation analysis: Pearson correlation coefficient matrices were calculated between samples to assess experimental repeatability; Intersection analysis: Common differentially expressed proteins were identified across multiple comparison groups using Venn diagrams. Final screening strategy: The following indicators were considered to establish a candidate protein set (size ≤20): fold change: proteins with high abundance variation were given priority; functional significance: node proteins enriched in disease-related pathways; interaction network: hub proteins located at the core of the PPI network.

[0052] 5.3 Results of DIA proteomics preliminary screening Peripheral venous blood was collected from healthy individuals in the community and outpatients or inpatients of Guangdong Provincial Hospital of Traditional Chinese Medicine according to standard operating procedures, and serum samples were collected. Five samples were from healthy individuals, five samples from patients with heart failure without blood stasis syndrome, and five samples from patients with heart failure with blood stasis syndrome. DDA library construction and DIA mass spectrometry analysis were performed on the serum samples using 4D-DIA proteomics technology. Data with at least half of the data being non-empty within each group were analyzed for significant differences. Data with expression fold changes >1.2 or <0.83 (up- or down-regulation) were selected. P Proteins with a T-value (T test) less than 0.05 were identified as differentially expressed proteins, and candidate proteins for heart failure and blood stasis syndrome were determined by combining bioinformatics GO and KEGG enrichment analyses.

[0053] The results are as follows Figures 1-17 As shown.

[0054] The results show: (1) Principal component analysis (PCA) was performed on the heart failure blood stasis syndrome group (HF-Y), the heart failure non-blood stasis syndrome group (HF), and the healthy control group (CON). The results showed that the heart failure blood stasis syndrome group and the healthy control group showed a significant spatial separation trend. Figure 1 The heart failure non-blood stasis syndrome group samples showed a unique distribution pattern: some data points overlapped with the healthy control group, while other individual data showed clustering with the heart failure blood stasis syndrome group. Figures 2-3 ).

[0055] (2) Volcano map ( Figures 4-6 The results showed that, using FC>1.2 or <0.83 and p-value<0.05 as screening criteria, there were 91 differentially expressed proteins between the HF-Y group and the CON group. Compared with the CON group, 77 proteins showed an increasing trend, and 14 proteins showed a decreasing trend. Figure 4 There were 77 differentially expressed proteins between the HF and CON groups. Compared with the CON group, 56 proteins showed an increasing trend, while 21 proteins showed a decreasing trend. Figure 6 Compared with the HF group, the HF-Y group had 50 differentially expressed proteins, with 33 showing an increasing trend and 17 showing a decreasing trend. Figure 5 ).

[0056] (3) Heatmap results: Differential proteins among the three groups showed a clustered distribution.

[0057] (4) GO and KEGG analyses were performed on the differentially expressed proteins between the HF-Y group and the CON group and the HF group, such as... Figures 7-12 As shown.

[0058] GO function analysis showed that the HF-Y group had gene functional differences in antifungal immunity and coagulation processes compared to the CON group. Figure 7 Compared with the HF group, the HF-Y group showed differences in the function of genes in melanosome and chromatin-related cellular components. Figure 8 Compared with the CON group, the HF group showed differences in gene function related to Toll-like receptor 4-associated immune signaling. Figure 9 ).

[0059] The bubble chart of the top 20 pathways in KEGG enrichment analysis is shown below. Figures 10-12 As shown.

[0060] (5) Venn diagram ( Figure 13The results showed that 10 differentially expressed proteins were significantly different between the HF-Y group and the CON group and the HF group, but there was no difference between the CON group and the HF group. Seven differentially expressed proteins (upregulated proteins UBB, PAM, AFM, PCSK9, B3GNT2, downregulated proteins APOC1, F13A1) were selected as candidate differentially expressed proteins for further validation with a larger sample size.

[0061] Example 2: Validation of candidate differentially expressed proteins The sample grouping criteria and sample collection and preparation methods are the same as in Example 1.

[0062] A total of 60 samples were collected, including 20 samples from patients with heart failure and blood stasis syndrome (HF-Y), 20 samples from patients with heart failure without blood stasis syndrome (HF), and 20 samples from healthy individuals (CON). None of the samples overlapped with the initial screening samples.

[0063] 1. ELISA validation of candidate differentially expressed proteins The relevant reagent kits are shown in Table 2.

[0064] Table 2 ELISA kits

[0065] The ELISA test procedure includes the following steps: (1) Move all reagents to room temperature (18~25℃) and equilibrate for at least 30 minutes. Prepare the reagents according to the kit instructions and set aside.

[0066] (2) Sample addition: Set up standard wells and test sample wells respectively. Add 100 µL of standard or test sample to each well, shake gently to mix, cover with plate, and incubate at 37°C for 2 hours.

[0067] (3) Discard the liquid, shake dry, no need to wash.

[0068] (4) Add 100 µL of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0069] (5) Discard the liquid in the wells, spin dry, and wash the plate 3 times. Soak for 2 minutes each time, 200 µL per well, and spin dry.

[0070] (6) Add 100 µL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0071] (7) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200 µL per well, and spin dry.

[0072] (8) Add 90 µL of substrate solution to each well in sequence and develop color at 37°C in the dark for 15-30 minutes.

[0073] (9) Add 50 µL of stop solution to each well in sequence to terminate the reaction.

[0074] (10) Within 5 minutes after the reaction is terminated, measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm using an ELISA reader. Other specific experimental procedures shall be performed in accordance with the instruction manual.

[0075] After removing outliers, the final number of groups included in the statistics for CON, HF, and HF-Y were as follows: UBB: n=14; PAM: n=16; AFM: n=19; PCSK9: n=18; B3GNT2: n=12; APOC1: n=20; F13A1: n=20.

[0076] The detection results for B3GNT2, AFM, APOC1, UBB, F13A1, PAM, and PCSK9 are as follows: Figure 14 As shown.

[0077] ELISA results showed that the expression levels of four of the seven candidate differentially expressed proteins (AFM, PCSK9, APOC1, and F13A1) in the serum samples of patients with heart failure and blood stasis syndrome were significantly different from their expression levels in the serum samples of patients with heart failure without blood stasis syndrome and healthy individuals; however, there was no significant difference between patients with heart failure without blood stasis syndrome and healthy individuals.

[0078] 2. ROC curve analysis Using the detection data of the samples in “1. ELISA Validation of Candidate Differential Proteins” above as the validation set, ROC curves were used to evaluate the sensitivity and specificity of the four candidate proteins (AFM, PCSK9, APOC1, F13A1) in predicting blood stasis syndrome in chronic heart failure.

[0079] The results are as follows Figure 15 As shown in Table 3, the ROC curves show that when AFM, PCSK9, and APOC1 are used individually to predict blood stasis syndrome in chronic heart failure, they can clearly distinguish patients with blood stasis syndrome from healthy individuals and patients without blood stasis syndrome in chronic heart failure. They have high sensitivity and specificity and can be used as specific biomarkers for patients with blood stasis syndrome in chronic heart failure.

[0080] A joint diagnostic model was constructed based on logistic regression, and the effectiveness of the model factors was evaluated using AUC. The joint diagnostic factors were compared between the HF-Y group and the CON and HF groups, respectively. The results are as follows: Figures 16-19 As shown in Table 3, the results indicate that any combination of two or more proteins in the biomarkers for blood stasis syndrome in chronic heart failure (AFM, PCSK9, APOC1) provided by this invention has good distinguishing ability between the HF-Y group and the other two groups.

[0081] Table 3. AUC values ​​of different biomarkers / biomarker combinations for distinguishing HF-Y from CON and HF.

[0082] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. Biomarkers for blood stasis syndrome in chronic heart failure, characterized in that, The biomarkers for blood stasis syndrome in chronic heart failure are selected from at least one of PCSK9, AFM, and APOC1.

2. The application of the biomarker for blood stasis syndrome in chronic heart failure according to claim 1 in the preparation of products for the diagnosis or auxiliary diagnosis of blood stasis syndrome in chronic heart failure.

3. The application of the product for detecting the content of the biomarker for chronic heart failure with blood stasis syndrome according to claim 1 in a sample in the preparation of products for the diagnosis or auxiliary diagnosis of chronic heart failure with blood stasis syndrome.

4. The application according to claim 3, characterized in that, The sample was peripheral blood serum.

5. The application according to claim 4, characterized in that, The product for detecting the content of the biomarker of chronic heart failure with blood stasis syndrome according to claim 1 in peripheral blood serum is a reagent, kit, chip, and / or instrument suitable for detecting the content of the biomarker of chronic heart failure with blood stasis syndrome in peripheral blood serum by at least one of the following methods: Western blotting, immunoturbidimetry, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, mass spectrometry, protein chip, or proteomics.

6. The application according to claim 5, characterized in that, The product for detecting the content of the biomarker of chronic heart failure with blood stasis syndrome according to claim 1 in peripheral blood serum is a reagent, kit, chip, and / or instrument suitable for detecting the content of the biomarker of chronic heart failure with blood stasis syndrome in peripheral blood serum by enzyme-linked immunosorbent assay (ELISA).

7. The application according to claim 6, characterized in that, The product for detecting the content of the biomarker for chronic heart failure with blood stasis syndrome according to claim 1 in peripheral blood serum is a kit suitable for detecting the content of the biomarker for chronic heart failure with blood stasis syndrome in peripheral blood serum by enzyme-linked immunosorbent assay (ELISA).

8. The application according to claim 7, characterized in that, The kit is an ELISA protein detection kit.

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