Application of peripheral blood protein marker FABP3 in diagnosis of obesity-induced cardiomyopathy and related kit

Abstract

The application provides a peripheral blood protein marker which can be used for early diagnosis, clinical identification and curative effect evaluation of myocardial disease caused by obesity, and the marker is fatty acid binding protein 3 (FABP3). The application also discloses application of the marker in preparation of an obesity-related myocardial disease diagnosis, differential diagnosis and curative effect evaluation kit. By establishing a disease diagnosis model based on FABP3, the overall prediction performance is excellent, the area under the curve (AUC) reaches 0.7881, the sensitivity is 0.778, the specificity is 0.720 and the difference has statistical significance (P<0.001). The experimental results prove that FABP3 is significantly highly expressed in peripheral blood of patients with obesity-related myocardial disease, and is positively correlated with the degree of myocardial injury, so that a new molecular target and solution are provided for early identification and accurate diagnosis and treatment of obesity-related myocardial disease.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of prevention and treatment of obesity-related cardiomyopathy and biological detection, and specifically relates to the application of peripheral blood protein marker FABP3 (fatty acid binding protein 3) as a marker for early diagnosis, clinical differentiation and treatment efficacy evaluation of obesity-induced cardiomyopathy. Background Technology

[0002] With changing lifestyles and dietary patterns worldwide, obesity has become a major public health challenge. Over the past few decades, the prevalence of obesity has exploded and is increasingly spreading to younger populations. Obesity is not only a significant risk factor for metabolic syndrome, type 2 diabetes, and hypertension, but it can also directly lead to abnormalities in myocardial structure and function, causing obesity-related cardiomyopathy (ORC). The core pathological mechanisms of this disease involve insulin resistance, lipotoxic damage, chronic inflammation, oxidative stress imbalance, and myocardial fibrosis. Early symptoms may include myocardial hypertrophy and diastolic dysfunction; as the disease progresses, it can gradually develop into heart failure, arrhythmias, and even sudden death, seriously threatening the patient's life and health.

[0003] However, the clinical diagnosis of obesity-related cardiomyopathy still faces many challenges, and the diagnostic system is not yet mature. On the one hand, the early symptoms of the disease lack specificity, often presenting as atypical symptoms such as fatigue and decreased exercise tolerance, which are easily confused with the physical changes caused by obesity itself and are often overlooked clinically. On the other hand, existing diagnostic techniques have significant limitations: although imaging examinations (such as echocardiography and cardiac MRI) can show changes in myocardial structure, they are greatly affected by equipment conditions and the operator's skill level, making them difficult to use as routine screening methods; invasive examinations (such as endomyocardial biopsy) are highly traumatic and risky, and are only applicable to difficult cases, and cannot be widely promoted.

[0004] Crucially, serological testing, a commonly used non-invasive diagnostic method in clinical practice, lacks clear and specific technical indicators in the diagnosis of obesity-related cardiomyopathy. Currently used myocardial injury biomarkers (such as creatine kinase isoenzymes and myoglobin) are mainly applicable to the diagnosis of acute coronary events such as acute myocardial infarction, exhibiting low sensitivity and specificity for obesity-related chronic myocardial injury, making it difficult to effectively distinguish cardiomyopathy patients from those with simple obesity in obese individuals. Furthermore, metabolic indicators (such as blood glucose and blood lipids) only reflect the body's metabolic disorder status and cannot directly correlate with the degree and progression of myocardial injury. Therefore, there is an urgent clinical need to find a highly specific and sensitive serum or peripheral blood biomarker to fill the technical gaps in the early diagnosis, disease monitoring, and efficacy evaluation of obesity-related cardiomyopathy, providing a reliable biological basis for the precise prevention and treatment of this disease. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a peripheral blood protein biomarker for the early diagnosis, clinical differentiation, and evaluation of treatment efficacy in obesity-related cardiomyopathy.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This study aims to provide peripheral blood protein marker FABP3 (fatty acid binding protein 3) as a marker for early diagnosis, clinical differentiation, and evaluation of treatment efficacy in obesity-related cardiomyopathy.

[0008] Application of a reagent for detecting FABP3 in peripheral blood in the diagnosis, differentiation, or efficacy evaluation of obesity-related cardiomyopathy.

[0009] The assay kit was used to detect the expression level of FABP3 in the peripheral blood of subjects using an ELISA method. The results showed that the FABP3 level in the peripheral blood of patients with obesity-induced cardiomyopathy was significantly higher than that of subjects with simple obesity. In animal models, the serum and tissue levels of Fap3 in high-fat diet (HFD) obese mice were significantly higher than those in control mice on a normal diet (NCD). In vitro cell experiments further confirmed that palmitic acid (PA) stimulation significantly increased the release of Fap3 from the cardiomyocyte supernatant. Clinical correlation analysis showed that FABP3 levels were significantly positively correlated with serum myocardial injury markers creatine kinase (CK) and myoglobin (MYO). These results collectively indicate that FABP3 can be used for the early diagnosis of obesity-induced cardiomyopathy and for the clinical differentiation from simple obesity.

[0010] The present invention also provides a reagent for the diagnosis, identification or efficacy evaluation of obesity-related cardiomyopathy, comprising a formulation capable of detecting FABP3 expression levels.

[0011] Meanwhile, the present invention provides a corresponding diagnostic, identification, or efficacy evaluation kit, which includes the above-mentioned detection reagents.

[0012] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0013] This invention provides a reliable peripheral blood protein biomarker for obesity-induced cardiomyopathy, which helps to achieve early diagnosis of the disease, accurately differentiate between simple obesity and obesity-induced cardiomyopathy, and provides an important basis for monitoring disease progression, evaluating treatment effects, and developing individualized treatment strategies. Attached Figure Description

[0014] Figure 1 Palmitic acid (PA) stimulation can lead to a significant increase in the release of FABP3 in the myocardial cell supernatant.

[0015] Figure 2The levels of Fap3 in the serum and heart tissue of obese mice on a high-fat diet (HFD) were significantly higher than those in control mice on a normal diet (NCD).

[0016] Figure 3 In patients with obesity-induced cardiomyopathy, serum FABP3 levels were significantly positively correlated with serum myocardial injury markers creatine kinase (CK) and myoglobin (MYO).

[0017] Figure 4 Serum FABP3 levels in patients with obesity-induced cardiomyopathy were significantly positively correlated with the inflammatory marker interleukin-6 (IL-6).

[0018] Figure 5 ROC curve analysis of the predictive model of this invention for diagnosing cardiomyopathy caused by obesity. Detailed Implementation

[0019] The present invention is further illustrated in the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] Example 1: This invention found that palmitic acid (PA) stimulation can significantly increase the release of Fabp3 in the supernatant of cardiomyocytes.

[0021] 1. Collection of cell supernatant

[0022] After HL-1 cardiomyocytes were resuscitated, they were seeded in 6-well plates with high-glucose DMEM medium containing 10% FBS and 1% penicillin-antibody solution at a seeding density of 5 × 10⁻⁶ cells / well. 5 cells / pores.

[0023] Incubate at 37℃ and 5% CO2 for 24 hours. When the cell confluence reaches 70%-80%, discard the original culture medium and wash the cells twice with PBS.

[0024] Palmitic acid stimulation treatment: Add PA working solution (0 μmol / L for control group, 200 μmol / L for experimental group) to each well, 1 mL per well, and set up 3 replicates per group; continue to incubate in 37℃, 5% CO2 incubator for 24 h.

[0025] Cell supernatant collection: After stimulation, carefully aspirate the cell supernatant from each well and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 4°C and 12,000 rpm for 10 min to remove any cell debris and PA precipitate that may be present in the supernatant. Transfer the centrifuged supernatant to a new centrifuge tube and use it immediately for FAP3 detection or freeze it at -80°C for later use (avoid repeated freeze-thaw cycles).

[0026] 2. Fap3 content detection (ELISA method):

[0027] Follow the instructions for the Fap3 ELISA kit and bring the kit to room temperature beforehand. Add 100 μL of standard (gradient concentration) and sample supernatant to each well of the ELISA plate, and add an equal volume of serum-free culture medium to the blank wells. Incubate at 37°C for 60 min.

[0028] Discard the liquid in the wells, wash the microplate 5 times with washing buffer, and discard the washing buffer after each 30-second stand.

[0029] Add 100 μL of biotin-labeled FBP3 detection antibody to each well, incubate at 37°C for 30 min, and repeat the washing steps.

[0030] Add 100 μL of horseradish peroxidase (HRP)-labeled avidin to each well, incubate at 37°C for 30 min, and repeat the washing step again.

[0031] Add 100 μL of substrate development solution (TMB) to each well and incubate at 37°C in the dark for 15 min. Once a clear gradient of colors appears in the standard wells, add 50 μL of stop solution to terminate the reaction.

[0032] The absorbance (OD value) of each well was measured using an ELISA reader at a wavelength of 450 nm, and the data was recorded.

[0033] 3. Data Processing and Statistical Analysis

[0034] Standard curve plotting: The standard curve is plotted using the concentration of the standard as the x-axis and the corresponding OD value as the y-axis. The four-parameter fitting method is used to calculate the regression equation (y=ax+b) and the correlation coefficient (R²≥0.95).

[0035] Calculation of Fap3 concentration in samples: Based on the sample OD value, substitute it into the regression equation to calculate the actual concentration of Fap3 in the cell supernatant of the experimental group and the control group.

[0036] The results are as follows Figure 1 It can be concluded that palmitic acid (PA) stimulation can lead to a significant increase in the release of Fap3 in the myocardial cell supernatant.

[0037] Example 2: This invention demonstrates that the levels of Fap3 in the serum and heart tissue of obese mice on a high-fat diet (HFD) are significantly higher than those in control mice on a normal diet (NCD).

[0038] Obesity-induced cardiac hypertrophy model: All experimental animals were housed at the Animal Center of Hangzhou Normal University. Twelve wild-type C57BL / 6 mice were used in the experiment and fed either a normal diet (NCD) or a high-fat diet (HFD) for 20 weeks. Both the normal diet (containing 10% fat, 20% protein, and 70% carbohydrates) and the high-fat diet (containing 60% fat, 20% protein, and 20% carbohydrates) were purchased from Shanghai Ruian Biotechnology Co., Ltd., China. Mouse weight was measured and recorded weekly. At the end of week 20, all mice were anesthetized and sacrificed, and their final weight was measured, and heart and blood samples were collected.

[0039] After homogenizing heart tissue, the sample was added to an appropriate proportion of SDS gel loading system. Following SDS-PAGE electrophoresis and membrane transfer, the membrane was blocked with 5% skim milk, incubated at room temperature for 1.5 hours, and washed three times with TBST. Then, primary antibodies Fap3 (1:1000) and GAPDH (1:10000) were added, and the membrane was incubated overnight at 4°C, washed three times with TBST, and incubated with a secondary antibody conjugated with horseradish peroxidase at room temperature for 1 hour. The membrane was washed three times, and the immunoreaction bands were detected using a high-sensitivity ECL chemiluminescence assay kit. Images were taken using a gel imaging system. Results are as follows: Figure 2 As shown in AB, the level of Fap3 in the heart tissue of obese mice on a high-fat diet (HFD) was significantly higher than that in control mice on a normal diet (NCD).

[0040] The expression of Fap3 in mouse serum and tissue lysates was detected using a Fap3 ELISA kit. The specific experimental procedures were as described in Example 1, and the results were as follows: Figure 2 As shown in Figure C, the levels of Fap3 in the serum and heart tissue of obese mice on a high-fat diet (HFD) were significantly higher than those in control mice on a normal diet (NCD).

[0041] Example 3: FABP3 levels were significantly positively correlated with serum myocardial injury markers creatine kinase (CK) and myoglobin (MYO).

[0042] Collect blood samples from qualified clinical patients:

[0043] Obese patients with cardiomyopathy (OCM, 27 cases):

[0044] Meets the diagnostic criteria for obesity (BMI ≥ 25 kg / m²); has been diagnosed with cardiomyopathy by echocardiography (left ventricular ejection fraction < 50%, or reduced left ventricular diastolic function, and other causes such as coronary heart disease, hypertensive heart disease, and congenital heart disease have been ruled out); and voluntarily signs informed consent.

[0045] Obese group without cardiomyopathy (OB, 25 cases):

[0046] Meets the diagnostic criteria for obesity (BMI ≥ 25 kg / m²); has normal cardiac function on echocardiography (LVEF ≥ 50%, with no abnormalities in left ventricular structure and diastolic function); is matched for age and sex with the obese cardiomyopathy group; has no history of other organic heart disease; and has signed informed consent.

[0047] Normal healthy individuals (NC, 20 cases):

[0048] Candidates must not meet the diagnostic criteria for obesity (BMI < 25 kg / m²); have normal cardiac function on echocardiography (LVEF ≥ 50%, with no abnormalities in left ventricular structure and diastolic function); be age and sex matched with the obese cardiomyopathy group; have no history of other organic heart disease; and have signed informed consent.

[0049] Sample collection and processing

[0050] Five mL of peripheral venous blood was collected from all subjects after fasting for 12 hours and placed in a vacuum blood collection tube without anticoagulant. The tube was then left to stand at room temperature for 30 minutes.

[0051] Centrifuge at 3000 rpm for 15 min (4℃) to separate the serum and aliquot it into 2 mL cryovials;

[0052] Store immediately in a -80°C freezer to avoid repeated freezing and thawing.

[0053] The expression level of FABP3 in human serum was detected using a FABP3 ELISA kit. The specific experimental procedures were as described in Example 1, and the results were as follows: Figure 3 As shown in Figure A, serum Fabp3 expression was increased in the obese group with cardiomyopathy (OCM) compared to the obese group without cardiomyopathy (OB). We also performed a correlation analysis between Fabp3 levels in obese patients with cardiomyopathy and serum myocardial injury markers creatine kinase (CK) and myoglobin (MYO). The results are as follows... Figure 3 BC:FABP3 levels were significantly positively correlated with serum myocardial injury markers creatine kinase (CK) and myoglobin (MYO).

[0054] Example 4: FABP3 level was significantly positively correlated with the inflammatory marker interleukin-6 (IL-6).

[0055] Follow the instructions for the IL-6 ELISA kit, specifically referring to the experimental procedure in Example 1. Results are as follows: Figure 4 As shown, FABP3 levels are significantly positively correlated with the inflammatory marker interleukin-6 (IL-6), indicating that changes in FABP3 levels can indirectly reflect the degree of disease-related inflammatory activation and can be used as an auxiliary indicator to assess the severity of the disease (e.g., the more severe the inflammation, the higher the FABP3 level may be, and the more obvious the myocardial damage).

[0056] Example 5:

[0057] Clinical sample test results: Serum FABP3 concentration values ​​(unit: ng / mL) in the obese cardiomyopathy group (OCM, n=27) and the obese non-cardiomyopathy group (OB, n=25). The data need to be verified by previous ELISA tests.

[0058] Open GraphPad Prism, import serum FABP3 concentration values ​​from the obese group with cardiomyopathy (OCM, n=27) and the obese group without cardiomyopathy (OB, n=25) to create ROC curves. Select the Youden index to determine the optimal cutoff value and calculate the 90% confidence interval of the AUC. Then optimize the curve style and label key results (AUC, sensitivity, specificity) to complete the curve creation. Results are as follows: Figure 5 As shown, a disease diagnostic model based on FABP3 was established. The model has excellent overall predictive performance, with an area under the curve (AUC) of 0.7881, a sensitivity of 0.778, and a specificity of 0.720, and the differences are statistically significant (P<0.001). This indicates that serum FABP3, as a peripheral blood protein marker, has high clinical translation and application value and can be used as an effective auxiliary tool for disease diagnosis.

Claims

1. A peripheral blood protein biomarker for the diagnosis of cardiomyopathy caused by obesity, characterized in that, The protein biomarker is protein FABP3.

2. The application of a detection reagent for peripheral blood protein markers in the preparation of diagnostic kits for cardiomyopathy, characterized in that, The detection reagents for peripheral blood protein markers include a reagent specifically for detecting the content of protein FABP3.

3. The application according to claim 2, characterized in that, The cardiomyopathy mentioned is cardiomyopathy caused by obesity.

4. The application according to claim 2, characterized in that, The detection reagents for peripheral blood protein markers include reagents for determining the FABP3 protein content in peripheral blood (serum) samples.

5. A medical auxiliary diagnostic system for cardiomyopathy caused by obesity, characterized in that, The diagnostic system includes a diagnostic model for cardiomyopathy caused by obesity, which includes protein FABP3, body mass index (BMI), waist circumference, and age composition.

6. The application of a detection agent for a blood biomarker in the preparation of a diagnostic and efficacy evaluation reagent for obesity-induced cardiomyopathy, characterized in that: The biomarker in the blood is FABP3.

7. The application according to claim 6, characterized in that: The diagnostic reagent detects the level of FABP3 in the blood of the patient to be diagnosed, which is significantly higher than that in obese patients without cardiomyopathy, thereby determining that the patient to be diagnosed has cardiomyopathy.

8. The application according to claim 6, characterized in that: The detection agent measures the level of FABP3 in the blood of patients with obesity-induced cardiomyopathy, and the significant decrease in FABP3 after treatment compared to before treatment is used to determine the efficacy of the treatment regimen.

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