Application of combination of natriuretic peptide and cardiac enzyme in diagnosis of diabetic cardiomyopathy

Abstract

The invention discloses an application of a natriuretic peptide combined cardiac enzyme in diagnosis of diabetic cardiomyopathy, which is implemented by detecting the change conditions of a natriuretic peptide MR-proANP and a cardiac enzyme Corin in the plasma of a patient with diabetic cardiomyopathy, and also discloses an application of the natriuretic peptide combined cardiac enzyme in preparation of a diagnostic reagent for diabetic cardiomyopathy. The problems that proANP is extremely unstable in peripheral blood, and missed diagnosis and misdiagnosis are easily caused are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of disease diagnosis, and specifically relates to the application of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy. Background Art

[0002] With the aging of the population and changes in lifestyle, the incidence and mortality of diabetes mellitus (DM) have increased year by year, and diabetic complications have received increasing attention. Diabetic cardiomyopathy (DCM) is one of the main complications of diabetic patients and the leading cause of death in diabetic patients, so it is particularly important to improve the diagnosis of diabetic cardiomyopathy.

[0003] Natriuretic peptides are a group of peptide substances with similar structures that play an important role in regulating blood pressure, fluid balance and cardiovascular function. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are closely related to the cardiovascular system. The precursors of ANP, proANP, and BNP, proBNP, are both upregulated and released into the peripheral blood when the heart is stretched. ProANP is mainly expressed in atrial cells, while proBNP is mainly expressed in ventricular cells. In the case of myocardial injury, the release of proANP is about 10-50 times that of proBNP. In theory, proANP is a more ideal marker for heart failure. However, proANP is extremely unstable in peripheral blood, which can easily lead to missed diagnosis and misdiagnosis, and has low practical application value. Summary of the invention

[0004] The first objective of the present invention is to provide the use of natriuretic peptides combined with cardiac enzymes in the diagnosis of diabetic cardiomyopathy, which solves the problem that proANP is extremely unstable in peripheral blood and easily causes missed diagnosis or misdiagnosis.

[0005] The second object of the present invention is to provide the use of natriuretic peptides combined with cardiac enzymes in the preparation of diagnostic reagents for diabetic cardiomyopathy.

[0006] The first technical solution adopted by the present invention is: application of natriuretic peptide combined with cardiac enzyme in diagnosis of diabetic cardiomyopathy.

[0007] The first technical solution adopted by the present invention is also characterized in that: Furthermore, the natriuretic peptide is MR-proANP, specifically the middle fragment of the 53rd to 90th amino acids of proANP, and the cardiac enzyme is specifically atrial natriuretic peptide converting enzyme Corin.

[0008] Furthermore, the application of natriuretic peptides combined with cardiac enzymes in the diagnosis of diabetic cardiomyopathy was implemented by detecting the changes of natriuretic peptide MR-proANP and cardiac enzyme Corin in the plasma of patients with diabetic cardiomyopathy.

[0009] Furthermore, the diagnostic value of the natriuretic peptide MR-proANP combined with the cardiac enzyme Corin in diabetic cardiomyopathy is higher than that of MR-proANP or Corin alone.

[0010] Furthermore, the natriuretic peptide MR-proANP was negatively correlated with cardiac contractile function.

[0011] Furthermore, cardiac enzyme Corin was positively correlated with cardiac contractile function.

[0012] The second technical solution adopted by the present invention is: application of natriuretic peptides combined with cardiac enzymes in the preparation of diagnostic reagents for diabetic cardiomyopathy.

[0013] The second technical solution adopted by the present invention is also characterized in that: Furthermore, the diagnostic reagent is implemented by detecting the levels of natriuretic peptides and cardiac enzymes in peripheral blood.

[0014] The beneficial effects of the present invention are: The present invention establishes the reference range of MR-proANP and Corin for the first time, and finds that the two indicators of MR-proANP combined with Corin have higher diagnostic value in diabetic cardiomyopathy than MR-proANP or Corin alone, thereby helping to improve the diagnostic accuracy of diabetic cardiomyopathy and reduce missed diagnosis and misdiagnosis rates, and therefore has good practical application value. On the other hand, the present invention finds that the change trends of the two indicators of MR-proANP and Corin in the myocardium and plasma of diabetic cardiomyopathy mice are consistent, which can provide powerful experimental reference data for the treatment of diabetic cardiomyopathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a comparison chart of left ventricular ejection fraction and plasma MR-proANP content in patients with diabetes and diabetic cardiomyopathy; Figure 2 This is a correlation analysis chart between left ventricular ejection fraction and MR-proANP in patients with diabetic cardiomyopathy; Figure 3 It is a graph of the diagnostic value of MR-proANP in plasma for patients with diabetic cardiomyopathy; Figure 4A is a comparison of plasma Corin levels in patients with diabetes and diabetic cardiomyopathy; Figure 4BThis is the correlation analysis between plasma Corin and left ventricular ejection fraction in patients with diabetic cardiomyopathy and the correlation analysis between plasma Corin and MR-proANP in patients with diabetic cardiomyopathy; Figure 5 This is a graph showing the diagnostic value of Corin in plasma for patients with diabetic cardiomyopathy; Figure 6 This is the effect diagram of MR-proANP combined with Corin in the diagnosis of diabetic cardiomyopathy; Figure 7 This is a graph showing changes in left ventricular ejection fraction, as well as plasma MR-proANP and Corin in mice with diabetic cardiomyopathy; Figure 8 This is a graph showing the changes in MR-proANP and Corin in the myocardial tissue of mice with diabetic cardiomyopathy. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0017] MR-proANP, as the intermediate fragment of the 53rd to 90th amino acids of proANP, can be stable for 48 hours at 4°C and for 6 months at -20°C. Moreover, its detection results are not significantly affected by severe hemolysis, jaundice, lipemia, etc. These superior properties have laid the foundation for the clinical application of MR-proANP. ANP is stored in the form of proANP in atrial myocyte granules. Pro-ANP is cleaved into two biologically active fragments, the C-terminal ANP (α-ANP) containing 28 peptides and the N-terminal ANP (NT-proANP) containing 98 peptides, under the activation and cleavage of the cardiac enzyme Corin. Plasma MR-proANP and Corin levels may be used as biological markers for the diagnosis and prognostic value of heart disease, but there is still a lack of effective indicators for clinical application in diabetic cardiomyopathy, especially diagnosis.

[0018] The invention discloses the application of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy, which is implemented by detecting the changes of natriuretic peptide MR-proANP and cardiac enzyme Corin in the plasma of diabetic cardiomyopathy patients.

[0019] The natriuretic peptide is MR-proANP, specifically the middle fragment of the 53rd-90th amino acids of proANP, and the cardiac enzyme is specifically the atrial natriuretic peptide converting enzyme Corin. The diagnostic value of the natriuretic peptide MR-proANP combined with the cardiac enzyme Corin in diabetic cardiomyopathy is higher than that of MR-proANP or Corin alone. The natriuretic peptide MR-proANP was negatively correlated with cardiac contractile function, while the cardiac enzyme Corin was positively correlated with cardiac contractile function.

[0020] The present invention also discloses the application of natriuretic peptide combined with cardiac enzyme in the preparation of diabetic cardiomyopathy diagnostic reagent, which is implemented by detecting the content of natriuretic peptide and cardiac enzyme in peripheral blood.

[0021] 1. Research plan 1.1 Clinical samples The present study selected patients with diabetes and diabetes complicated with heart failure who were initially diagnosed in the Department of Endocrinology and Department of Cardiology of the Second Affiliated Hospital of Xi'an Medical College from November 2022 to November 2023. This study was approved by the hospital ethics committee (X2Y202208). The final participants included 30 patients with diabetes and 30 patients with diabetes with myocardial damage. The remaining plasma samples were obtained and stored at -80°C.

[0022] 1.2 Experimental methods 1.2.1 Determination of MR-proANP content in human plasma The enzyme linked immunosorbent assay (ELISA) was used to detect the plasma content of MR-proANP in diabetic patients and 30 patients with diabetic cardiomyopathy. The ELISA kit was purchased from Ruixin Biotechnology (No. RX101781H). The assay was performed according to the instructions for use of the kit. The specific steps are as follows: After preparing the working solution of the various components of the kit, add the standard or sample to a 96-well microplate, incubate at 37°C in the dark for 60 minutes, discard the liquid, wash 5 times with washing solution, and pat the reaction plate dry; add the substrate mixture, incubate at 37°C in the dark for 15 minutes, and then add the stop solution; measure the absorbance at a wavelength of 450nm on an ELISA reader, create a standard curve equation based on the standard curve, and use the equation to calculate the sample concentration value through the absorbance (OD value) of the sample.

[0023] 1.2.2 Determination of Corin Content in Human Plasma ELISA kit was used to detect the plasma content of Corin in diabetic patients and 30 patients with diabetic cardiomyopathy. The ELISA kit was purchased from Jianglai Biological (No. JL14844). The specific operation steps are as follows: first, prepare the working solution of various components of the kit, then add the standard or sample to a 96-well microplate, incubate at 37°C in the dark for 60 minutes, discard the liquid, add the biotinylated antibody working solution and incubate at 37°C for 60 minutes, discard the liquid, add the washing solution and wash 3 times, add the enzyme conjugate working solution, incubate at 37°C in the dark for 30 minutes, wash the plate 5 times, add the substrate and incubate at 37°C in the dark for 15 minutes, add the stop solution; measure the absorbance at a wavelength of 450nm on an enzyme reader, create the standard curve equation according to the standard curve, and calculate the sample concentration value through the absorbance (OD value) of the sample.

[0024] 1.2.3 Establishment of diabetic cardiomyopathy model in mice Twenty and sixteen 4-week-old SPF-grade C57BL / 6 male mice were selected and randomly divided into a control group and a diabetic model group, with 8 mice in each group. The control group was fed with ordinary feed, and the diabetic model group was fed with high-fat feed for 4 weeks. After fasting for 12 hours, the diabetic model group was intraperitoneally injected with 100 mg / kg streptozotocin-sodium citrate buffer and continued to be fed with high-fat feed for 12 weeks. The control group was intraperitoneally injected with an equal volume of citric acid-sodium citrate buffer. At the 16th week, the fasting blood glucose was measured to be >13.89 mmol / L, which was considered to be a successful model of diabetic mice. The heart function was detected using a small animal ultrasound detector, and the left ventricular ejection fraction was measured and recorded.

[0025] 1.2.4 Determination of blood glucose in mice, sampling of orbital blood and heart samples The blood glucose of mice was measured with a Roche blood glucose meter. After the experiment, sodium pentobarbital was injected intraperitoneally for anesthesia. Blood was collected from the eye sockets to separate the serum and stored at -80°C. The heart was quickly removed, and the excess blood stains were washed with saline. The heart was dried with absorbent paper and then frozen at -80°C.

[0026] 1.2.5 Determination of MR-proANP and Corin Content in Mouse Plasma The ELISA kit was used to detect the levels of MR-proANP and Corin in mouse plasma. The specific operating steps were as follows: prepare the working solutions of various components of the kit, then add the standards or samples to a 96-well microplate, incubate at 37°C in the dark for 60 minutes, discard the liquid, add the biotinylated antibody working solution and incubate at 37°C for 60 minutes, discard the liquid, add the washing solution and wash 5 times, then add the substrate mixture, incubate at 37°C in the dark for 15 minutes, and then add the stop solution; measure the absorbance at a wavelength of 450nm on an enzyme reader, create the standard curve equation based on the standard curve, and calculate the sample concentration value through the absorbance (OD value) of the sample.

[0027] 1.2.6 Determination of MR-proANP and Corin Content in Mouse Myocardial Tissue ELISA kits were used to detect the MR-proANP and Corin levels in mouse myocardial tissue. The heart was rinsed with pre-cooled PBS to remove residual blood. The tissue was chopped up after weighing, and a certain proportion of PBS containing protease inhibitors was added. The tissue was fully ground on ice, and the homogenate was centrifuged at 5000g for 10 minutes. The supernatant was collected for detection. The remaining specific operation steps were basically similar to the above-mentioned steps for determining the MR-proANP and Corin levels in mouse plasma.

[0028] 1.2.7 Data processing methods The content level was expressed as mean ± standard deviation, and the Student's t-test was used to compare the differences in protein content between different groups. The correlation between MR-proANP, left ventricular ejection fraction and Corin was analyzed by using the Pearson correlation coefficient. The diagnostic value of MR-proANP and Corin was evaluated by drawing the receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC) and its 95% confidence interval.

[0029] 2. Research Results Comparison of general data between patients with diabetes and patients with diabetic cardiomyopathy By comparing the general clinical data of diabetic patients and diabetic cardiomyopathy patients, as shown in Table 1, including gender ratio, BMI, fasting blood glucose, glycosylated hemoglobin, systolic blood pressure, diastolic blood pressure, total cholesterol and triglyceride content, there was no statistical difference in the above indicators.

[0030] Table 1 Comparison of general clinical data

[0031] Example 1 Patients with diabetic cardiomyopathy have impaired cardiac function and elevated plasma MR-proANP levels.

[0032] like Figure 1 As shown, the left side shows the ultrasound index left ventricular ejection fraction, which is a reliable indicator of cardiac contractile function. The results show that the heart function of patients with diabetic cardiomyopathy is impaired. Elisa is further used to detect the MR-proANP content in the patient's plasma. The results are as follows Figure 1 As shown on the right, the plasma MR-proANP level in patients with diabetes mellitus was significantly higher than that in patients with diabetes mellitus ( P <0.05).

[0033] Example 2 There is a certain correlation between plasma MR-proANP and cardiac contractile function in patients with diabetic cardiomyopathy.

[0034] In order to explore the relationship between MR-proANP and cardiac contractile function, the correlation between the two indicators was tested. Figure 2 As shown in Figure 2, the plasma MR-proANP level in patients with diabetic cardiomyopathy was strongly correlated with the left ventricular systolic function index EF (R 2 =0.7624, P <0.0001), and they were negatively correlated.

[0035] Example 3 Plasma MR-proANP content has a certain diagnostic value for patients with diabetic cardiomyopathy.

[0036] like Figure 3 As shown in the data, through ROC curve analysis, the AUC of MR-proANP for diagnosing diabetic cardiomyopathy was 0.92, and the 95% confidence interval was 0.8509-0.9891 (P<0.0001), indicating that the MR-proANP content in plasma has a high diagnostic value for patients with diabetic cardiomyopathy.

[0037] Example 4 Plasma Corin is decreased in patients with diabetic cardiomyopathy and has a certain correlation with MR-proANP.

[0038] Corin can highly specifically convert pro-atrial natriuretic peptide (pro-ANP) into biologically active atrial natriuretic peptide (ANP). Corin is associated with impaired cardiac function, but its role in diabetic cardiomyopathy is still unclear. This study investigated the plasma Corin content in diabetic cardiomyopathy. Figure 4A As shown, the plasma Corin level in patients with diabetic cardiomyopathy is significantly reduced ( P <0.05, and in patients with diabetic cardiomyopathy, Corin content was negatively correlated with MR-proANP, with a certain correlation ( P <0.05), Corin content was positively correlated with EF in patients with diabetic cardiomyopathy, and there was a certain correlation ( P <0.05), but the correlation was lower than that of MR-proANP, such as Figure 4B shown.

[0039] Example 5 Plasma Corin has a high diagnostic value in patients with diabetic cardiomyopathy.

[0040] To further explore the diagnostic value of Corin in diabetic cardiomyopathy, the ROC curve analysis showed that the AUC of Corin in diagnosing diabetic cardiomyopathy was 0.92 ( P <0.0001), indicating that the level of Corin in plasma has a high diagnostic value for patients with diabetic cardiomyopathy, such as Figure 5 shown.

[0041] Example 6 The detection of plasma MR-proANP combined with Corin has a high diagnostic value for patients with diabetic cardiomyopathy.

[0042] The diagnostic value of MR-proANP combined with Corin in diabetic cardiomyopathy was further explored. Through ROC curve analysis, the AUC of MR-proANP combined with Corin for diabetic cardiomyopathy was 0.982 (P<0.0001), indicating that MR-proANP combined with Corin has a high diagnostic value for patients with diabetic cardiomyopathy, and is much higher than the diagnostic value of MR-proANP and Corin alone for diabetic cardiomyopathy. Figure 6 shown.

[0043] Example 7 The cardiac function of mice with diabetic cardiomyopathy was reduced, the plasma MR-proANP content gradually increased, the plasma Corin content decreased, and the relative activity of myocardial Corin decreased.

[0044] In order to verify the condition of diabetic cardiomyopathy in mice, the left ventricular ejection fraction of mice was first tested, and the cardiac function of diabetic cardiomyopathy mice was verified to be reduced, indicating that the model was successful. The levels of MR-proANP and Corin in mouse plasma were tested, and the results showed that the plasma MR-proANP level of diabetic cardiomyopathy mice was higher than that of normal mice ( P <0.01), the plasma Corin content of diabetic cardiomyopathy mice was lower than that of normal mice ( P <0.01), the relative activity of Corin in myocardial tissue of diabetic cardiomyopathy was lower than that in myocardial tissue of normal mice ( P <0.01), such as Figure 7 shown.

[0045] Example 8 The relative levels of MR-proANP and Corin in myocardial tissue of diabetic cardiomyopathy mice were lower than those in myocardial tissue of normal mice ( P <0.01).

[0046] In order to verify the content of myocardial tissue, the content of MR-proANP and Corin in the myocardial tissue of mice was detected, and the relative content was calculated. The results showed that the content of MR-proANP in the myocardial tissue of diabetic cardiomyopathy mice was higher than that in normal mice ( P <0.01), the Corin content in myocardial tissue of diabetic cardiomyopathy mice was lower than that of normal mice ( P <0.01), such as Figure 8 shown.

Claims

1. Application of natriuretic peptides combined with cardiac enzymes in the diagnosis of diabetic cardiomyopathy.

2. The use of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy according to claim 1, characterized in that: The natriuretic peptide is MR-proANP, specifically the middle fragment of the 53rd to 90th amino acids of proANP, and the cardiac enzyme is specifically the atrial natriuretic peptide converting enzyme Corin.

3. The use of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy according to claim 1, characterized in that: The application of the natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy is implemented by detecting the changes of the natriuretic peptide MR-proANP and the cardiac enzyme Corin in the plasma of patients with diabetic cardiomyopathy.

4. The use of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy according to claim 3, characterized in that: The diagnostic value of the two indicators of natriuretic peptide MR-proANP combined with cardiac enzyme Corin in diabetic cardiomyopathy is higher than that of MR-proANP or Corin alone.

5. The use of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy according to claim 3, characterized in that: The natriuretic peptide MR-proANP is negatively correlated with cardiac contractile function.

6. The use of natriuretic peptide combined with cardiac enzyme in the diagnosis of diabetic cardiomyopathy according to claim 3, characterized in that: The cardiac enzyme Corin is positively correlated with cardiac contractile function.

7. Application of natriuretic peptides combined with cardiac enzymes in the preparation of diagnostic reagents for diabetic cardiomyopathy.

8. The use of natriuretic peptide combined with cardiac enzyme in the preparation of a diagnostic reagent for diabetic cardiomyopathy according to claim 7, characterized in that: The diagnostic reagent is implemented by detecting the content of natriuretic peptides and cardiac enzymes in peripheral blood.

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