Application of n-heptadecanoic acid MA as diagnostic marker and therapeutic drug for diabetic cardiomyopathy

Abstract

The invention prompts that n-heptadecanoic acid MA can be used as a marker, a diagnostic kit and a therapeutic drug for diagnosis of diabetic cardiomyopathy. MA can be extracted from dairy products such as milk and can be used as a nutritional supplement for treating diabetic cardiomyopathy.

Description

Application of heptadecanoate (MA) as a diagnostic marker and therapeutic agent for diabetic cardiomyopathy Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of heptadecanoate (MA) as a diagnostic marker and therapeutic agent for diabetic cardiomyopathy. Background Technology

[0002] Diabetic cardiomyopathy (DCM) refers to a heart disease caused by non-ischemic damage in patients with diabetes mellitus (DM) without coronary heart disease, hypertension, valvular heart disease, or congenital heart disease, where there are abnormalities in the structure and function of the myocardium. DCM is a major cardiovascular complication of diabetes, characterized by myocardial fibrosis, ventricular remodeling, and heart failure. According to the condition of the heart structure and function, diabetic cardiomyopathy can be divided into three stages: (1) the subclinical stage, mainly manifested by myocardial cell structural damage induced by high glucose; (2) decreased myocardial compliance and systolic-diastolic dysfunction; and (3) increased myocardial mass, increased stiffness, and other manifestations of heart failure. Ventricular remodeling, characterized by excessive deposition of extracellular matrix in the myocardial interstitium, is the main cause of heart failure and is also the end-stage adverse outcome of diabetic cardiomyopathy. According to statistics, the number of diabetic patients worldwide may reach 700 million by 2045, and my country, as a "diabetic superpower," will bear a huge social burden and economic loss. Studies have shown that for every 1% increase in the baseline level of glycated hemoglobin, the risk of heart failure in patients increases by 15%. However, specific treatments for diabetic cardiomyopathy are currently lacking. Therefore, there is an urgent need for more in-depth research into the mechanisms of diabetic cardiomyopathy to develop targeted treatment strategies, which undoubtedly has significant clinical and social value.

[0003] Furthermore, although the cardiac dysfunction and even heart failure caused by diabetes are increasingly attracting attention, there is still a lack of effective diagnostic strategies and accurate biomarkers for diabetic cardiomyopathy, which affects the detection and treatment of the disease. When patients are diagnosed with the disease through indirect diagnosis by echocardiography, cardiac MRI and routine myocardial enzyme spectrum, irreversible changes in cardiac function and structural changes have often occurred, ultimately leading to heart failure.

[0004] Margarine acid (MA, C17), also known as heptadecanoic acid, is an odd-chain fatty acid. MA has a molecular weight of 270.4507 Da and contains 13–21 carbon atoms. MA is primarily involved in lipid peroxidation, fatty acid metabolism, and antioxidant processes, and can be detected in blood, saliva, and urine. However, the prevalence of serum MA in patients with diabetic cardiomyopathy-related diastolic dysfunction is unclear, and whether serum MA has any impact on diabetic cardiomyopathy outcomes has not been investigated. Summary of the Invention

[0005] This invention aims to provide a long-chain fatty acid (MA) as a biomarker and diagnostic kit for diabetic myocardial damage; its use as a nutritional supplement for the treatment of diabetic cardiomyopathy; and a system for assessing cardiac damage in target subjects based on the MA biomarker.

[0006] This invention provides the application of a metabolite in peripheral blood in the preparation of a tool to distinguish between diabetic cardiomyopathy and non-diabetic cardiomyopathy, characterized in that: the metabolite in peripheral blood is n-heptadecanoic acid.

[0007] Furthermore, the application provided by this invention is characterized by the method of using a tool to distinguish between diabetic cardiomyopathy and non-diabetic cardiomyopathy, comprising the following steps:

[0008] The expression level of n-heptadecanoic acid in the test sample was detected to determine and predict the risk of diabetic cardiomyopathy.

[0009] The judgment criterion is: if the relative abundance of n-heptadecanoic acid in the sample is less than 0.604, it indicates a high risk of diabetic cardiomyopathy;

[0010] If not, it suggests a low risk of diabetic cardiomyopathy.

[0011] Furthermore, the application provided by the present invention is characterized in that the above-mentioned n-heptadecanoic acid is also jointly evaluated in conjunction with the NT-pIoBNP level, LVEF% and E / e' indicators.

[0012] Furthermore, the application provided by the present invention is characterized in that the tool includes a detection reagent, a detection kit, and a detection chip.

[0013] Furthermore, this invention also provides the use of n-heptadecanoic acid in the preparation of pharmaceuticals / dietary supplements for treating and alleviating diabetic cardiomyopathy.

[0014] Furthermore, this invention also provides the application of n-heptadecanoic acid in the preparation of pharmaceuticals / dietary supplements that inhibit the transdifferentiation level of cardiac fibroblasts.

[0015] Furthermore, this invention also provides the application of n-heptadecanoic acid in the preparation of pharmaceuticals / dietary supplements that improve ventricular remodeling in diabetic cardiomyopathy.

[0016] The function and effects of this invention:

[0017] This invention, through clinical observational studies, confirms a close correlation between decreased plasma MA levels and the incidence of diabetic cardiomyopathy. MA, an odd-chain fatty acid, is readily and rapidly detectable qualitatively and quantitatively using mass spectrometry, and can be obtained from blood, urine, and saliva. It can serve as a biomarker and diagnostic kit for diabetic cardiomyopathy. Furthermore, MA can be extracted from dairy products such as milk and can be used as a nutritional supplement for the treatment of diabetic cardiomyopathy. Attached Figure Description

[0018] Figure 1. Non-targeted metabolomics analysis based on liquid chromatography-high resolution mass spectrometry (LC-HRMS);

[0019] Wherein, A is the principal component analysis (PCA) score of QC samples under positive ion mode; B is the principal component analysis (PCA) score of QC samples under negative ion mode; C is the principal component analysis (PCA) score of samples from the healthy group, T2D group, and DCM group; D is the principal component analysis (PCA) score of samples from the healthy group and DCM group; E is the principal component analysis (PCA) score of samples from the T2D group and DCM group; F is the orthogonal partial least squares discriminant analysis (OPLS-DA) score of samples from the healthy group, T2D group, and DCM group; G is the orthogonal partial least squares discriminant analysis (OPLS-DA) score of samples from the healthy group and DCM group; H is the orthogonal partial least squares discriminant analysis (OPLS-DA) score of samples from the T2D group and DCM group; I is the differential lipid metabolites (free fatty acids, MA, and 19) between the DCM group and the combined group of healthy group + T2D group. The relative abundance heatmap of various lipids; JK represents the receiver operating characteristic (ROC) curve analysis results; L represents the bee colony plot analysis of serum MA concentration in the healthy group, T2D group, and DCM group; M represents the scatter plot of the correlation between serum MA concentration and Log (NT-roBNP) level in the DCM and T2D groups; N represents the scatter plot of the correlation between serum MA concentration and LVEF% level in the DCM and T2D groups; O represents the scatter plot of the correlation between serum MA concentration and E / e' level in the DCM and T2D groups.

[0020] Figure 2. Study of the effect of lipid metabolite MA on transdifferentiation of cardiac fibroblasts at the cellular level;

[0021] In this table, A represents the results of fibroblasts induced by TGF-β1; B represents the effect of MA on the proliferation function of primary cardiac fibroblasts; CK represents the results of real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot experiments; LM represents the results of EdU detection; and NP represents the results of immunofluorescence analysis.

[0022] Figure 3. Effects of lipid metabolite MA on mitochondrial respiratory dysfunction;

[0023] In this diagram, AB represents the results of immunofluorescence analysis; CD represents the immunofluorescence imaging map of mitochondrial membrane potential (MMP) detection; and EG represents the results of Western blot analysis.

[0024] Figure 4. Effect of lipid metabolite MA on ventricular remodeling in diabetic cardiomyopathy;

[0025] In this diagram, A represents the design of the animal model; B represents representative M-mode echocardiogram images of the heart; CD represents the results of left ventricular ejection fraction and fractional shortening; EF represents the results of left ventricular end-systolic diameter and left ventricular end-diastolic diameter; G represents the trend of body weight change in mice in each experimental group; HK represents the results of plasma triglycerides, total cholesterol, low-density lipoprotein cholesterol, and blood glucose in each experimental group; and LO represents the results of pathological morphology and molecular indicators.

[0026] Figure 5. Effects of lipid metabolite MA on cardiac fibrosis;

[0027] In this figure, AD represents the results of immunohistochemistry detection of fibrosis markers, and E represents the experimental results of cardiomyocyte apoptosis and ultrastructure in the DCM model.

[0028] Figure 6. Key target proteins of the lipid metabolite MA;

[0029] AE represents the results of transcriptomics analysis.

[0030] Figure 7. Regulatory mechanism of lipid metabolite MA on Nqo1;

[0031] Figure 8 shows the correlation between the lipid metabolite MA and DCM using molecular docking technology in 3D simulation. Figure A represents the docking site of Nqo1 simulated using molecular docking technology. Figure B and C represent the results of Western blot analysis of Nqo1 expression in mouse myocardial tissue. Figure D and E represent the results of Western blot analysis of Tgfβ1 and Smad4 protein phosphorylation levels. Figure 8 illustrates the correlation between the content of lipid metabolite MA and DCM. Figure A shows the comparison of serum MA levels in different populations. Figure B shows the correlation analysis between MA and Ln (NT-proBNP). Figure C shows the correlation analysis between MA and LVEF. Figure D shows the correlation analysis between MA and E / e'. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Example 1. Correlation between lipid metabolite MA and diabetic cardiomyopathy

[0034] 1.1. With the approval of the Ethics Committee of Tongren Hospital affiliated to Shanghai Jiao Tong University School of Medicine and the informed consent of the patients, patients were enrolled according to the diagnostic criteria for diabetic cardiomyopathy. Peripheral blood samples were collected from 50 patients with diabetic cardiomyopathy and 50 patients with diabetes as references. Metabolites in peripheral blood were extracted and subjected to targeted mass spectrometry detection to quantitatively detect the content of lipid metabolite MA in patients with diabetic cardiomyopathy. At the same time, the correlation between the patients' laboratory test and imaging data indicators, survival prognosis and other indicators reflecting the efficacy was analyzed.

[0035] More than 4,000 positive ion features and more than 2,000 negative ion features were obtained by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Principal component analysis (PCA) was performed on the normalized peaks obtained from the quality control (QC) samples for quality control testing. All samples were extracted and analyzed, and the results are shown in Figure 1.

[0036] Figures A and B show that the QC samples clustered tightly in both positive and negative ion modes, indicating the reproducibility of the experiment. Figures C, D, and E show that the three groups of samples were separated, with the DCM group separated from the healthy group and the T2D group, respectively. This finding indicates that there are differences in lipid metabolism between the healthy group and the DCM group in both positive and negative ion modes of LC-HRMS. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to model the samples. An OPLS-DA model containing one principal component and one orthogonal component was established, which could effectively distinguish between three or two groups of samples (Figures F, G, and H). In this embodiment, 34 differentially expressed metabolites were screened using the variable importance projection value of the first principal component of the model (threshold > 1) and the p-value of the one-dimensional test (threshold < 0.05). By setting the p-value threshold to approximately 0.01, another 20 metabolites were identified (Figure I, Table 4). The heatmap (Figure I) shows the relative abundance of free fatty acids, MA, and 19 other lipid metabolites that were expressed differently in the DCM group compared to the healthy group and the T2D group.

[0037] Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of MA in distinguishing patients with DCM from those with T2D only. As shown in Figure JK, MA exhibited excellent discriminatory ability against DCM, with an AUC of 0.901 (95% confidence interval: 0.8246–0.9776, p < 0.0001), indicating high accuracy. The optimal cutoff value was 0.604 ng / mL (Youden index = 0.70), with corresponding sensitivity and specificity of 0.73 and 0.96, respectively. Furthermore, the combined AUC of MA with NT-proBNP level, LVEF% and E / e' was 0.976 (95% confidence interval: 0.943–1.000, p < 0.0001), indicating that this combination was more effective than single traditional heart failure biomarkers and demonstrated superior predictive performance.

[0038] To investigate the relationship between MA levels and heart failure-related biomarkers, this study performed a correlation analysis between serum MA concentration and common heart failure indicators (NT-proBNP, LVEF%, and E / e') in the DCM group (Figure LO). Serum MA levels were negatively correlated with NT-proBNP levels (DCM patients: R = -0.65, 95% confidence interval [CI]: -0.82 to -0.37, p < 0.0001; healthy participants: R = -0.32, 95% CI: -0.62 to -0.04, p = 0.075), indicating a strong association between MA and cardiac damage in DCM. A moderate positive correlation was found between MA and LVEF% (DCM patients: R = 0.62, 95% CI: 0.33–0.81, p < 0.001; healthy participants: R = 0.15, 95% CI: -0.23 to 0.49, p = 0.431), suggesting that MA may be associated with myocardial injury. Furthermore, a significant negative correlation was found between MA and E / e' (DCM patients: R = -0.60, 95% CI: -0.79 to -0.29, p < 0.001; healthy participants: R = -0.13, 95% CI: -0.47 to 0.25, p = 0.505).

[0039] The results, as shown in the figure above, indicate the correlation between the content of the lipid metabolite MA and diabetic cardiomyopathy.

[0040] Example 2. Systematic evaluation of the function of the lipid metabolite MA in primary cardiac fibroblasts and animal models.

[0041] 2.1 Studying the effect of lipid metabolite MA on transdifferentiation of cardiac fibroblasts at the cellular level

[0042] 2.1.1. Effects of the lipid metabolite MA on the proliferative function of primary cardiac fibroblasts: Primary cardiac fibroblasts (cell culture medium: DMEM + 10% FBS + 1% PS) were used as the research subject. After treating cells with MA at gradient concentrations of 0, 1, 10, and 20 μM for 24 and 48 hours, changes in cell proliferation were assessed using the CCK-8 assay. After treating cells with Tgfβ1 at gradient concentrations of 0, 1, 10, and 20 ng / mL for 24 and 48 hours, changes in cell proliferation were assessed using the CCK-8 assay.

[0043] 2.1.2. Study on the transdifferentiation effect of lipid metabolite MA on primary cardiac fibroblasts: Primary cardiac fibroblasts were treated with 10 μM MA in 50 ng / mL Tgf-β1 for 24 hours. The changes in the levels of fibrosis-related genes and proteins such as α-SMA, COL-1, FN, and MMP9 were detected by RT-qPCR and Western blot experiments. Cell proliferation was detected by EdU fluorescence staining. The expression of α-SMA and COL-1 in cells was detected by immunofluorescence staining.

[0044] 2.1.3. Study on the effect of lipid metabolite MA on mitochondrial function of primary cardiac fibroblasts: Primary cardiac fibroblasts were treated with 10 μM MA in 50 ng / mL Tgf-β1 culture for 24 hours. The generation of ROS in fibroblasts was detected by Mitotracker fluorescence staining; changes in mitochondrial function of fibroblasts were detected by JC-1 fluorescence staining; and the expression of TOM20 and COXIV mitochondrial-related proteins was detected by Western blot.

[0045] To investigate the direct effects of myofibroblasts (CFs) on fibrotic stimulation, this study established a TGF-β1-induced fibroblast transdifferentiation model and evaluated myofibroblast activation markers and mitochondrial function indicators. The results are shown in Figures 2-3.

[0046] In Figure 2, TGF-β1 induction reduced fibroblast viability in a time- and concentration-dependent manner (Figure A). Treatment with 10 ng / mL TGF-β1 for 48 hours was chosen as the optimal pro-fibrotic condition because it induced significant cytotoxicity without causing complete cell death, facilitating subsequent rescue experiments. Treatment with MA (10 μM) for 24 hours restored fibroblast viability (Figure B). This optimized model lays the foundation for studying the anti-fibrotic and mitochondrial function-promoting effects of MA in fibroblasts.

[0047] Results from real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot experiments showed that TGF-β1 stimulation significantly increased the mRNA and protein expression levels of α-SMA, collagen I, fibronectin (FN), and Mmp9, while MA treatment reversed these effects (Figure CK). EdU assays showed that TGF-β1 significantly promoted CF proliferation, while MA treatment inhibited this effect (Figure LM). Immunofluorescence analysis confirmed that MA inhibited TGF-β1-induced upregulation of α-SMA (a myofibroblast marker) and collagen I (an extracellular matrix component) (Figure NP).

[0048] These findings are consistent with the antifibrotic effects of MA in vivo, confirming its potential as a therapeutic agent targeting CF activation in DCM.

[0049] Furthermore, in Figure 3, TGF-β1 led to significant MMP depolarization (reduced red / green ratio), while MA maintained mitochondrial membrane integrity (Figure AB). TGF-β1-induced mitochondrial dysfunction was associated with increased apoptosis, which MA alleviated (Figure CD). Western blot analysis showed that TGF-β1 stimulation significantly downregulated the expression of TOM20 and COXIV, indicating increased mitochondrial volume. MA treatment restored these levels to normal (Figure EG). These findings may represent a compensatory response to mitochondrial respiratory dysfunction.

[0050] 2.2 Using animal models to study the effects of lipid metabolite MA on ventricular remodeling in diabetic cardiomyopathy

[0051] The animal experiments were approved by the Animal Ethics Committee of the Hongqiao Research Institute affiliated with Tongren Hospital. Male homozygous db / db mice with leptin receptor deficiency were used as a diabetic animal model (n=5 mice / group).

[0052] Establishment of a mouse model of diabetic cardiomyopathy: db / db mice were continuously infused subcutaneously with AngII 1000 ng / kg / min or 0.9% saline for 4 consecutive weeks. Subsequently, the oral glucose tolerance test (OGTT) was used to confirm the successful establishment of the diabetic model; blood glucose and lipid levels were measured to determine if metabolic abnormalities were present; and echocardiography was used to determine if cardiac function and structure were affected. After confirming successful establishment of diabetic cardiomyopathy, mice were randomly divided into three groups: the sham group (n=5), the diabetic cardiomyopathy model group (DCM, n=5), and the diabetic cardiomyopathy model + MA treatment group (DCM+MA, n=5). MA was continuously injected intraperitoneally, and the effect of the lipid metabolite MA on improving ventricular remodeling in vivo was observed. The observations included: (1) measuring cardiac function-related indicators of mice by echocardiography; (2) observing the morphology of mouse cardiomyocytes by HE staining of myocardial tissue; (3) observing the accumulation of myocardial glycogen in mice by PAS staining; (4) detecting the content of heart failure-related proteins such as ANP and BNP by Western blot; (5) quantitatively analyzing the degree of fibrosis in mouse myocardial tissue by Masson, α-SMA, and COLI immunohistochemical staining; (6) observing the apoptosis of mouse cardiomyocytes by TUNEL fluorescence staining; and (7) analyzing the morphological changes of mitochondria in mouse cardiomyocytes by transmission electron microscopy. The results are shown in Figure 4.

[0053] The experimental timeline is shown in Figure A. Consistent with previously reported results, the cardiomyopathy mice exhibited typical pathological changes, including impaired systolic / diastolic function, cardiac hypertrophy, and fibrosis. Figure B shows representative M-mode echocardiographic images of the heart. Quantitative analysis showed that, compared with the cardiomyopathy group, mice treated with MA showed significantly improved left ventricular systolic function (Figures C-D). Simultaneously, MA reduced left ventricular dilation (Figures E-F).

[0054] Following assessment of cardiac function, this embodiment also evaluated metabolic parameters, including body weight, glycemic control, and lipid levels, to comprehensively describe the therapeutic effect of MA. As shown in Figure G, mice with DCM had significantly higher body weight than the sham-operated group, consistent with the metabolic disturbances associated with diabetic cardiomyopathy. Notably, MA treatment significantly reduced the body weight of DCM mice, bringing it close to the physiological weight range of the sham-operated group. Compared to the sham-operated group, DCM mice exhibited severe hyperglycemia. MA intervention significantly improved the hyperglycemic state, thereby reducing blood glucose levels. Compared to the sham-operated group, DCM mice showed lipid dyslipidemia, with significantly elevated levels of plasma triglycerides, total cholesterol, and low-density lipoprotein cholesterol, while MA treatment effectively restored lipid balance (Figure HJ). Glucose metabolism was assessed using random blood glucose measurements (Figure K).

[0055] H&E staining was used to assess the pathological condition of myocardial tissue. The cardiomyopathy group showed severe pathological changes, including enlarged cardiomyocytes and disordered myofibrous arrangement. However, the mice were healthy, and myocardial injury was significantly improved after MA intervention. To further explore the structural basis of the cardioprotective effect mediated by MA, histological staining and molecular analysis were performed on cardiac tissue. However, after MA treatment, the PAS-positive area was significantly reduced, indicating that myocardial glycogen metabolism was improved (Figure L). Western blotting results for myocardial injury markers ANP and BNP showed that ANP and BNP expression was significantly increased in the DCM group, suggesting myocardial injury / heart failure. After MA treatment, ANP and BNP expression was reduced compared to the DCM group, indicating that MA alleviated myocardial injury (Figure M). Quantitative protein analysis showed that after MA treatment, ANP level was significantly lower than in the DCM group, and BNP level also showed a decreasing trend, further verifying the ameliorative effect of MA (Figure NO).

[0056] Furthermore, as shown in Figure 5, to assess the effect of MA on cardiac fibrosis, this embodiment detected the expression of key fibrosis markers (α-SMA and collagen I) using immunohistochemistry and quantitatively measured myocardial collagen deposition using Massen's trichrome staining. Representative immunohistochemical images are shown in Figure A. Compared to sham-operated mice, the expression of both α-SMA and collagen I was upregulated in DCM mice. Conversely, MA treatment significantly inhibited the expression of both α-SMA and collagen I. As shown in Figures BD, extensive myocardial fibrosis was observed in the DCM model mice. MA treatment significantly reduced collagen deposition, confirming the improvement in cardiac structural remodeling.

[0057] To investigate the antioxidant effect of amino acids (MA) in diabetic cardiomyocytes (DCM), this study evaluated mitochondrial function using histopathological, molecular, and biochemical methods (Figure E). Mice with DCM had a higher proportion of TUNEL-positive cardiomyocytes, which was significantly improved after MA treatment. Furthermore, mice with DCM exhibited mitochondrial dysfunction, and MA treatment significantly restored mitochondrial homeostasis.

[0058] Example 3. Target and molecular mechanism of lipid metabolite MA in reversing cardiac fibroblast transdifferentiation

[0059] 3.1 Nqo1 was confirmed to be a target protein of the lipid metabolite MA.

[0060] Transcriptomic analysis revealed that Nqo1 may be a direct target protein of the lipid metabolite MA. Therefore, this embodiment further confirms in vitro that the lipid metabolite MA can interact with its target protein Nqo1 and explores the binding site.

[0061] 3.1.1. Utilizing transcriptomics to analyze key target proteins of the lipid metabolite MA

[0062] As shown in Figure 6, STZ-induced mice exhibited diabetic cardiomyopathy in both the compensation and decompensation phases, demonstrating a typical pattern of cardiac function changes. Transcriptomic analysis of cardiac tissue from mice displaying DCM and treated with MA identified a total of 16,549 genes. Principal component analysis (PCA) highlighted significant differences in gene expression profiles between the groups, indicating substantial transcriptomic changes regulated by DCM intervention and MA administration (Figure A). When p-values ​​< 0.05 and fold changes ≤ 0.67 or ≥ 1.5 were considered acceptable thresholds, 416 differentially expressed proteins (DEPs) were identified between the DCM and MA groups. Hierarchical clustering and heatmap visualization of these DEPs revealed distinct expression patterns, indicating the impact of MA on transcriptomic signatures (Figure B). These DEPs were subsequently visualized using a volcano plot (Figure C). Compared to DCM mice, mice treated with MA exhibited 189 upregulated genes and 227 downregulated genes. Furthermore, gene ontology (GO) analysis revealed a significant upregulation of antioxidant activity (Figure D). Wikipedia pathway enrichment analysis of differentially expressed proteins (DEPs) after MA treatment (Figure E) highlighted the regulatory roles of metabolic pathways and the TGF-β signaling pathway, indicating a holistic impact on cellular processes. This phenomenon was further confirmed by the increased expression of Nqo1 (a quinone oxidoreductase) observed after MA treatment (Figure FG). Taken together, these transcriptomic insights highlight the potential of MA to mitigate DCM-induced changes by regulating fibrosis-related signaling and oxidative stress, thus demonstrating its therapeutic potential for DCM.

[0063] 3.1.2. Regulation mechanism of lipid metabolite MA on Nqo1: (1) The docking site between lipid metabolite MA and Nqo1 was simulated in 3D using molecular docking technology; (2) The expression of Nqo1 in mouse myocardial tissue was detected by Western blot experiment. At the same time, the protein phosphorylation levels of Tgfβ1 and Smad4 were detected.

[0064] The transcriptomic analysis described above indicated that the expression level of Nqo1 increased after MA treatment in vivo. To further assess the potential interaction between MA and Nqo1, molecular docking analysis was performed, and the results are shown in Figure 7.

[0065] MA exhibits a Vina contact score of -5.1 kcal / mol, indicating strong affinity binding. MA binds to the Nqo1 substrate binding pocket via hydrogen bonding with TYR-127 and ALA-123, with bond lengths of 3.0 Å, 2.0 Å, and 2.8 Å, respectively (Figure A).

[0066] To delve into the molecular mechanisms underlying the therapeutic effects of fibrosis cytokines (MA), the influence of MA on the Nqo1 signaling pathway during TGF-β1-induced fibrosis activation was investigated. As shown in Figures BC, Nqo1 expression levels increased after MA treatment. Furthermore, MA treatment also reduced the expression of downstream target proteins, including the inflammatory fibrosis factors TGF-β1 and Smad4 (Figure DF).

[0067] Example 4. Using MA as a biomarker to assess diabetic cardiomyopathy

[0068] 4.1. Differences between diabetes mellitus and diabetic cardiomyopathy

[0069] The echocardiographic measurement results are summarized in Table 1. The table shows no significant difference between the DCM group and the T2D group in terms of LVIDd or LVIDs (p > 0.05).

[0070] However, the LVEF in the DCM group was significantly lower than that in the T2D group (56.46% ± 9.40% vs. 63.91% ± 4.33%, p < 0.001), indicating that DCM patients have left ventricular systolic dysfunction.

[0071] After evaluating other echocardiographic abnormalities in the DCM group, it was found that: (1) the FS in the DCM group was lower (29.71% ± 1.5% compared to 31.80% ± 1.80% in the T2D group, p = 0.013), consistent with the LVEF results; (2) the E / e′ ratio in the DCM group was significantly higher than that in the T2D group (10.9 ± 3.87 compared to 8.66 ± 2.26, p = 0.002), indicating impaired left ventricular diastolic function; (3) the E / A ratio in the DCM group was lower than that in the T2D group (0.7 ± 0.24 compared to 0.9 ± 0.40, p = 0.014), further supporting diastolic dysfunction.

[0072] Table 1. Echocardiographic Measurement Results

[0073]

[0074] The laboratory parameters are listed in Table 2. As can be seen from Table 2, there were no significant differences between the DCM group and the T2D group in terms of cardiac injury markers (high-sensitivity troponin I, creatine kinase isoenzyme, myoglobin level), renal function (serum creatinine level), lipid profile (total cholesterol, triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol levels) or natriuretic peptide (NT-proBNP) (all p values ​​were greater than 0.05).

[0075] However, compared with the T2D group, the DCM group showed significant abnormalities in metabolism and myocardial stress: (1) In terms of glycemic control, the DCM group had higher glycated hemoglobin levels (8.62% ± 2.19% vs. 7.63% ± 1.38%, p < 0.001) and higher fasting blood glucose (7.66 ± 2.89 vs. 7.05 ± 2.35 mmol / L, p = 0.003), indicating poorer long-term and acute glycemic control; (2) In terms of assessing myocardial injury / metabolism, the DCM group had elevated serum lactate dehydrogenase levels (188.47 ± 55.82 vs. 178.20 ± 40.40 units / L, p = 0.019), indicating increased myocardial cell turnover or enhanced oxidative stress.

[0076] Table 2. Results of cardiac injury markers

[0077]

[0078] 4.2. Clinical validation of the diagnostic efficacy of MA in diabetic cardiomyopathy

[0079] To further validate the clinical effectiveness of myocardial infarction (MA) in real-world patients, blood samples were randomly collected from 300 hospitalized patients between March 2025 and January 2026. MA parameters were measured using targeted mass spectrometry. Based on the MA cut-off values ​​obtained in previous studies, patients were divided into high-MA and low-MA groups. Baseline clinical data were collected, including duration of diabetes, myocardial injury markers, and echocardiographic assessment of cardiac function severity in all enrolled patients. Multiple linear regression models were used to analyze the predictive value of these indicators for cardiac function deterioration (as shown in the table).

[0080]

[0081] The echocardiographic measurements are summarized in the table. There was no significant difference in LVIDd or LVIDs between the MA high and MA low groups (p > 0.05). However, the LVEF in the MA low group was significantly lower than that in the MA high group (60.31% ± 7.11% vs. 64.01% ± 4.86%, p < 0.001), indicating left ventricular systolic dysfunction in patients in the MA low group. After evaluating other echocardiographic abnormalities in the MA low group, it was found that: (1) the FS in the MA low group was lower (28.13% ± 1.4% vs. 32.8% ± 1.6% in the MA high group, p < 0.001), consistent with the LVEF results; (2) the E / e′ ratio in the MA low group was significantly higher than that in the MA high group (9.5 ± 3.09 vs. 7.9 ± 2.34, p < 0.001), indicating impaired left ventricular diastolic function; (3) the E / A ratio in the MA low group was lower than that in the MA high group (0.59 ± 0.34 vs. 0.89 ± 0.33, p < 0.001), further supporting diastolic dysfunction.

[0082]

[0083] The laboratory parameters are listed in the table. There were no significant differences between the MA low and MA high groups in cardiac injury markers (creatine kinase isoenzymes), lipid profiles (total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels), and renal function (serum creatinine level) (all p values ​​were greater than 0.05). However, compared with the MA high group, the MA low group showed significant differences in myocardial stress, such as elevated NT-proBNP (56.12 vs. 32.36 ng / L, p < .001), high-sensitivity troponin (0.0198 vs. 0.012 μg / L, p < 0.008), myoglobin levels (47.52 vs. 39.04 μg / L, p < 0.027), and elevated serum lactate dehydrogenase levels (169 vs. 158 U / L, p = 0.003), indicating increased cardiomyocyte turnover or enhanced oxidative stress in the MA low group.

[0084] Based on the above results, we believe that the MA low group has lower cardiac function and can predict the patient profile of DCM (Figure 8). When we define the MA low group as the DCM patient group and the MA high group as the T2D patient group, the results of targeted mass spectrometry quantitative analysis show that the expression of MA concentration is significantly reduced in DCM patients, which demonstrates the diagnostic value of MA in distinguishing patients with DCM from those with only T2D.

[0085] Furthermore, to explore the relationship between MA levels and heart failure-related biomarkers, this study performed a correlation analysis on common serum heart failure markers (NT-proBNP, LVEF%, and E / e') in the DCM group (Figure BD). Serum MA levels were negatively correlated with NT-proBNP levels (DCM patients: R = -0.62, 95% confidence interval [CI]: -0.71 to -0.5, p < 0.0001; T2D patients: R = -0.16, 95% CI: -0.31 to -0.01, p = 0.052), indicating a strong association between MA and cardiac damage in DCM. A moderate positive correlation was found between MA and LVEF% (DCM patients: R = 0.58, 95% CI: 0.46 to 0.67, p < 0.0001; T2D patients: R = 0.04, 95% CI: -0.12 to 0.21, p = 0.603), suggesting that MA may be associated with myocardial injury. Furthermore, a significant negative correlation was found between MA and E / e' (DCM patients: R = -0.66, 95% CI: -0.74 to -0.56, p < 0.0001; T2D patients: R = -0.10, 95% CI: -0.26 to 0.06, p = 0.219). The results, as shown in the figure above, indicate the correlation between the lipid metabolite MA content and DCM. This is consistent with our previous small-sample cross-sectional results.

Claims

1. The application of peripheral blood metabolites in the preparation of tools to differentiate between diabetic cardiomyopathy and non-diabetic cardiomyopathy, characterized in that: The metabolite in the peripheral blood is n-heptadecanoic acid.

2. The application as described in claim 1, characterized in that, The method of using the tool to distinguish between diabetic cardiomyopathy and non-diabetic cardiomyopathy includes the following steps: detecting the expression level of n-heptadecanoic acid in the test sample to determine and predict the risk of diabetic cardiomyopathy; the judgment criteria are: if the relative abundance of n-heptadecanoic acid in the test sample is less than 0.604, it indicates a high risk of diabetic cardiomyopathy; otherwise, it indicates a low risk of diabetic cardiomyopathy.

3. The application of peripheral blood metabolites in the preparation of tools to differentiate between diabetic cardiomyopathy and non-diabetic cardiomyopathy, characterized in that: The heptadecanoic acid is also associated with NT-pIoBNP levels, LVEF% and E / e' indicators.

4. The application as described in claim 1, characterized in that, The tools include detection reagents, detection kits, and detection chips.

5. Application of n-Heptadecanoic acid in the preparation of drugs / dietary supplements for the treatment and relief of diabetic cardiomyopathy.

6. Application of n-Heptadecanoic acid in the preparation of drugs / dietary supplements that inhibit the transdifferentiation of cardiac fibroblasts.

7. Application of n-Heptadecanoic acid in the preparation of drugs / dietary supplements for improving ventricular remodeling in diabetic cardiomyopathy.

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