Metabolic biomarkers for the diagnosis of systemic lupus erythematosus and their applications

Abstract

The application provides metabolic biomarkers for diagnosing systemic lupus erythematosus and application thereof, and belongs to the technical field of biological medicine. The metabolic biomarker is at least one metabolite of sarsasurine, ceramide phosphate and high quercetin acid. A reagent for detecting the expression amount of the metabolic biomarker can be used for preparing a product for diagnosing systemic lupus erythematosus. The application determines that the expression amounts of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, sarsasurine, ceramide phosphate and high quercetin acid metabolites in blood samples of SLE patients and healthy people change significantly, so the metabolites are proposed to be used as biomarkers for diagnosing SLE diseases alone or in combination, and the biomarkers have the advantages of high accuracy, high sensitivity and high specificity, and provide new targets for the diagnosis and intervention improvement of SLE patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to metabolic biomarkers for the diagnosis of systemic lupus erythematosus and their applications. Background Technology

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple systems. Its pathogenesis is complex, and its clinical manifestations are highly heterogeneous. Early or atypical cases are easily missed or misdiagnosed.

[0003] Metabolomics is a systems biology approach that uses omics detection methods, primarily blood, urine, and feces, to identify specific metabolites or metabolomes. Currently, the pathogenesis of SLE is generally believed to be related to multiple factors, including familial genetic susceptibility, environmental changes, immune influences, and estrogen levels. Metabolomics participates in the pathogenesis of SLE through multiple pathways. Among these, blood lipid metabolism and amino acid metabolism have revealed significantly altered metabolite classes in SLE patients, such as lysophosphatidylethanolamine and taurine, which have been identified as potential biomarkers for SLE. However, these biomarkers generally suffer from insufficient sensitivity or specificity. Therefore, there is an urgent need for biomarkers with high accuracy, sensitivity, and specificity for the diagnosis of systemic lupus erythematosus (SLE). Summary of the Invention

[0004] Therefore, the present invention aims to provide metabolic biomarkers for the diagnosis of systemic lupus erythematosus and their applications, in order to solve at least one of the technical problems in the background art.

[0005] This invention is implemented as follows:

[0006] The first aspect of the present invention provides a metabolic biomarker for diagnosing systemic lupus erythematosus, said metabolic biomarker being at least one metabolite selected from sasolline, ceramide phosphate, and homosaccharide.

[0007] Furthermore, the metabolic biomarker also includes 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine.

[0008] Furthermore, the metabolic biomarker is a combination of at least two metabolites selected from sasolline, ceramide phosphate, and succinate.

[0009] Furthermore, the metabolic biomarker is a combination of at least three metabolites selected from 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine, sasolene, ceramide phosphate, and homosaccharide.

[0010] Furthermore, the metabolic biomarker is a combination of 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine, sasolene, ceramide phosphate and homosaccharide.

[0011] A second aspect of the present invention provides the use of a reagent for detecting the expression levels of the above-mentioned metabolic biomarkers for diagnosing systemic lupus erythematosus in the preparation of products for diagnosing systemic lupus erythematosus.

[0012] Furthermore, the product is based on mass spectrometry to detect the expression level of the metabolic biomarker in blood; the product is a kit or reagent.

[0013] A third aspect of the present invention provides a product for diagnosing systemic lupus erythematosus, comprising a reagent for detecting the expression levels of the aforementioned metabolic biomarkers for diagnosing systemic lupus erythematosus.

[0014] Furthermore, the product is based on mass spectrometry to detect the expression levels of the metabolic biomarkers in the blood.

[0015] Furthermore, the product is a kit or reagent.

[0016] This invention identifies significant differences in the expression levels of metabolites such as 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine, sasolene, ceramide phosphate, and homoscarpic acid in blood samples from SLE patients and healthy individuals. Therefore, this invention proposes that these metabolites, alone or in combination, serve as biomarkers for SLE diagnosis, offering advantages of high accuracy, high sensitivity, and high specificity, providing new targets for the diagnosis and intervention of SLE patients. The development of corresponding auxiliary early diagnostic reagents and kits based on these biomarkers, alone or in combination, has broad research value and clinical applications, providing significant convenience for early screening, clinical diagnosis, and intervention treatment. Attached Figure Description

[0017] Figure 1 Box plots showing the differential expression of four metabolites in the blood of the SLE group and the HC group in the experimental set;

[0018] Figure 2 ROC curves of four metabolites used as biomarkers in the experimental group in the SLE and HC groups;

[0019] Figure 3 ROC curves for the SLE group and HC group, showing any two combinations of the four metabolites in the experimental set as biomarkers.

[0020] Figure 4 ROC curves for the SLE group and HC group, showing any three combinations of four metabolites from the experimental set as biomarkers.

[0021] Figure 5 ROC curves of four metabolite combinations used as biomarkers in the experimental group were presented in the SLE and HC groups.

[0022] Figure 6 To validate the box plots showing the differential expression of four metabolites in the blood of the SLE group and the HC group;

[0023] Figure 7 To verify the ROC curves of the four metabolites as biomarkers in the SLE and HC groups;

[0024] Figure 8 To verify the ROC curves of any two combinations of the four metabolites as biomarkers in the SLE and HC groups;

[0025] Figure 9 To verify the ROC curves of any three combinations of the four metabolites as biomarkers in the SLE and HC groups;

[0026] Figure 10 To verify the ROC curves of the combination of four metabolites as biomarkers in the SLE and HC groups. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0028] I. Experimental Subjects

[0029] The experimental set included 138 patients with systemic lupus erythematosus (SLE) as the SLE group and 58 healthy individuals without SLE as the HC group; the validation set included 51 patients with SLE as the SLE group and 42 healthy individuals without SLE as the HC group; the characteristic information is shown in Table 1 below.

[0030] Table 1

[0031]

[0032] Some patients with systemic lupus erythematosus lack clinical information on indicators such as occult blood in urine, protein in urine, anti-double-stranded DNA antibody, antinuclear antibody, and anti-Sm antibody. Therefore, when calculating the positive rate of the above indicators, the total number of patients with clinical information on the above indicators is used as the benchmark.

[0033] II. Screening for differentially expressed metabolites

[0034] 1. Sample pretreatment

[0035] (1) Take out the blood samples of the experimental subjects from -80℃, thaw them slowly, and then vortex them thoroughly to mix them.

[0036] (2) Take out 100 μL of the original sample from each sample, and then add 400 μL of extraction buffer containing internal standard, methanol MeOH / acetonitrile ACN (1:1, v / v), vortex thoroughly for 30 s, and then sonicate for 1 min.

[0037] (3) The sample was placed at -20℃ for 1 hour to precipitate;

[0038] (4) Centrifuge at 18000g for 15 min at 4℃ to remove protein precipitate, and transfer the supernatant to a new centrifuge tube;

[0039] (5) After drying at room temperature using a concentrator, add 100 μL of ACN:H2O (1:1, v / v), vortex thoroughly for 30 seconds, and then sonicate for 10 minutes to reconstitute.

[0040] (6) Centrifuge at 18000g for 15 min at 4℃, and transfer the supernatant to a new centrifuge tube for LC / MS analysis.

[0041] 2. Liquid chromatography-mass spectrometry analysis

[0042] Metabolites were separated using a Waters UPLC ultra-high performance liquid chromatography system with a Waters ACQUITY UPLC BEH C18 column (1.7µm, 2.1mm×100mm). The injection volume was 10µL, and elution was performed at a flow rate of 400µl / min and a column temperature of 45℃.

[0043] Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is acetonitrile containing 0.1% formic acid. Liquid phase gradient settings: 0 min–11 min, 2%B–98%B; 11.0 min–12.0 min, 98%B; 12.0 min–12.1 min, 98%B–2%B; 12.1 min–15.0 min, 2%B.

[0044] Metabolites were separated by an ultra-high performance liquid chromatography (UHPLC) system and then injected into an ESI ion source for ionization before analysis by a timsTOFPro mass spectrometer. The ion source voltage was set to 4.5 kV, and high-resolution TOF was used to detect and analyze both the peptide precursor ion and its secondary fragments. The mass spectrometry scan range was set to 50 m / z to 1300 m / z. The parallel accumulation serial fragmentation (PASEF) mode was used for data acquisition. After primary mass spectrometry acquisition, two PASEF scans were performed to acquire secondary spectra with precursor ion charges ranging from 0 to 1. The dynamic exclusion time for tandem mass spectrometry was set to 6 s to avoid duplicate scanning of the precursor ion.

[0045] 3. Database search

[0046] Mass spectrometry data were extracted, aligned, and retained time corrected using MetaboScape 2022. The primary and secondary mass errors were controlled within 20 ppm to ensure the accuracy of the identification results. The structure and annotation information of metabolites were obtained by comparing spectra from NIST, HMDB, our own database, and integrated public databases.

[0047] III. Verifying Diagnostic Efficiency

[0048] Figure 1 and Figure 6 The figures show comparisons of the expression levels of four metabolites in the blood of SLE and HC subjects in the experimental and validation sets, respectively. The relationship between the differences in the expression levels of these four metabolites in the blood of SLE and HC subjects and clinical parameters was evaluated using statistical tests and regression analysis. Diagnostic efficacy was evaluated using the receiver operating characteristic (ROC) curve. A diagnostic indicator was considered "highly accurate" when the area under the ROC curve (AUC) ≥ 0.9, "accurate" when 0.8 ≤ AUC < 0.9, and "moderately accurate" when 0.7 ≤ AUC < 0.8.

[0049] The four metabolites are 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine, sasolene, ceramide phosphate, and homoveratric acid. 1-Stearyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine is a lysophosphatidylethanolamine. Sasolene is a tetrahydroisoquinoline neurotoxin. Ceramide phosphate is a phosphorylated derivative of ceramide and an important molecule in the sphingomyelin metabolic pathway. Homoveratric acid, also known as homometa-vanillic acid, is the main metabolic end product of dopamine.

[0050] The above four metabolites, alone or in combination, were used as serum diagnostic markers to distinguish between the SLE and HC groups. The ROC curves for SLE and HC in the experimental set are shown below. Figures 2 to 5 As shown, the ROC curves of SLE and HC in the validation set are as follows: Figures 7 to 10 As shown, AUC, sensitivity, and specificity are shown in Tables 2 to 5.

[0051] Table 2

[0052]

[0053] Table 3

[0054]

[0055] Table 4

[0056]

[0057] Table 5

[0058]

[0059] Depend on Figure 1 and Figure 6 As can be seen, the expression levels of 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine, sasolline, ceramide phosphate, and homosaccharide in the blood of patients with systemic lupus erythematosus (SLE) were all higher than those in healthy individuals (HC), and the differences were highly significant (p < 0.001).

[0060] Figure 2 , Figure 7 The results in Table 2 show that the differentially expressed metabolites can effectively distinguish between SLE and HC on their own (AUC>0.8), but their sensitivity and specificity need to be improved. Among them, 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphate ethanolamine has lower sensitivity and specificity when used alone as a biomarker.

[0061] like Figures 3 to 5 , Figures 8 to 10 As shown in Tables 3 to 5, any two, three, or all combinations of the four metabolites can effectively distinguish between SLE and HC as biomarkers, and their effects are significantly better than those of individual metabolites. Among them, the advantages of three and all combinations are more obvious, with significantly improved AUC, sensitivity, or specificity.

[0062] Experimental results show that the four metabolites can serve as blood diagnostic biomarkers to distinguish healthy individuals from those with SLE, and can be used to prepare diagnostic products for systemic lupus erythematosus, such as kits and reagents. Combining these metabolites as biomarkers demonstrates higher sensitivity, specificity, and accuracy. This provides important evidence for further clinical research and offers new insights into SLE diagnosis and treatment.

[0063] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Use of a metabolic biomarker for the manufacture of a product for diagnosing systemic lupus erythematosus, characterized in that, The metabolic biomarker is at least one of sarsasapogenin, ceramide phosphate, and glycyrrhizic acid.

2. Use according to claim 1, characterized in that, The metabolic biomarker further comprises 1-stearoyl-2-hydroxysn-glycero-3-phosphoethanolamine.

3. Use according to claim 1, characterized in that, The metabolic biomarker is a combination of at least two of sarsasapogenin, ceramide phosphate, and glycyrrhizic acid.

4. Use according to claim 2, characterized in that, The metabolic biomarker is a combination of at least three of 1-stearoyl-2-hydroxysn-glycero-3-phosphoethanolamine, sarsasapogenin, ceramide phosphate, and glycyrrhizic acid.

5. Use according to claim 4, characterized in that, The metabolic biomarker is a combination of 1-stearoyl-2-hydroxysn-glycero-3-phosphoethanolamine, sarsasapogenin, ceramide phosphate, and glycyrrhizic acid.

6. Use according to claim 1, characterized in that, The product detects the expression amount of the metabolic biomarker in blood based on mass spectrometry; the product is a kit or a reagent.

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