Metabolic biomarker for diagnosing systemic lupus erythematosus and application thereof

Abstract

The invention provides a metabolic biomarker for diagnosing systemic lupus erythematosus and application of the metabolic biomarker, and belongs to the technical field of biological medicines. The metabolic biomarker is at least one metabolite of sasol, ceramide phosphate and homoveratric acid. A reagent for detecting the expression quantity of the metabolic biomarker can be used for preparing a product for diagnosing systemic lupus erythematosus. The invention determines that the expression quantities of metabolites such as 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphoethanolamine, sasol, ceramide phosphate, homoveratric acid and the like in blood samples of an SLE patient and a healthy person have significant changes, so that the metabolite is put forward to be used as a biomarker for SLE disease diagnosis independently or in combination; the kit has the advantages of high accuracy, high sensitivity and high specificity, and provides a new target for diagnosis, intervention improvement and the like of SLE patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to metabolic biomarkers for diagnosing systemic lupus erythematosus and application thereof. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple systems, and its pathogenesis is complex. The clinical manifestations are highly heterogeneous, and early or atypical cases are prone to be missed or misdiagnosed.

[0003] Metabolomics is a systems biology method that uses blood, urine, feces and other samples as the main research specimens to find specific metabolites or metabolites. It is generally believed that the pathogenesis of SLE is related to family genetic susceptibility, environmental changes, immune effects and estrogen levels. Metabolomics is involved in the pathogenesis of SLE through multiple pathways, and the lipid metabolism and amino acid metabolism of blood in SLE patients are found to have significantly changed metabolite categories, such as lysophosphatidyl ethanolamine and taurine, which are identified as potential biomarkers for SLE. However, there is a common problem of insufficient sensitivity or specificity. Therefore, there is an urgent need for biomarkers with high accuracy and high sensitivity and high specificity for diagnosing systemic lupus erythematosus. SUMMARY

[0004] In view of this, the present application aims to provide metabolic biomarkers for diagnosing systemic lupus erythematosus and application thereof, and to solve at least one technical problem in the background art.

[0005] The present application is implemented as follows: The present application provides metabolic biomarkers for diagnosing systemic lupus erythematosus, and the metabolic biomarkers are at least one metabolite of sarsorine, ceramide phosphate and kuh-safflower acid.

[0006] Further, the metabolic biomarker further includes 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine.

[0007] Further, the metabolic biomarker is a combination of at least two metabolites of sarsorine, ceramide phosphate and kuh-safflower acid.

[0008] Further, the metabolic biomarker is a combination of at least three metabolites of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, sarsorine, ceramide phosphate and kuh-safflower acid.

[0009] Further, the metabolic biomarker is a combination of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, sarsorine, ceramide phosphate and kuh-safflower acid.

[0010] The second aspect of the present application provides use of a reagent for detecting expression of the metabolic biomarker for diagnosing systemic lupus erythematosus in the preparation of a product for diagnosing systemic lupus erythematosus.

[0011] Further, the product detects the expression of the metabolic biomarker in blood based on mass spectrometry; and the product is a kit or a reagent.

[0012] The third aspect of the present application provides a product for diagnosing systemic lupus erythematosus, which comprises a reagent for detecting expression of the metabolic biomarker for diagnosing systemic lupus erythematosus.

[0013] Further, the product detects the expression of the metabolic biomarker in blood based on mass spectrometry.

[0014] Further, the product is a kit or a reagent.

[0015] The present application determines that the expression of metabolites such as 1-stearoyl-2-hydroxyl-sn-glycero-3-phosphoethanolamine, sarsorine, ceramide phosphate, and soladulcidum acid in blood samples of SLE patients and healthy people changes significantly, and therefore proposes the metabolites alone or in combination as biomarkers for the diagnosis of SLE disease, with the advantages of high accuracy, high sensitivity, and high specificity, providing new targets for the diagnosis and intervention improvement of SLE patients. The corresponding auxiliary early diagnosis reagents and kits based on the biomarkers of the metabolites alone or in combination have wide scientific research value and clinical effect, and provide great convenience for early screening, clinical diagnosis, and intervention treatment. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 FIG. 4 is a box plot of the differential expression of four metabolites in blood of SLE group and HC group in the experiment; Figure 2 FIG. 5 is a ROC curve of four metabolites alone as biomarkers in SLE group and HC group in the experiment; Figure 3 FIG. 6 is a ROC curve of any two of the four metabolites combined as biomarkers in SLE group and HC group in the experiment; Figure 4 FIG. 7 is a ROC curve of any three of the four metabolites combined as biomarkers in SLE group and HC group in the experiment; Figure 5 FIG. 8 is a ROC curve of the four metabolites combined as biomarkers in SLE group and HC group in the experiment; Figure 6 FIG. 9 is a box plot of the differential expression of four metabolites in blood of SLE group and HC group in the verification; Figure 7 ROC curve of the 4 metabolites in the validation set as biomarkers in the SLE group and the HC group; Figure 8 ROC curve of any two of the 4 metabolites in the validation set as biomarkers in the SLE group and the HC group; Figure 9 ROC curve of any three of the 4 metabolites in the validation set as biomarkers in the SLE group and the HC group; Figure 10 ROC curve of the combination of the 4 metabolites in the validation set as biomarkers in the SLE group and the HC group. DETAILED DESCRIPTION

[0017] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0018] I. Experimental subjects 138 patients with systemic lupus erythematosus were selected as the SLE group in the experimental set, and 58 healthy people without systemic lupus erythematosus were selected as the HC group. 51 patients with systemic lupus erythematosus were selected as the SLE group in the validation set, and 42 healthy people without systemic lupus erythematosus were selected as the HC group. The characteristic information is shown in Table 1.

[0019] Table 1

[0020] Among them, part of the systemic lupus erythematosus patients lack the clinical information of urinary occult blood, urinary protein, anti-double-stranded DNA antibody, anti-nuclear antibody, anti-Sm antibody and the like, so when calculating the positive proportion of the above indexes, the total number of patients with clinical information of the index is taken as the basis.

[0021] II. Screening of differentially expressed metabolites 1. Sample pretreatment (1) The blood samples of the experimental subjects were taken out from -80℃, and after slow thawing, each sample was fully vortexed and mixed; (2) 100 μL of the original sample was taken out from each sample, and 400 μL of the extraction buffer methanol MeOH / acetonitrile ACN (1:1, v / v) containing an internal standard was added, and after fully vortexing for 30 s, ultrasonic lysis was performed for 1 min; (3) The sample was placed at -20℃ for 1 h; (4) Centrifugation at 4℃, 18000g for 15 min, remove the protein precipitate, and transfer the supernatant to a new centrifuge tube; (5) After the concentrator was dried at room temperature, 100 μL of ACN:H2O (1:1, v / v) was added, vortexed thoroughly for 30 s, and then redissolved by ultrasonic for 10 min. (6) Centrifugation at 4°C, 18000g for 15 min, and then the supernatant was transferred to a new centrifuge tube for LC / MS analysis.

[0022] 2. Liquid chromatography-mass spectrometry analysis The metabolites were separated by Waters UPLC ultra-high performance liquid system combined with Waters ACQUITY UPLC BEH C18 Column (1.7 μm, 2.1 mm x 100 mm), the injection volume was 10 μL, eluted at a flow rate of 400 μl / min, and the column temperature was 45°C.

[0023] Among them, the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is 0.1% formic acid acetonitrile. The liquid phase gradient setting: 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.

[0024] After the metabolites were separated by the ultra-high performance liquid system, they were injected into the ESI ion source for ionization and then analyzed by timsTOF Pro mass spectrometry. The ion source voltage was set to 4.5 kV, and the parent ions and their secondary fragments of the peptide segments were detected and analyzed by high-resolution TOF. The mass spectrometry scan range was set to 50 m / z~1300 m / z. The data acquisition mode used the parallel accumulation serial fragmentation (PASEF) mode. After the first mass spectrometry acquisition, two PASEF mode acquisitions of the secondary spectrum of the parent ion charge number in the range of 0~1 were performed, and the dynamic exclusion time of tandem mass spectrometry scanning was set to 6 s to avoid repeated scanning of the parent ion.

[0025] 3. Database search The mass spectrometry data was used for peak extraction, alignment, and retention time correction of the raw data by MetaboScape 2022, and the first and second mass errors were controlled within 20 ppm to ensure the accuracy of the identification results. The structure and annotation information of the metabolites were obtained by NIST, HMDB, self-database, and integrated public database spectrum comparison.

[0026] III. Verification of diagnostic efficiency Figure 1 and Figure 6The comparison chart of the expression of four metabolites in the blood of SLE and HC groups in the experimental set and the validation set respectively, according to the relationship between the difference of the expression of four metabolites in the blood of SLE and HC experimental objects and the clinical parameters, the statistical test, regression analysis is carried out to evaluate. The diagnostic efficiency is evaluated by the receiver operating characteristic curve (ROC curve). When the area-under-the-curve (AUC) of the ROC curve is greater than or equal to 0.9, the diagnostic index is considered to be “highly accurate”, when 0.8≤AUC<0.9, the diagnostic index is considered to be “accurate”, when 0.7≤AUC<0.8, the diagnostic index is considered to be “moderately accurate”.

[0027] The four metabolites are 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, salsolinol, ceramide phosphate and high-chenopodin acid respectively; 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine is a lysophosphatidyl ethanolamine; salsolinol is a tetrahydroisoquinoline neurotoxin; ceramide phosphate is a phosphorylated derivative of ceramide, which is an important molecule in the sphingomyelin metabolic pathway; high-chenopodin acid is also known as homoveratric acid, which is the main metabolic end product of dopamine.

[0028] Using the above four metabolites alone or in combination as serum diagnostic markers for distinguishing SLE group and HC group, the ROC curve of SLE and HC in the experimental set is shown in Figures 2 to 5 , the ROC curve of SLE and HC in the validation set is shown in Figures 7 to 10 , and the AUC, sensitivity and specificity are shown in Tables 2 to 5.

[0029] Table 2

[0030] Table 3

[0031] Table 4

[0032] Table 5

[0033] As can be seen from Figure 1 and Figure 6 , the expression of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, salsolinol, ceramide phosphate and high-chenopodin acid in the blood of systemic lupus erythematosus patients (SLE) is higher than that of healthy people (HC), and the difference is very significant (p less than 0.001).

[0034] Figure 2 、 Figure 7 and Table 2 show that the above-mentioned differentially expressed metabolites can effectively distinguish SLE from HC (AUC>0.8) alone, but the sensitivity and specificity need to be improved, wherein 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine has lower sensitivity and specificity as a biomarker alone.

[0035] As Figures 3 to 5 、 Figures 8 to 10 , Table 3 to Table 5 show that any two combinations, three combinations, and all combinations of the four metabolites can effectively distinguish SLE from HC as biomarkers, and the effect is obviously better than that of the metabolites alone; wherein the advantages of the three combinations and all combinations are more obvious, and the AUC, sensitivity or specificity is significantly improved.

[0036] The experimental results show that the four metabolites can be used as blood diagnostic markers for distinguishing healthy people from SLE, and are used for preparing products for diagnosing systemic lupus erythematosus, such as kits and reagents. The combination of the metabolites as markers has higher sensitivity, specificity and accuracy. It provides an important basis for further clinical research and a new idea for the diagnosis and treatment of SLE.

[0037] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A metabolic biomarker for diagnosing systemic lupus erythematosus, characterized in that: The metabolic biomarker is at least one metabolite of sarsolin, ceramide phosphate, and homoveratric acid.

2. The metabolic biomarker for diagnosing systemic lupus erythematosus according to claim 1, characterized in that: The metabolic biomarkers also include 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine.

3. The metabolic biomarker for diagnosing systemic lupus erythematosus according to claim 1, characterized in that: The metabolic biomarker is a combination of at least two metabolites of sarsolin, ceramide phosphate, and homoveratric acid.

4. The metabolic biomarker for diagnosing systemic lupus erythematosus according to claim 2, characterized in that: The metabolic biomarker is a combination of at least three metabolites of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, sarsolin, ceramide phosphate, and homoveratric acid.

5. The metabolic biomarker for diagnosing systemic lupus erythematosus according to claim 4, characterized in that: The metabolic biomarker is a combination of 1-stearoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine, sarsolin, ceramide phosphate and homoveratric acid.

6. Use of a reagent for detecting the expression level of a metabolic biomarker for diagnosing systemic lupus erythematosus according to any one of claims 1 to 5 in the preparation of a product for diagnosing systemic lupus erythematosus.

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

8. A product for diagnosing systemic lupus erythematosus, characterized in that: The product comprises a reagent for detecting the expression level of the metabolic biomarker for diagnosing systemic lupus erythematosus according to any one of claims 1 to 5.

9. The product for diagnosing systemic lupus erythematosus according to claim 8, characterized in that: The product detects the expression level of the metabolic biomarker in the blood based on mass spectrometry.

10. The product for diagnosing systemic lupus erythematosus according to claim 8, characterized in that: The product is a kit or a reagent.

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