Serum exosome metabolism markers for identifying SLE and nephritis implication and application of serum exosome metabolism markers

Abstract

The invention discloses a group of serum exosome metabolism markers for identifying SLE (systemic lupus erythematosus) and nephritis implication, wherein the serum exosome metabolism markers comprise one or more of Pro-Asn-Gln-Met-Ser, C24: 1 spingolipid and protoporphyrin IX. The invention further discloses a kit for identifying the SLE and the nephritis implication, and the kit for identifying the SLE and the nephritis implication comprises one or more of the Pro-Asn-Gln-Met-Ser, the C24: 1 spingolipid and the protoporphyrin IX. The invention provides three serum exosome metabolic markers, namely Pro-Asn-Gln-Met-Ser, C24: 1 spingolipid and protoporphyrin IX, which are high in sensitivity and strong in specificity, and the serum exosome metabolic markers can be used for identifying the systemic lupus erythematosus and distinguishing whether the systemic lupus erythematosus has lupus nephritis or not. According to the invention, the identification accuracy can be further improved, timely early warning can be realized clinically, and new standards for clinical identification of systemic lupus erythematosus can be determined.

Description

Technical Field

[0001] The present invention belongs to the technical field of the detection and identification of systemic lupus erythematosus, and particularly relates to a group of serum exosome metabolic markers for identifying SLE and identifying nephritis involvement and their applications. Background Art

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by a breakdown of self-tolerance, leading to the production of nuclear autoantigens and immune complexes, which drive inflammation in multiple organ systems. Lupus nephritis (LN) is an important cause of morbidity and mortality in SLE, and many patients ultimately progress to chronic kidney disease or end-stage kidney disease despite effective anti-inflammatory and immunosuppressive treatment. Therefore, accurate and timely identification of SLE and the presence or absence of kidney involvement is crucial for improving patient prognosis. However, due to the lack of highly sensitive and specific biomarkers and the heterogeneity of the disease itself, this remains a challenge in clinical practice. Traditional serological biomarkers, such as anti-dsDNA and anti-Smith (anti-Sm) antibodies, show high specificity for SLE, but their sensitivity is significantly limited. Similarly, although renal biopsy is considered the gold standard for the identification of LN, its invasiveness, bleeding risk, and limitations in patient dynamic monitoring bring many inconveniences. Therefore, there is an urgent need to develop novel identification biomarkers with higher accuracy to improve the early detection and disease monitoring of SLE and LN.

[0003] Exosomes are key mediators of intercellular communication, carrying a large number of bioactive molecules, including proteins, lipids, metabolites, and nucleic acids. Exosomes are considered to be highly stable in a variety of biological fluids, including blood, urine, and saliva, making them excellent candidates for non-invasive identification methods. A large amount of evidence indicates that exosomes play a key role in the course of multiple autoimmune diseases (AID), mainly by affecting intercellular communication and the functions of immune cells to participate in the pathogenesis of AID. The concentration of exosomes themselves and the expression of nucleic acids, proteins, and metabolites encapsulated therein can reflect the degree of disease progression and are good biomarkers for AID. However, current articles on exosomal markers mainly focus on the RNA and protein levels, and there is currently no report on the study of SLE exosome metabolic markers. Summary of the Invention

[0004] Based on the above invention purpose, the present invention discloses a group of serum exosome metabolic markers for identifying SLE and identifying nephritis involvement and their applications.

[0005] On the one hand, the present invention provides a set of serum exosome metabolic markers for identifying SLE and nephritis involvement, and the serum exosome metabolic markers include one or more of Pro-Asn-Gln-Met-Ser, C24:1 sphingolipid, and protoporphyrin IX.

[0006] Preferably, the method for selecting the serum exosome metabolic markers includes the following steps: S1. Collect serum samples: Collect serum exosome samples from patients with systemic lupus erythematosus and healthy subjects undergoing physical examinations, respectively. S2. Perform non-target metabolomics sequencing on the serum exosome samples collected in S1, and screen out differential exosome metabolites. S3. Use the receiver operating characteristic curve ROC to analyze the diagnostic value of the differential metabolites in S2 for systemic lupus erythematosus and the discrimination value for distinguishing the presence or absence of nephritis.

[0007] On the other hand, the present invention provides a kit for identifying systemic lupus erythematosus, and the kit includes the serum exosome metabolic markers described in claim 1 or their detection reagents.

[0008] Compared with the prior art, the present invention has the following beneficial effects: 1. Provide biomarkers with high sensitivity and specificity: The present invention provides three serum exosome metabolic markers, namely Pro-Asn-Gln-Met-Ser, C24:1 sphingolipid, and protoporphyrin IX, which have high sensitivity and strong specificity, and can identify systemic lupus erythematosus and distinguish the presence or absence of lupus nephritis.

[0009] 2. Improve the discrimination accuracy and early warning: The present invention can further improve the discrimination accuracy, contribute to realizing timely early warning clinically, and help establish a new standard for clinical discrimination of systemic lupus erythematosus.

[0010] 3. Provide ideas and methods for screening serum exosome metabolic markers: The present invention provides an idea and method for screening serum exosome metabolic markers for LN identification, providing a reference for subsequent research and clinical applications.

[0011] 4. Meet some clinical discrimination needs: The differential exosome metabolites of the present invention can meet the current clinical discrimination effect to a certain extent, can be used as a supplement to existing clinical discrimination indicators, and have certain clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0013] Figure 1 It is the design diagram of the solution of the present invention.

[0014] Figure 2 It is the identification result diagram of serum exosomes in the HC and SLE groups in Example 1 of the present invention: Among them, A is the morphological structure diagram of exosomes under a transmission electron microscope; B is the particle size analysis diagram of exosomes; C is the Western Blot identification result of exosomes.

[0015] Figure 3 It is the K-means clustering analysis result of serum exosome-derived metabolites in three groups of healthy physical examination subjects (HC), patients with systemic lupus erythematosus without nephritis (SLE-LN), and patients with systemic lupus erythematosus complicated with nephritis (SLE+LN) in Example 1 of the present invention.

[0016] Figure 4 It is the characterization and metabolomics sequencing analysis of exosomes in the HC and SLE groups in Example 2 of the present invention: Among them, A is the PCA analysis; B is the volcano plot analysis; C is the KEGG pathway enrichment analysis of differential metabolites showing a linear increase and decrease in Cluster 1 and Cluster 3.

[0017] Figure 5 It is the differential analysis and discriminant performance analysis of exosome metabolites among three groups of healthy physical examination subjects (HC), patients with systemic lupus erythematosus without nephritis (SLE-LN), and patients with systemic lupus erythematosus complicated with nephritis (SLE+LN) in Example 2 of the present invention: Among them, A is the differential analysis of the expression level of protoporphyrin IX derived from EV in the HC, SLE, and LN groups; B is the differential analysis of the expression level of Pro-Asn-Gln-Met-Ser derived from EV in the HC, SLE, and LN groups; C is the differential analysis of the expression level of C24:1 sphingomyelin derived from EV in the HC, SLE, and LN groups. D is the discriminant efficacy analysis of differential exosome metabolites between the SLE and HC groups; E is the discriminant efficacy analysis of differential exosome metabolites between the SLE with and without nephritis groups.

[0018] Figure 6 It is the correlation analysis of key clinical indicators of differential exosome metabolites between the SLE with and without nephritis groups in Example 2 of the present invention: Among them, A is the correlation heat map analysis between differential exosome metabolites and clinical parameters; B is the analysis of the expression level of differential exosome metabolites between severe and non-severe SLE; C is the top 10 features selected by random forest analysis, sorted by overall importance; D is the comparison of the discriminant ability of the combined discrimination of three key metabolites and clinical parameters in distinguishing SLE with and without nephritis. Detailed implementation mode

[0019] To understand the features and technical content of the present invention in more detail, the implementation of the present invention will be described in detail below with reference to the accompanying drawings. The attached drawings are for reference and explanation only and are not used to limit the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above content of the present invention. In the following embodiments, unless otherwise specified, the reagents used can be obtained from commercial channels.

[0020] Example 1: Metabolomics sequencing screening of serum exosomes.

[0021] 1.1 Collection of serum samples: Collect 30 patients with systemic lupus erythematosus without nephritis (SLE-LN), 28 patients with systemic lupus erythematosus combined with nephritis (SLE+LN), and 33 healthy controls (HC) diagnosed by the Department of Rheumatology and Immunology of Nanjing Drum Tower Hospital Affiliated to Nanjing University School of Medicine, and retain their serum specimens during routine tests.

[0022] Inclusion criteria: The inclusion criteria for the systemic lupus erythematosus group (SLE) need to meet at least 4 of the 11 SLE classification criteria established by the American College of Rheumatology (ACR) in 1997; the inclusion of the lupus nephritis group (LN) requires a clear kidney biopsy pathological result as the gold standard.

[0023] The age range is 18-60 years old; Sign the informed consent form and be willing to provide serum samples.

[0024] Exclusion criteria: Any of the following criteria will be excluded: 1) Suffering from other autoimmune diseases except SLE: including patients with dermatomyositis / polymyositis, mixed connective tissue disease, systemic sclerosis, rheumatoid arthritis, etc.; 2) Complicated with various infections.

[0025] The disease activity index score of SLE patients is based on the systemic lupus erythematosus disease activity index (systemiclupus erythematosus disease activity index-2000, SLEDAI-2K) scale, and the SLEDAI-2K score is calculated. This invention has been reviewed and approved by the Ethics Committee of Nanjing Drum Tower Hospital Affiliated to Nanjing University School of Medicine (Project No.: 2023-568-01).

[0026] 1.2 Extraction and characterization of serum exosomes: 1.2.1 Extraction of exosomes In the present invention, all samples were processed by ultracentrifugation for exosome isolation. Briefly, 2 mL of frozen serum was thawed at 4 ºC and then transferred to a new centrifuge tube. It was centrifuged at 12,000 × g for 20 minutes at 4 ºC to remove insoluble impurities. Then the supernatant was diluted with PBS at a ratio of 1:4, filtered through a 0.22 μm filter, and centrifuged again at 12,000 × g for 1 hour at 4 ºC to remove larger vesicles and residual contaminants. Next, the sample was ultracentrifuged at 120,000 × g for 90 minutes. After discarding the supernatant, the pellet was resuspended in pre-cooled particle-free PBS and centrifuged at 100,000 × g for 90 minutes at 4 ºC for a second round of ultracentrifugation. The final supernatant was removed, and the pellet was resuspended in 100 μL of pre-cooled particle-free PBS. Then the purified exosomes were stored at 4 ºC for future use in subsequent experiments within no more than three days.

[0027] 1.2.2 Characterization of exosomes In the present invention, the main population of cup-shaped vesicles was determined by transmission and scanning electron microscopy. As shown in Figure 2A, the exosomes were surrounded by a lipid bilayer and had a particle size of approximately 150 nm, which was Figure 2 consistent with the NTA analysis results of B. Western blot analysis in Figure 2C identified three positive exosome markers, CD9, CD63, and TSG101, as well as the negative marker Calnexin. The above results indicated that the exosomes were successfully extracted.

[0028] 1.3 Serum exosome metabolome sequencing: 1.3.1 Chromatographic conditions: 10 μL of the sample was separated by an HSS T3 chromatographic column (100 mm × 2.1 mm i.d., 1.8 μm) and then entered mass spectrometry detection. Mobile phase A was a solution of water / acetonitrile (95 / 5, v / v) containing 0.1% formic acid, and mobile phase B was a solution of acetonitrile / isopropanol / water (47.5 / 47.5 / 5, v / v / v) containing 0.1% formic acid. Positive ion mode separation gradient: 0 - 3 min, mobile phase B increased from 0% to 20%; 3 - 4.5 min, mobile phase B increased from 20% to 35%; 4.5 - 5 min, mobile phase B increased from 35% to 100%; 5 - 6.3 min, mobile phase B was maintained at 100%; 6.3 - 6.4 min, mobile phase B decreased from 100% to 0%; 6.4 - 8 min, mobile phase B was maintained at 0%. Negative ion mode separation gradient: 0 - 1.5 min, mobile phase B increased from 0 to 5%; 1.5 - 2 min, mobile phase B increased from 5% to 10%; 2 - 4.5 min, mobile phase B increased from 10% to 30%; 4.5 - 5 min, mobile phase B increased from 30% to 100%; 5 - 6.3 min, mobile phase B was linearly maintained at 100%; 6.3 - 6.4 min, mobile phase B decreased from 100% to 0%; 6.4 - 8 min, mobile phase B was linearly maintained at 0%. The flow rate was 0.40 mL / min and the column temperature was 40 °C.

[0029] 1.3.2 Mass spectrometry conditions: The mass spectrometry signals of the sample were collected in positive and negative ion scanning modes, and the mass scanning range was 70 - 1050 m / z. The sheath gas flow rate was 60 psi, the auxiliary gas flow rate was 20 psi, the auxiliary gas heating temperature was 350 °C, the capillary temperature was 320 °C, the positive mode ion spray voltage was set at 3400 V, the negative mode ion spray voltage was set at -3000 V, and the normalized collision energy was 20 - 40 - 60 eV cyclic collision energy. The resolution of the first - stage mass spectrometry was 60000, the resolution of the second - stage mass spectrometry was 15000, and data was collected in DDA mode.

[0030] 1.3.3 Substance identification and analysis After the instrument run was completed, the LC - MS raw data was imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, a data matrix of retention time, mass - to - charge ratio, and peak intensity was obtained. At the same time, metabolite information was obtained by comparing the MS and MSMS mass spectrometry information with the metabolic public databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ).

[0031] Example 2: Clinical Application of Serum Differential Exosomal Metabolic Markers in Lupus Nephritis LN 2.1 Identification and Analysis of Serum Exosomal Metabolomics Sequencing Results To identify exosome-derived metabolic biomarkers associated with disease progression, especially the development of nephritis, the present invention conducted a comprehensive trend analysis of differential exosomal metabolites, revealing ten different trend changes, as shown in Figure 3. To identify biomarkers associated with disease severity, the present invention selected differential metabolites from Group 1 (sub class 1) and Group 3 (sub class 3), which showed decreasing and increasing trends, respectively, with the progression of disease severity. To identify novel metabolic biomarkers in exosomes derived from the sera of SLE patients, the present invention performed LC-MS / MS on exosomes from the SLE and HC groups. Endogenous metabolites were identified using the HMDB and Pubchem databases. Figure 4 The PCA of A effectively separated the SLE and HC samples, highlighting the metabolic differences between the two groups; the volcano plot in Figure 4B showed 586 differentially expressed serum exosome-derived metabolites, of which 225 were significantly upregulated, 88 were downregulated, and 273 showed no significant change; the KEGG pathway enrichment analysis of differential metabolites in Figure 4C highlighted a series of disturbed metabolic pathways, among which glycerolipid metabolism became the most significantly disrupted pathway in SLE. The above results indicate that there is a unique exosomal metabolic expression profile in the sera of SLE patients.

[0032] 2.2 Discriminatory Value of Exosome-Derived Metabolites in Lupus Nephritis LN and Systemic Lupus Erythematosus Patients SLE To evaluate the discriminatory value of these three differential exosome-derived metabolites, the present invention performed differential expression analysis on three groups. The results are shown in Figures 5A-5C. Protoporphyrin IX and Pro-Asn-Gln-Met-Ser showed an increasing trend in the HC, SLE, and LN groups, while C24:1 sphingolipid showed a decreasing trend. Figure 5 D-5E was the ROC curve analysis. The AUCs of the three exosomal metabolites in distinguishing SLE and HC were 0.998, 0.992, and 0.969, respectively, and the combined AUC was 1.0; the AUC values in distinguishing LN and SLE were 0.920, 0.893, and 0.865, respectively, and the combined AUC was 0.931. The above results indicate that these three differential exosome-derived metabolites have excellent discriminatory value.

[0033] 2.3 Correlation and discriminative efficacy of differential exosome-derived metabolites with clinical parameters Proteinuria, ACR, 24-hour urinary protein, hematuria, urinary casts, eGFR, and serum albumin are key clinical biomarkers for evaluating renal involvement in SLE patients. To explore the correlation between these differential exosome metabolites and clinical parameters, the present invention performed Spearman correlation analysis. The results in Figure 6A showed that there was a strong correlation between three metabolites and clinical indicators such as SLEDAI, serum albumin, ACR, 24-hour urinary protein, proteinuria, hematuria, urinary casts, and eGFR.

[0034] To further investigate whether these metabolites have the potential to be used as early discriminative indicators for severe lupus, the present invention used the SLEDAI score as a threshold to perform differential analysis on the two groups. The results in Figure 6B showed that the expression levels of serum exosome protoporphyrin IX and Pro-Asn-Gln-Met-Ser in patients with severe lupus were significantly increased, while C24:1 sphingolipid was significantly decreased. Figure 6C is the association heat map analysis between differential exosome metabolites and clinical parameters. The importance ranking of the three differential exosome metabolites and clinical parameters is as follows: ACR, urinary protein, Pro-Asn-Gln-Met-Ser, serum albumin, protoporphyrin IX, C24:1 sphingolipid, eGFR, hematuria, cylindruria, and pyuria.

[0035] Currently, the commonly used clinical indicators for differentiating LN are 24-hour urinary protein and the ratio of urinary microalbumin to creatinine, that is, ACR. However, the 24-hour urinary protein quantification requires patients to accurately collect all urine within 24 hours, which is a cumbersome process and is prone to inaccurate results due to improper collection; ACR evaluates renal function by measuring the ratio of albumin to creatinine in urine. This test is more convenient than 24-hour urinary protein quantification, but the results of ACR may be affected by the degree of urine concentration. The present invention compared three exosome-derived metabolites with existing clinical parameters. The results in Figure 6D showed that ACR and urinary protein were still the best discriminative indicators, with AUC values of 0.99 and 0.957 respectively, while the combined exosome metabolites followed closely, with an AUC of 0.931. The above results indicate that differential exosome metabolites can, to a certain extent, meet the current clinical discrimination effect.

[0036] The present invention provides a screening idea and method for serum exosome metabolic markers for LN identification. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A set of serum exosome metabolite markers for identifying SLE and nephritis involvement, characterized in that: The serum exosome metabolic markers include one or more of Pro-Asn-Gln-Met-Ser, C24:1 sphingolipid, and protoporphyrin IX.

2. The serum exosome metabolic marker according to claim 1, characterized in that The method for selecting serum exosome metabolic markers comprises the following steps: S1. Collect serum samples: collect serum exosome samples from patients with systemic lupus erythematosus and healthy subjects; S2, perform non-targeted metabolomics sequencing on the serum exosome samples collected in S1 to screen for differential exosome metabolites; S3. Receiver operating characteristic (ROC) curve was used to analyze the differential metabolites in S2 for the diagnosis of systemic lupus erythematosus and the differentiation of the presence or absence of nephritis.

3. A kit for identifying systemic lupus erythematosus, characterized in that: The kit comprises the serum exosome metabolic marker or a detection reagent thereof according to claim 1.

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