A biomarker for diagnosing rheumatoid arthritis and its application

Abstract

This invention relates to a biomarker for diagnosing rheumatoid arthritis (RA) and its application, belonging to the field of biomedical technology. This invention discovered that the concentrations of lipopolysaccharide (LPS) and diamine oxidase (DAO) in the plasma of RA patients are significantly higher than those in healthy controls. Furthermore, this invention also found that three proteins derived from plasma exosomes—phospholipid invertase 1 (PLSCR1), keratin 1 (KRT1), and complement C1QC chain (C1QC)—have good diagnostic effects on RA. Combining LPS and DAO with these three proteins can significantly improve the diagnostic accuracy of RA. Therefore, applying the biomarker of this invention to the preparation of RA diagnostic products can provide a core basis for the accurate diagnosis of RA and the generation of risk assessment systems, and has translational application value in the early diagnosis of RA and the screening and precise prevention and treatment of high-risk groups.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomarker for diagnosing rheumatoid arthritis and its application. Background Technology

[0002] The pathogenesis of rheumatoid arthritis (RA) is closely related to immune cell dysfunction. T cell abnormalities are a core element of RA immune dysregulation. Studies have found a significant increase in citrullinated proteoglycan (citPG)-specific T cells in the synovium of RA patients, and their reactivity is correlated with disease activity. B cells participate in RA pathogenesis through antigen presentation, cytokine secretion, and autoantibody production. B cells present citrullinated antigens to follicular helper T cells (Tfh) or peripheral helper T cells (Tph), promoting the secretion of interleukin-21 (IL-21) and CXC motif chemokine 13 (CXCL13), driving B cells to differentiate into plasma cells and produce anti-cyclic citrullinated (CCP) antibodies. Macrophages, as core effector cells of innate immunity, play a dual role in rheumatoid arthritis (RA): microorganisms or their metabolites (such as lipopolysaccharide) activate macrophages through pattern recognition receptors such as Toll-like receptor 4 (TLR4), triggering the cascade release of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), recruiting and activating T / B cells, initiating inflammation, and forming a positive inflammatory feedback loop; macrophages infiltrating the synovium of RA highly express molecules such as interleukin-17 (IL-17) and matrix metalloproteinases (MMPs), directly destroying cartilage and bone matrix, while promoting osteoclast maturation and bone tissue destruction by secreting nuclear factor κB receptor activator ligand (RANKL).

[0003] Currently, the clinical diagnosis of rheumatoid arthritis (RA) mainly relies on clinical symptoms, imaging examinations, and serological biomarkers, such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). However, the sensitivity and specificity of RF and anti-CCP are limited, with a negative rate of 20%-30% in RA patients, leading to delayed or missed diagnoses and missed opportunities for optimal treatment. The average delay from symptom onset to diagnosis in RA patients is approximately 6-12 months, and the inadequacy of diagnostic methods significantly impacts treatment outcomes and disease burden. Therefore, the search for new, highly sensitive, and highly specific RA biomarkers is an urgent need in current clinical practice. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem of poor sensitivity and specificity of existing rheumatoid arthritis diagnostic methods.

[0005] To address the aforementioned technical problems, this invention provides a biomarker for diagnosing rheumatoid arthritis and its application. This invention found that the concentrations of lipopolysaccharide (LPS) and diamine oxidase (DIO) in the plasma of rheumatoid arthritis (RA) patients are significantly higher than those in healthy controls. Furthermore, this invention also discovered that three proteins derived from plasma exosomes—phospholipid invertase 1 (PLSCR1), keratin 1 (KRT1), and complement C1QC chain (C1QC)—have good diagnostic effects on rheumatoid arthritis. Combining LPS and DIO with these three proteins for diagnosis can significantly improve the diagnostic efficacy for rheumatoid arthritis.

[0006] The first objective of this invention is to provide an application of a reagent for detecting the content of a biomarker in the preparation of a diagnostic product for rheumatoid arthritis, wherein the biomarker is selected from one or more of keratin 1, complement C1Q C chain and phospholipid invertase 1.

[0007] Furthermore, the biomarkers also include lipopolysaccharide and diamine oxidase, which are derived from plasma.

[0008] Furthermore, the biomarkers are selected from any one of the following groups:

[0009] (1) Keratin 1;

[0010] (2) Complement C1Q C chain;

[0011] (3) Phospholipid invertase 1;

[0012] (4) Lipopolysaccharide, diamine oxidase and complement C1Q C chain;

[0013] (5) Lipopolysaccharide, diamine oxidase and keratin 1;

[0014] (6) Lipopolysaccharide, diamine oxidase and phospholipid invertase 1;

[0015] (7) Lipopolysaccharide, diamine oxidase, keratin 1 and complement C1Q C chain;

[0016] (8) Lipopolysaccharide, diamine oxidase, keratin 1 and phospholipid invertase 1;

[0017] (9) Lipopolysaccharide, diamine oxidase, complement C1Q C chain and phospholipid invertase 1;

[0018] (10) Lipopolysaccharide, diamine oxidase, keratin 1, complement C1Q C chain and phospholipid invertase 1.

[0019] Furthermore, the UniProt number of the diamine oxidase is P19801, the UniProt number of keratin 1 is P04264, the UniProt number of the complement C1Q C chain is P02747, and the UniProt number of the phospholipid invertase 1 is O15162.

[0020] Furthermore, the samples for the rheumatoid arthritis diagnostic product are derived from plasma and / or plasma exosomes.

[0021] Furthermore, lipopolysaccharide and diamine oxidase are derived from plasma, while keratin 1, complement C1Q C chain, and phospholipid invertase 1 are derived from plasma exosomes.

[0022] A second objective of this invention is to provide a diagnostic product for rheumatoid arthritis, the diagnostic product comprising a reagent for detecting the content of a biomarker selected from one or more of lipopolysaccharide, diamine oxidase, keratin 1, complement C1QC chain, and phospholipid invertase 1.

[0023] Furthermore, the diagnostic product also includes reagents for separating plasma from blood.

[0024] Furthermore, the diagnostic product also includes reagents for extracting exosomes from plasma.

[0025] Furthermore, the diagnostic product also includes reagents for detecting the concentration or content of the biomarker in the sample to be tested by enzyme-linked immunosorbent assay (ELISA), chromatography, spectroscopy, mass spectrometry, or a combination thereof.

[0026] A third objective of this invention is to provide a reagent for detecting the concentration or content of a biomarker in a sample to be tested, wherein the biomarker is selected from one or more of lipopolysaccharide, diamine oxidase, keratin 1, complement C1Q C chain, and phospholipid invertase 1.

[0027] The fourth objective of this invention is to provide an application of a biomarker in the preparation of a diagnostic product for rheumatoid arthritis, wherein the biomarker is selected from one or more of lipopolysaccharide, diamine oxidase, keratin 1, complement C1QC chain, and phospholipid invertase 1.

[0028] Furthermore, the rheumatoid arthritis diagnostic product includes antibodies, probes, or aptamers that can specifically recognize and bind to the biomarkers.

[0029] The fifth objective of this invention is to provide an application of a biomarker in constructing a predictive model for rheumatoid arthritis, wherein the biomarker is selected from one or more of lipopolysaccharide, diamine oxidase, keratin 1, complement C1QC chain, and phospholipid invertase 1.

[0030] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0031] This invention provides a set of characteristic biomarkers for diagnosing rheumatoid arthritis (RA): keratin 1, complement C1Q C chain, and phospholipid invertase 1. These biomarkers exhibit excellent diagnostic efficacy for RA. When lipopolysaccharide, diamine oxidase, keratin 1, complement C1Q C chain, and phospholipid invertase 1 are used in combination to construct an RA diagnostic model, the area under the curve (AUC) of the RA diagnostic model reaches as high as 0.906, demonstrating excellent diagnostic sensitivity and specificity. Therefore, applying the biomarkers of this invention to the preparation of RA diagnostic products can provide a core basis for the accurate diagnosis of RA and the generation of risk assessment systems, and has translational application value in the early diagnosis of RA and the screening and precise prevention and treatment of high-risk populations. Attached Figure Description

[0032] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0033] Figure 1 This is a bar chart showing the expression levels of plasma lipopolysaccharide (LPS) and diamine oxidase (DAO) in the case group and control group in Example 1. ** indicates P < 0.01, and *** indicates P < 0.001.

[0034] Figure 2 Volcano plot showing the differential expression of plasma exosomal proteins between the case group and the control group in Example 1;

[0035] Figure 3 The image shows the screening results of plasma exosomal differentially expressed proteins that are significantly associated with plasma LPS and DAO. In the image, A is the intersection plot of plasma exosomal differentially expressed proteins that are significantly associated with LPS and DAO, B is the plasma exosomal differentially expressed proteins based on A that are involved in the immune response, and C is the correlation heatmap of plasma exosomal differentially expressed proteins and membrane proteins based on B. In the image, * indicates P < 0.05.

[0036] Figure 4 The graph shows the differences in expression of the three key plasma exosomal proteins identified in Example 1 between the case group and the control group, where * indicates P < 0.05;

[0037] Figure 5 ROC curves were plotted using the case and control groups from Example 1;

[0038] Figure 6 The bar chart shows the plasma LPS and DAO expression levels in the RA validation group and the control validation group in Example 2.

[0039] Figure 7This is a graph showing the expression differences of the three key plasma exosomal proteins identified in Example 2 between the RA validation group and the control validation group.

[0040] Figure 8 Receiver operating characteristic curves for three proteins derived from plasma exosomes, alone (AC) and in combination with LPS and DAO (DI);

[0041] Figure 9 The receiver operating characteristic curves were constructed using LPS, DAO, C1QC, KRT1, and PLSCR1 in combination. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0043] Example 1: Screening of differentially expressed protein markers from plasma exosomal sites in rheumatoid arthritis patients based on lipopolysaccharide (LPS) and diamine oxidase (DAO).

[0044] 1. Subject characteristics in the example

[0045] This embodiment includes two groups of subjects, both of whom are middle-aged and elderly Han Chinese women:

[0046] (1) Case group: Includes 18 patients with rheumatoid arthritis (recruited from the First Affiliated Hospital of Soochow University between December 2014 and July 2024). The diagnostic criteria adopted the RA classification criteria jointly launched by the American College of Rheumatology and the European Association for Rheumatology in August 2010. This classification criteria uses a scoring system, mainly including four items: number of affected joints, serological antibody detection (serum rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) levels), duration of synovitis, and levels of acute phase reactants (erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)). A cumulative maximum score of ≥6 points for all the above items is sufficient for a diagnosis of RA.

[0047] (2) Control group: including 83 healthy individuals from the prospective cohort for "Prevention and Control of Osteoporosis" being developed by the project team (Chinese Clinical Trial Registry - Registration No.: ChiCTR2000040832). 83 healthy individuals whose background information was matched with that of the case group served as controls.

[0048] In this case group, patients with a history of cancer, severe infection, recent use of antibiotics, use of probiotics, known history of inflammatory bowel disease, other autoimmune diseases (such as systemic lupus erythematosus, multiple sclerosis, etc.) and diabetes, as well as other diseases that affect immune or metabolic status, were excluded.

[0049] All participants signed informed consent forms before participating in the study. Venous blood was collected from participants in the morning on an empty stomach, and the plasma was separated and stored at -80°C for later use.

[0050] 2. The levels of plasma LPS and DAO were quantitatively detected using enzyme-linked immunosorbent assay (ELISA).

[0051] Commercially available ELISA kits were used to detect plasma LPS and DAO levels using a microplate reader. Results showed that LPS and DAO levels in the case group were significantly higher than those in the control group (see...). Figure 1 ).

[0052] 3. Isolation and identification of plasma exosomes

[0053] Exosomes in plasma samples were separated using a plasma exosome affinity extraction kit, and their size was calculated using nanoparticle tracking analysis. The morphology of the exosomes was then detected by transmission electron microscopy.

[0054] 4. Total protein extraction from exosomes

[0055] Total exosome protein was extracted by heating using SDT lysis buffer (sodium dodecyl sulfate (SDS), 1,4-dithiothreitol (DTT), and Tris-HCl buffer). The protein concentration of the total exosome protein samples was determined using a quinolinic acid (BCA) protein assay kit and an ELISA reader.

[0056] 5. Four-dimensional data-independent acquisition technology (4D-DIA) for proteomics analysis

[0057] (1) Sample preparation and chromatographic separation: The proteomic expression profile of total protein samples from exosomal samples was detected using a liquid chromatography-mass spectrometry (LC-MS / MS) system. The total protein samples were digested into peptide fragments by trypsin. The mobile phases A (0.1% formic acid aqueous solution) and B (0.1% formic acid acetonitrile solution) used for liquid chromatography analysis were used for gradient elution of peptide fragments. 400 ng of the trypsin-digested sample was dissolved in 10 μL of solution A, centrifuged at 14000×g for 20 min at 4℃, and the supernatant was injected. The chromatographic separation conditions were as follows: the proportion of mobile phase B was gradually increased from 5% to 95% within 0-60 min, and the elution peaks were efficiently separated by optimizing the gradient (24% solution B at 46 min, 36% solution B at 53 min).

[0058] (2) Mass spectrometry data acquisition: Data was acquired using two modes: data-dependent acquisition (DDA) and data-independent acquisition (DIA). DDA mode: Data was acquired using a timsTOF_HT mass spectrometer (captive spray ion source), with a mass-to-charge ratio (m / z) of 100-1700, a primary mass spectrometry resolution of 60,000 (1222 m / z), ion mobility of 0.6-1.6 cm² / (V), a cumulative time of 100 ms, a capillary voltage of 1.6 kV, a total cycle time of 1.1 seconds, and a total of 10 PASEF cycles. DIA mode: The scan range was 300-1500 m / z, with a mobility of 0.7-1.3 cm² / (V), a cumulative time of 50 ms, a voltage of 1.5 kV, and a cycle time of 1.23 seconds, ensuring high sensitivity detection of low-abundance proteins.

[0059] (3) Data Analysis and Database Retrieval: Raw data were processed using Spectronaut 16 software and retrieved from the Human Protein Database (Homo sapiens SP). Parameters were set as follows: trypsin digestion, fixed carboxymethylation modification, and variable modifications including methionine oxidation (+15.995 Da) and N-terminal acetylation, allowing a maximum of two missed cleavage sites. The DDA mass spectrometry library and DIA quantitative data were integrated using the Pulsar algorithm to screen for non-empty quantitative proteins and perform cross-sample statistics.

[0060] (4) Constructing differential protein expression profiles: Differential expression analysis of plasma exosome proteome data was performed using R software. The T-test was used to screen differentially expressed proteins between groups. Differentially expressed proteins needed to meet the following conditions: log2(FC) > 1 or log2(FC) < -1; FDR value < 0.05. Here, FC refers to the fold change, and FDR value refers to the false positive rate of peak detection. Results are shown below. Figure 2 As shown.

[0061] 6. Exosome membrane protein chip analysis

[0062] Using EVArray antibody chip technology from Beijing E-Microbes Technology Co., Ltd., we achieved high-sensitivity (fg / mL) and high-throughput detection and analysis of exosomal membrane proteins in a small amount of plasma (10μL).

[0063] Specific operations:

[0064] Remove plasma samples from the -80°C freezer, thaw on ice, and then perform initial centrifugation (4°C, 3000g for 20 minutes). Transfer the supernatant to a new centrifuge tube. Take 10 μL of each sample and dilute it to a 100 μL system with 1×PBS.

[0065] (1) Blocking reaction: Add 100 μL of blocking solution to each well of the antibody chip and incubate at room temperature for 30 min. Remove the blocking solution from each well.

[0066] (2) Hybridization reaction: Add 100 μL of diluted sample to each well and incubate at room temperature for 30 min.

[0067] (3) Antibody incubation: Remove the reaction solution from each well, add 200 μL of 1×PBS solution to each well to wash the chip (PBS: phosphate buffer), repeat 3 times. Then add 100 μL of biotin-conjugated detection antibody to each well (dilute the antibody with 1×PBS at a ratio of 1:1000), and incubate at room temperature for 30 min.

[0068] (4) Remove the reaction solution from each well, add 200 μL of 1×PBS to each well, wash the chip, and repeat 3 times. Then add 100 μL of anthocyanin dye 3 / anthocyanin dye 5 labeled streptavidin (Cy3 / Cy5-Streptavidin) to each well (diluted with 1×PBS at a ratio of 1:1000), and incubate at room temperature in the dark for 30 min.

[0069] (5) Detection: Remove the supernatant, add 200 μL of 1×PBS solution to each well to wash the chip, repeat 3 times. Then dry the chip, scan the signal using a laser scanner at 532 nm, and read the fluorescence value of the antibody chip using GenePix software.

[0070] (6) Quality control and processing of offline data: Calculate the mean and median of the three replicate points, and then subtract the reading of the PBS antibody point in the well to obtain the reading of the antibody point. When the antibody point value is less than 100, it is supplemented with 100.

[0071] Gene ontology (GO) analysis was performed on differentially expressed plasma exosomal proteins to screen pathways closely related to immune metabolism, such as: negative regulation of immune effector processes, lymphocyte-mediated immunity, humoral immune responses, and immunoglobulin-mediated immune responses (see...). Figure 3 ). Figure 3 The protein abbreviations and their Chinese translations are shown in Tables 1 and 2.

[0072] Table 1 Figure 3 The abbreviations and Chinese translations of proteins related to the letter B in the text.

[0073]

[0074] Table 2 Figure 3 The C in the text refers to protein abbreviations and their Chinese translations.

[0075]

[0076] Spearman correlation analysis was applied to examine the correlations between the above-mentioned plasma exosome proteins and plasma LPS, DAO levels, as well as their correlations with plasma exosome membrane protein levels. A significance test (p < 0.05) was used to determine whether the correlations were statistically significant (see [link to analysis]). Figure 3 ).

[0077] Three plasma exosomal proteins that may originate from immune cells (macrophages and T cells) were ultimately screened: phospholipid invertase 1 (PLSCR1), keratin 1 (KRT1), and complement C1QC chain (C1QC) (Table 3). Their levels were significantly positively correlated with plasma LPS and DAO levels, and showed significant differences in expression levels between the RA case group and the control group (see Table 3). Figure 4 ).

[0078] Using the data from Example 1 and constructing a model using the logistic regression method, the ROC curve was plotted and the AUC value was calculated. The results are as follows: Figure 5 As shown.

[0079] Table 3. Plasma exosomal proteins that are significantly positively correlated with microbial component entry into the blood (LPS) and their possible cell sources.

[0080]

[0081] Example 2: Validation and diagnostic performance evaluation of plasma exosome differential protein markers and plasma LPS and DAO in patients with rheumatoid arthritis.

[0082] 1. Validation of plasma exosome differential protein markers and plasma LPS and DAO in patients with rheumatoid arthritis

[0083] This embodiment included 8 independent RA patients (RA validation group) and 100 healthy controls (control validation group). Patients and controls were matched for age and sex. Plasma LPS and DAO levels were measured in both the RA validation group and the control validation group, and plasma exosomes were isolated and total exosome protein was extracted. Four-dimensional data-independent acquisition (4D-DIA) proteomics analysis was performed.

[0084] The specific experimental and analytical procedures were the same as in Example 1. The analytical results showed that there were significant differences in plasma LPS and DAO levels between the RA validation group and the control validation group (P < 0.05) (see Example 1). Figure 6 ). Figure 7 The results showed that the levels of plasma exosomal proteins KRT1, C1QC, and PLSCR1 in the RA validation group were also different from those in the control validation group.

[0085] 2. Evaluation of the efficacy of plasma exosomal proteins PLSCR1, KRT1, and C1QC combined with plasma LPS and DAO in the diagnosis of RA: Receiver operating characteristic (ROC) curve analysis

[0086] Using the data from Example 2 and employing a 10x cross-validation approach to select models, the ROC curve of the models was plotted, and the area under the curve (AUC) value was calculated. The results are as follows: Figure 8 As shown, the AUCs for the three proteins are: KRT1 (AUC=0.873), C1QC (AUC=0.743), and PLSCR1 (AUC=0.792). When proteins KRT1, C1QC, and PLSCR1 are combined with LPS and DAO, the areas under the curves also increase to above 0.8, specifically 0.833, 0.824, and 0.865, respectively, demonstrating high accuracy and discriminative ability. The RA diagnostic model constructed by combining C1QC, KRT1, PLSCR1, LPS, and DAO further improves the AUC value to 0.906 (see...). Figure 9 The diagnostic efficacy is excellent (model: logit(P(Y=1))=-1.7976+0.7458×X). LPS -0.4171×X DAO +0.6627×X C1QC +0.3913×X KRT1 +0.9250×X PLSCR1 , where X represents the standardized value of the corresponding plasma exosome protein measured in mass spectrometry analysis.

[0087] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of a reagent for detecting the content of a biomarker in the manufacture of a diagnostic product for rheumatoid arthritis, characterized in that, The biomarkers are selected from any one of the following groups: (1) Keratin 1; (2) Complement C1Q C chain; (3) Lipopolysaccharide, diamine oxidase and complement C1Q C chain; (4) Lipopolysaccharide, diamine oxidase and keratin 1; (5) Lipopolysaccharide, diamine oxidase and phospholipid invertase 1; (6) Lipopolysaccharide, diamine oxidase, keratin 1 and complement C1Q C chain; (7) Lipopolysaccharide, diamine oxidase, keratin 1 and phospholipid invertase 1; (8) Lipopolysaccharide, diamine oxidase, complement C1Q C chain and phospholipid invertase 1; (9) Lipopolysaccharide, diamine oxidase, keratin 1, complement C1Q C chain and phospholipid invertase 1; The lipopolysaccharide and diamine oxidase are derived from plasma, and the keratin 1, complement C1Q C chain and phospholipid invertase 1 are derived from plasma exosomes.

2. A diagnostic product for rheumatoid arthritis, characterized in that, The diagnostic product includes reagents for detecting the levels of the biomarkers in claim 1.

3. The diagnostic product according to claim 2, characterized in that, The diagnostic products also include reagents for separating plasma from blood.

4. The diagnostic product according to claim 2, characterized in that, The diagnostic products also include reagents for extracting exosomes from plasma.

5. The diagnostic product according to claim 2, characterized in that, The diagnostic products also include reagents for detecting the concentration or content of the biomarkers in the sample to be tested by enzyme-linked immunosorbent assay (ELISA), chromatography, spectroscopy, mass spectrometry, or a combination thereof.

6. A reagent for detecting the concentration or content of a biomarker in a test sample, characterized in that, The biomarker is the biomarker described in claim 1.

7. The application of biomarkers in the preparation of diagnostic products for rheumatoid arthritis, characterized in that, The biomarker is the biomarker described in claim 1.

8. The application of biomarkers in constructing predictive models for rheumatoid arthritis, characterized in that, The biomarker is the biomarker described in claim 1.

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