Biomarker for auxiliary diagnosis of systemic lupus erythematosus and application thereof

Abstract

The invention provides a biomarker for auxiliary diagnosis of systemic lupus erythematosus and application thereof, belongs to the field of biomedicine, and finds that the protein expression level of ALOX15 is significantly reduced in SLE neutrophils. The invention also finds that the protein expression level of the neutrophil ALOX15 is in negative correlation with the disease activity of an SLE patient, and ROC curve analysis shows that the protein expression level of the ALOX15 has better sensitivity and specificity for diagnosing the SLE. Therefore, the neutrophile granulocyte ALOX15 can be used as the biomarker of the SLE in clinical application, and has important significance on clinical auxiliary diagnosis of the SLE and disease evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a biomarker for auxiliary diagnosis of systemic lupus erythematosus and its application. Background Art

[0002] Systemic lupus erythematosus (SLE) is a typical autoimmune disease, which is clinically manifested as multi-system and multi-organ involvement to varying degrees, with high disability and mortality rates. In terms of diagnosis, SLE still lacks biomarkers with strong sensitivity and specificity, so the misdiagnosis rate and missed diagnosis rate are high. Therefore, in-depth understanding of the pathogenesis of SLE and then finding new biomarkers with better diagnostic effects are important research directions to improve the diagnosis level of SLE.

[0003] At present, the etiology of SLE is still unclear. Previous studies have mostly focused on the involvement of B lymphocytes, T lymphocytes, and dendritic cells in the pathogenesis of SLE. However, in recent years, the important role played by neutrophils in the occurrence of SLE has attracted more attention from researchers. Neutrophils are the most numerous white blood cells in peripheral blood and are also important innate immune cells. Under normal physiological conditions, neutrophils play a variety of biological functions such as chemotaxis, phagocytosis, and bactericidal, and participate in the first line of defense after pathogens invade the body. Studies have found that the number of neutrophils in the peripheral blood of SLE patients is significantly reduced, and the main reason for the reduction is that neutrophils undergo ferroptosis. Ferroptosis is an iron-dependent programmed cell death method, and its core feature is that metabolic abnormalities and redox imbalance cause excessive accumulation of lipid peroxides on the cell membrane, ultimately leading to cell ferroptosis. The expression of glutathione peroxidase 4 (GPX4) in neutrophils of SLE patients is significantly reduced, so that the lipid peroxides produced are not cleared by GPX4 in time and accumulate excessively in the cell, causing ferroptosis. Ferroptosis-induced neutrophils play an important role in the pathogenesis of SLE: in lupus mouse models, the use of ferroptosis inhibitors can alleviate the disease; and after conditional knockdown of GPX4 in neutrophils, knockout mice spontaneously developed a lupus-like phenotype. However, there are currently no biomarkers related to neutrophil lipid peroxide metabolism that can assist in the detection of systemic lupus erythematosus, and further research is still needed. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a biomarker for auxiliary diagnosis of systemic lupus erythematosus and its application.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of arachidonic acid-15-lipoxygenase ALOX15 gene as a biomarker in the preparation of a reagent for assisting the diagnosis of systemic lupus erythematosus.

[0007] The present invention provides application of a reagent for detecting the expression amount of arachidonic acid-15-lipoxygenase ALOX15 gene in preparing a kit for assisting the diagnosis of systemic lupus erythematosus.

[0008] Preferably, the method for detecting the expression level of arachidonic acid-15-lipoxygenase ALOX15 gene is RNA sequencing.

[0009] The invention provides application of a reagent for detecting the content of arachidonic acid-15-lipoxygenase ALOX15 in preparing a kit for assisting the diagnosis of systemic lupus erythematosus.

[0010] The present invention provides a kit for auxiliary diagnosis of systemic lupus erythematosus, the kit comprising a reagent for detecting the content of arachidonic acid-15-lipoxygenase ALOX15; the arachidonic acid-15-lipoxygenase ALOX15 content is detected by a Western blot method, and the reagent comprises a capture reagent for specifically identifying arachidonic acid-15-lipoxygenase ALOX15 and a capture reagent for specifically identifying GAPDH protein;

[0011] The capture reagent that specifically recognizes arachidonic acid-15-lipoxygenase ALOX15 includes ALOX15 primary antibody and anti-rabbit-derived antibody secondary antibody;

[0012] The capture reagent that specifically recognizes the GAPDH protein includes a primary GAPDH antibody and a secondary antibody against mouse-derived antibodies.

[0013] Preferably, the kit assists in the diagnosis of systemic lupus erythematosus by detecting the relative content of arachidonic acid-15-lipoxygenase ALOX15 in neutrophils;

[0014] The relative content is the ratio of the arachidonic acid-15-lipoxygenase ALOX15 protein content of neutrophils to the GAPDH protein content of neutrophils;

[0015] If the ratio is less than 0.5719, it is determined that there is a risk of developing systemic lupus erythematosus.

[0016] Preferably, the neutrophils are derived from the peripheral blood of the sample to be tested.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention found that the protein expression level of ALOX15 was significantly decreased in SLE neutrophils. The present invention also found that the protein expression level of ALOX15 in neutrophils was negatively correlated with the disease activity of SLE patients, and the protein expression level of ALOX15 in SLE patients with renal involvement was significantly lower than that in SLE patients without renal involvement, indicating that a low ALOX15 protein expression level may indicate lupus disease activity and a potential risk of combined lupus nephritis. ROC curve analysis showed that the protein expression level of ALOX15 had good sensitivity and specificity for diagnosing SLE. It shows that neutrophil ALOX15 can be used as a biomarker for SLE in clinical practice, which is of great significance for clinical auxiliary diagnosis of SLE and disease evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The expression of lipid peroxide metabolism enzymes in neutrophils in a large sample transcriptomic study;

[0020] Figure 2 The relative expression of ALOX15 on neutrophils in healthy people and patients with different autoimmune diseases is compared in Figure A, wherein A is the relative expression of ALOX15 on neutrophils in healthy people and patients with SLE, B is the relative expression of ALOX15 on neutrophils in healthy people and patients with adult Still's disease, C is the relative expression of ALOX15 on neutrophils in healthy people and patients with rheumatoid arthritis, and D is the relative expression of ALOX15 on neutrophils in healthy people and patients with fibromyalgia.

[0021] Figure 3 Comparison of ALOX15 protein expression in neutrophils between healthy people and SLE patients, where A is the comparison of ALOX15 protein expression in neutrophils between healthy people and SLE patients on NC membrane, and B is the statistical analysis of ALOX15 protein expression in neutrophils between healthy people and SLE patients after relative quantification;

[0022] Figure 4 This is a correlation analysis chart between the relative protein expression of ALOX15 in neutrophils and disease activity in SLE patients;

[0023] Figure 5 This is a comparison of the relative protein expression of ALOX15 in neutrophils of SLE patients with and without renal involvement;

[0024] Figure 6 Schematic diagram of the area under the ROC curve of relative protein expression of ALOX15 in neutrophils. DETAILED DESCRIPTION

[0025] The present invention provides the use of arachidonic acid-15-lipoxygenase ALOX15 gene as a biomarker in the preparation of a reagent for assisting the diagnosis of systemic lupus erythematosus. Systemic lupus erythematosus can be assisted in the detection of systemic lupus erythematosus by the relative expression level of the arachidonic acid-15-lipoxygenase ALOX15 gene.

[0026] The present invention provides application of a reagent for detecting the expression amount of arachidonic acid-15-lipoxygenase ALOX15 gene in preparing a kit for assisting the diagnosis of systemic lupus erythematosus.

[0027] In the present invention, the method for detecting the expression level of arachidonic acid-15-lipoxygenase ALOX15 gene is preferably RNA sequencing.

[0028] The invention provides application of a reagent for detecting the content of arachidonic acid-15-lipoxygenase ALOX15 in preparing a kit for assisting the diagnosis of systemic lupus erythematosus.

[0029] The present invention provides a kit for auxiliary diagnosis of systemic lupus erythematosus, wherein the kit preferably comprises a reagent for detecting the content of arachidonic acid-15-lipoxygenase ALOX15;

[0030] The detection of arachidonic acid-15-lipoxygenase ALOX15 content is preferably carried out by Western blot, and the reagents are preferably capture reagents that specifically recognize arachidonic acid-15-lipoxygenase ALOX15 and capture reagents that specifically recognize GAPDH protein;

[0031] The capture reagent that specifically recognizes arachidonic acid-15-lipoxygenase ALOX15 is preferably ALOX15 primary antibody: abcam, secondary antibody against rabbit-derived antibody; the capture reagent that specifically recognizes GAPDH protein is preferably GAPDH primary antibody: MBL, secondary antibody against mouse-derived antibody;

[0032] In the present invention, the kit also includes a film, a blocking agent, a color developer and a buffer; the film is preferably an NC film; the blocking agent is preferably a 5% solution prepared from skimmed milk powder; the color developer is preferably an ECL color developer; and the buffer is preferably a 5% BSA solution and / or TBST buffer.

[0033] In the present invention, the kit assists in the diagnosis of systemic lupus erythematosus by detecting the relative content of arachidonic acid-15-lipoxygenase ALOX15 in neutrophils;

[0034] The relative content is the ratio of the arachidonic acid-15-lipoxygenase ALOX15 protein content of neutrophils to the GAPDH protein content of neutrophils;

[0035] If the ratio is less than 0.5719, it is determined that there is a risk of developing systemic lupus erythematosus.

[0036] In the present invention, the neutrophils are preferably derived from the peripheral blood of the sample to be detected.

[0037] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Sources

[0039]

[0040]

[0041] Example 1: Comparison of ALOX15 expression levels in neutrophils of SLE patients and healthy subjects (HC)

[0042] According to the results of a large sample transcriptomic study of 136 SLE patients and 89 healthy controls in the reference Nakano M, Ota M, Takeshima Y, et al. Distinct transcriptome architectures underlying lupus establishment and exacerbation. Cell. 2022; 185(18): 3375-3389. e21. Figure 1 The expression of neutrophil lipid peroxide metabolic enzymes was analyzed, and ALOX15 was screened out as the only enzyme molecule with significant abnormal expression changes (|logFC|≧1.5, P<0.05).

[0043] Further RNA-seq was performed using peripheral blood neutrophil samples from SLE patients and healthy controls to verify the conclusion:

[0044] Inclusion criteria for healthy controls: healthy people aged 18-65 years, regardless of gender;

[0045] Exclusion criteria: Subjects with any of the following conditions cannot be included:

[0046] a) Healthy controls who had taken antibiotics, anticonvulsants or had gastrointestinal colds in the past month;

[0047] b) Those who have recently suffered from serious underlying diseases such as infectious diseases, malignant tumors, leukemia, acute heart failure, acute coronary syndrome, etc.;

[0048] c) have symptoms or signs of severe, progressive or uncontrolled renal, hepatic, hematological, gastrointestinal, endocrine, pulmonary, cardiac, neurological, psychiatric or brain disease;

[0049] d) Organ transplantation surgery;

[0050] e) Pregnant women.

[0051] Inclusion criteria for SLE patients: SLE patients who meet the 2010 classification diagnostic criteria of the American College of Rheumatology (ACR) and have not taken glucocorticoids or immunosuppressants within three months;

[0052] Exclusion criteria: patients with chronic or acute infection or serious underlying diseases.

[0053] Isolate fresh neutrophils from the peripheral blood of HC or SLE patients: Use 9ml EDTA anticoagulation tubes to collect peripheral blood from HC and SLE patients. Place the anticoagulation tube containing whole blood in a centrifuge, centrifuge at 24℃, 2000rpm for 10min, and aspirate the upper light yellow plasma into 4 ep tubes, 1ml per tube, which is plasma. Add PBS equal to the amount of aspirated plasma to the anticoagulation tube, and blow up the cells with a pipette. Take a 15ml centrifuge tube and add 5ml human lymphocyte separation solution. Tilt the centrifuge tube and use a pipette to slowly spread the blood in the anticoagulation tube on the lymphocyte separation solution. After spreading, quickly centrifuge at 24℃, 1800rpm, 9 ups and 1 downs, centrifuge for 20 minutes, and then carefully remove it. The thin white layer on the upper layer of red blood cells in the centrifuge tube is neutrophils. Use a new pipette to draw neutrophils into a new centrifuge tube, add 13ml of lysate, place on a horizontal shaker to mix, keep at room temperature for 10 minutes, observe the liquid in the centrifuge tube change from turbid to transparent, centrifuge at 4℃, 1800rpm for 8-10 minutes, and discard the supernatant. Add 5ml of lysate again, blow up the cells, centrifuge at 4℃, 1800rpm for 5 minutes, and discard the supernatant. The neutrophils at this time can be directly used to extract protein and RNA. Flow cytometry experiments using CD16 and CD11b antibodies confirmed that the purity of neutrophils (CD11b+CD16+) extracted by this method is above 98%.

[0054] Total RNA from these neutrophils was extracted using TRIzo1 and subjected to RNA-seq analysis. RNA sequencing was performed by the Novogene Experimental Department using the I11umina HiSeq 4000 platform. The raw reads in fastq format were first processed by an in-house perl script, and all downstream analyses were based on the cleaned data. The reference genome index was established by using bowtie2v2.2.8, and then the paired clean data were aligned with the reference genome (version number hg38) using HISAT2 (v2.0.4) to test the relative expression of ALOX15 in neutrophils. The mapped reads of each sample were assembled by StringTie (v1.3.1). Cuffdiff (v2.1.1) was used to calculate the FPKMs of coding genes in each sample. Figure 2 As shown in the figure, the relative expression of ALOX15 in neutrophils of SLE patients is significantly lower than that in neutrophils of healthy people. At the same time, the RNA-seq data of peripheral blood neutrophils of three other autoimmune diseases in the GEO (Gene Expression Omnibus) database were referenced (GSE150996, GSE93776, GSE229750). Figure 2 As shown, there was no significant difference in the relative expression of ALOX15 in neutrophils in adult-onset Still's disease (AOSD), rheumatoid arthritis (RA), and fibromyalgia (FM), suggesting that the decreased relative expression of ALOX15 in neutrophils in SLE patients is disease-specific.

[0055] Neutrophils were isolated from the patient's biological samples by the above density gradient centrifugation method. Centrifuge at 5,000rpm and 4℃ for 5min, discard the supernatant, add 110μl 1X RIPA cell lysis buffer to lyse the cells, centrifuge at 13,600rpm and 4℃ for 20min, aspirate 102.5μl supernatant and transfer to a new 1.5ml EP tube, take 2.5μl of it and dilute it 10 times with double distilled water and add it to the ELISA plate, add 200μl BCA reaction solution (A solution: B solution = 50:1), incubate at 37℃ for 30min, measure the OD value with an ELISA instrument, and calculate the protein concentration according to the standard curve. Add 25μl 5X Loading Buffer to the remaining 100μl protein sample and boil it at 100℃ for 10min. Take 10μg and load it on the SDS precast gel, add the electrophoresis solution and run at a constant voltage of 80V for 30min, and change to a constant voltage of 120V until bromophenol blue runs out of the SDS gel. The protein was transferred to the NC membrane using a transfer solution at 220 mA constant flow for 90 min. The NC membrane containing the protein was removed and stained with Ponceau red solution to determine the overall transfer effect. The membrane was then blocked in a 5% skim milk powder solution at room temperature for 1 h. The primary antibody of ALOX15, abcam antibody, and the primary antibody of the internal reference protein GAPDH, MBL antibody, were diluted 1:1000 with a 5% BSA solution. The diluted primary antibodies were added dropwise to the NC membrane containing the protein until the diluted primary antibodies evenly covered the entire NC membrane containing the protein, and incubated at 4°C overnight. Wash with 1X TBST solution for 3 times, 10 minutes each time, dilute the secondary antibodies against rabbit and mouse antibodies (Bio-Edge, BE0101, BE0102) with 5% BSA solution at 1:5000, add the diluted secondary antibodies to the NC membrane containing protein, until the diluted secondary antibodies evenly cover the entire NC membrane containing protein, incubate at room temperature for 1 hour, and then wash with 1X TBST solution for 3 times, 10 minutes each time, add ECL color development solution to the NC membrane containing protein for color development, use gel imaging to take pictures of protein bands, use ImageJ software to quantitatively analyze the grayscale values ​​of the bands, detect the expression levels of ALOX15 protein and internal reference protein GAPDH protein, and compare them with the relative expression level of ALOX15 in neutrophils of healthy people. The results are shown in Figure 3 As shown in the figure, the protein expression level of ALOX15 in neutrophils of SLE patients was significantly lower than the relative expression level of ALOX15 in neutrophils of healthy people, which was consistent with the trend of RNA-seq results.

[0056] Example 2: Neutrophil ALOX15 expression level is negatively correlated with SLE disease activity

[0057] The disease activity of SLE patients was evaluated according to the collected clinical information and related examination results of SLE patients, with reference to the SLE disease activity index-2000 (SLEDAI 2000). The relative expression of ALOX15 gene in neutrophils of SLE patients measured by western blot was analyzed with SLEDAI 2000 for linear correlation. Figure 4 As shown in Figure 2, the relative expression of ALOX15 was significantly correlated with the SLEDAI 2000 score, and the correlation was negative (P = 0.0150, R 2 =0.4015).

[0058] Example 3: Comparison of ALOX15 expression levels in peripheral blood of SLE patients with different clinical characteristics

[0059] SLE patients were divided into SLE patients with renal involvement and SLE patients without renal involvement according to whether they had proteinuria, hematuria, pyuria and / or tubular urine. The expression levels of ALOX15 in peripheral blood of SLE patients with and without renal involvement were compared (the relative protein expression of ALOX15 gene in neutrophils of SLE patients was compared by western blot). Figure 5 As shown in the figure, the relative expression of ALOX15 gene in neutrophils of SLE patients with renal involvement was significantly lower than that of SLE patients without renal involvement, indicating that the decreased expression level of ALOX15 protein may indicate the potential risk of SLE patients with lupus nephritis.

[0060] Example 4: Study on the sensitivity and specificity of relative expression of ALOX15 in neutrophils in diagnosing SLE

[0061] The 100 subjects were randomly divided into a training set and a validation set in a ratio of 7:3, including 70 cases in the training set (36 cases in the SLE group and 34 cases in the HC group) and 30 cases in the validation set (15 cases in the SLE group and 15 cases in the HC group). Figure 6 As shown, the ROC curve of neutrophil ALOX15 relative protein expression for diagnosing SLE was drawn, and the AUC of the training set was 0.7582 (P=0.0002), and the AUC of the validation set was 0.7200 (P=0.0401). When the cutoff value of the training set was 0.5719, the specificity was 79.41% and the sensitivity was 72.22%.

[0062] The above results indicate that neutrophil ALOX15 may have certain clinical application value in the clinical diagnosis and disease activity evaluation of SLE, especially when there is renal involvement.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of arachidonic acid-15-lipoxygenase ALOX15 gene as a biomarker in the preparation of reagents to assist in the diagnosis of systemic lupus erythematosus.

2. Use of a reagent for detecting the expression of arachidonic acid-15-lipoxygenase ALOX15 gene in the preparation of a kit for assisting the diagnosis of systemic lupus erythematosus.

3. The use according to claim 2, characterized in that: The method for detecting the expression level of arachidonic acid-15-lipoxygenase ALOX15 gene is RNA sequencing.

4. Use of a reagent for detecting the content of arachidonic acid-15-lipoxygenase ALOX15 in the preparation of a kit for assisting the diagnosis of systemic lupus erythematosus.

5. A kit for assisting diagnosis of systemic lupus erythematosus, characterized in that: The kit includes a reagent for detecting the content of arachidonic acid-15-lipoxygenase ALOX15; the content of arachidonic acid-15-lipoxygenase ALOX15 is detected by Western blot, and the reagent includes a capture reagent for specifically identifying arachidonic acid-15-lipoxygenase ALOX15 and a capture reagent for specifically identifying GAPDH protein; The capture reagent that specifically recognizes arachidonic acid-15-lipoxygenase ALOX15 includes ALOX15 primary antibody and anti-rabbit-derived antibody secondary antibody; The capture reagent that specifically recognizes the GAPDH protein includes a primary GAPDH antibody and a secondary antibody against mouse-derived antibodies.

6. The kit according to claim 5, characterized in that The kit assists in the diagnosis of systemic lupus erythematosus by detecting the relative content of arachidonic acid-15-lipoxygenase ALOX15 in neutrophils; The relative content is the ratio of the arachidonic acid-15-lipoxygenase ALOX15 protein content of neutrophils to the GAPDH protein content of neutrophils; If the ratio is less than 0.5719, it is determined that there is a risk of developing systemic lupus erythematosus.

7. The kit according to claim 6, characterized in that The neutrophils are derived from the peripheral blood of the sample to be detected.

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