Application of SBDP145 and calpain in preparation of systemic lupus erythematosus diagnostic product

Abstract

The invention discloses application of SBDP145 and calpain in preparation of a systemic lupus erythematosus diagnosis product, and belongs to the technical field of disease detection. Specifically, through comparison of transcriptome data of a healthy control and a systemic lupus erythematosus (SLE) patient from a database, it is found that calpain has a significant difference in the two groups of data; and further comparing the calpain and the cracking product SBDP145 of the substrate of the calpain in the serum of the healthy control and the SLE patient, and finding that the calpain and the SBDP145 are up-regulated in the patient group, so that the calpain and the SBDP145 are used as candidate diagnostic markers. Then, an actual serum sample is adopted to explore the expression difference of calpain in healthy control and patients, the disease correlation between SBDP145 and calpain and systemic lupus erythematosus and the like, and the feasibility of the marker in SLE diagnosis and detection is verified. The invention provides a new SLE detection strategy, which is expected to overcome the defects of the traditional auxiliary diagnosis mode, and provides a new choice for clinical SLE diagnosis, illness monitoring and prognosis evaluation.

Description

Technical Field

[0001] The invention relates to application of SBDP145 and calpain in preparing a systemic lupus erythematosus diagnosis product, belonging to the technical field of disease detection. Background Art

[0002] Systemic lupus erythematosus (SLE) is an autoimmune disease that affects multiple systems of the body. Its pathogenesis is complex. SLE is characterized by the presence of multiple pathogenic autoantibodies and a large amount of immune complex deposition. Its onset is mainly considered to be the result of abnormal immune regulation. According to incomplete estimates, the global prevalence of SLE is approximately 30 to 150 per 100,000 people, and it is increasing over time. If the diagnosis is not timely or the disease activity is not controlled in time, tissues and organs will suffer irreversible damage. The clinical manifestations of SLE are heterogeneous, and a specific biomarker may only reflect a specific aspect of SLE, but not the overall status of the disease. In addition, the sensitivity or specificity of traditional laboratory indicators is not high, and there are many factors affecting the detection, which still have limitations for clinical diagnosis, disease monitoring and prognosis evaluation. Therefore, it is particularly important to find serum markers that are more convenient and well correlated in the clinical diagnosis and treatment of SLE patients. Summary of the invention

[0003] To solve the above problems, the present invention provides an application of a peripheral blood marker with high sensitivity and specificity in clinical condition monitoring and prognosis evaluation of systemic lupus erythematosus, specifically the application of peripheral blood SBDP145 and calpain activity in clinical diagnosis, condition monitoring and prognosis evaluation of systemic lupus erythematosus.

[0004] The first object of the present invention is to provide the use of calpain and / or SBDP145 in preparing a diagnostic product for systemic lupus erythematosus.

[0005] Furthermore, the calpain includes at least one of calpain-1 and calpain-2.

[0006] Furthermore, the diagnostic product is used to detect the activity of calpain and / or the content of SBDP145 in a sample.

[0007] Furthermore, the test samples include but are not limited to serum samples.

[0008] The second object of the present invention is to provide a product for detecting systemic lupus erythematosus, wherein the product contains a first reagent and / or a second reagent, wherein the first reagent is used to detect the activity of calpain, and the second reagent is used to detect the content of SBDP145.

[0009] Furthermore, the product is used for the clinical diagnosis of systemic lupus erythematosus.

[0010] Furthermore, the product is used for detecting the condition of systemic lupus erythematosus.

[0011] Furthermore, the product is used for the prognosis assessment of systemic lupus erythematosus.

[0012] Furthermore, the product can be used to differentiate between high and low activity levels of systemic lupus erythematosus.

[0013] Furthermore, the first reagent is used to detect the activities of calpain-1 and calpain-2 separately or simultaneously.

[0014] Furthermore, the product also contains a third reagent, and the third reagent is used for detecting anti-double-stranded DNA antibodies.

[0015] Beneficial effects of the present invention:

[0016] At present, there is no clinically available biodiagnostic marker for systemic lupus erythematosus that has both sensitivity and specificity. The present invention has discovered a new marker, calpain, and its substrate cleavage product SBDP145 through high-throughput screening, and it has been verified that the sensitivity of the biomarker for systemic lupus erythematosus detection is 63.1% and 88.7%, respectively, and the specificity is 81.1% and 80.0%, respectively. When the two are used in combination, the sensitivity and specificity reach 86.3% and 88%, and are further improved when used in combination with other diagnostic methods. At the same time, the marker discovered by the present invention can also distinguish the active stage of the disease, which is impossible to achieve with existing diagnostic methods. Therefore, the present invention realizes detection and diagnosis through the SBDP145 content and calpain activity in serum, and the index is easy to detect and standardize, and has great application potential in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 There were significant differences in calpain activity and SBDP145 content in peripheral blood mononuclear cells between SLE patients and healthy controls.

[0018] Figure 2 Serum samples were validated for significant differences in calpain activity and SBDP145 content between SLE patients and healthy controls, as well as their association with disease activity.

[0019] Figure 3 To investigate the correlation between serum SBDP145 levels and calpain activity and clinical parameters in SLE patients.

[0020] Figure 4Serum SBDP145 content and calpain activity are independent risk factors for SLE.

[0021] Figure 5 To investigate the diagnostic value of serum SBDP145 content and calpain activity in patients with SLE.

[0022] Figure 6 To investigate the relationship between serum SBDP145 levels and calpain activity and organ involvement in SLE patients. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0024] The scheme involved in the present invention is as follows:

[0025] Biomarkers play a vital role in the diagnosis of SLE, classification of SLE complications, assessment of SLE disease activity, and reflection of the effect of SLE intervention treatment. The diagnosis and classification of SLE are based on the patient's clinical symptoms, signs, and laboratory biomarkers that reflect the immune reactivity and inflammation of various organs. It is necessary to develop consistent SLE classification criteria for research and clinical diagnosis. The most widely used SLE classification criteria were established by the American College of Rheumatology (ACR) and include laboratory biomarkers, including proteinuria, urine pattern, hemolytic anemia with reticulocytosis, white blood cells, lymphocytes, platelets, anti-Smith (Sm) antibodies, antinuclear antibodies (ANA), anti-DNA antibodies, total complement activity, complement (2, 3, 4), and lupus erythematosus (LE) cells. The sensitivity and specificity of common detection indicators are as follows:

[0026] (1) Anti-double-stranded DNA antibodies: Anti-double-stranded DNA (dsDNA) antibodies have high specificity for SLE and are closely related to the activity of SLE, especially the activity of lupus nephritis. About 40% to 75% of SLE patients are positive for anti-ds-DNA antibodies;

[0027] (2) Anti-Sm antibodies: Sm antigens appear in U-family small molecule nuclear ribonucleoproteins (UsnRNPs), such as U2, U4 / U6, and U5, with a specificity of 97% in SLE. However, the sensitivity is very low, only 20% to 30%. The titer of anti-Sm antibodies may be related to the activity of SLE, and an increase in the titer may indicate a relapse of SLE.

[0028] (3) Anti-ribonucleoprotein antibodies: also known as anti-RNP antibodies, are a type of autoantibody, including anti-U1RNP antibodies, anti-U2RNP antibodies, anti-U5RNP antibodies, anti-U7RNP antibodies, etc. Since they mainly precipitate the U1 part of the ribonucleoprotein antigen, the clinical detection of anti-U1RNP antibodies is mainly based on the detection of anti-U1RNP antibodies, which has a certain significance for the diagnosis of SLE, with a positive rate of 35% to 45%.

[0029] (4) Anti-nucleosome antibody: Its antigen is the nucleosome complex. Anti-nucleosome antibody AnuA contains anti-double-stranded DNA antibody and anti-histone antibody. It plays an important role in the induction and pathogenesis of SLE. It is a specific antibody for SLE, with a positive rate of 50% to 60% and a specificity of 95%. However, its increase is not related to the activity of SLE. The combined detection of AnuA, anti-dsDNA, and anti-Sm antibodies can effectively improve the experimental diagnosis rate of SLE.

[0030] (5) Anti-ribosomal P protein: Anti-ribosomal P protein mainly recognizes the three phosphoprotein subunits P0, P1 and P2 on the 60S ribosome. This antibody is of reference value for the diagnosis of SLE patients who are negative for anti-dsDNA antibodies or anti-Sm antibodies, and is related to the mental symptoms of SLE, but the positive rate in SLE is about 20% to 30%.

[0031] (6) Antibody against proliferating cell nuclear antigen (PCNA): Proliferating cell nuclear antigen is a protein involved in eukaryotic cell DNA replication. It is a cyclic trimer composed of three identical subunits. It was first discovered and named by Miyachi et al. in the serum of SLE patients in 1978. Later studies found that PCNA is closely related to cell DNA synthesis and is a highly specific antibody for SLE. It can be used as a marker antibody for SLE, but its positive rate is only 1% to 5%.

[0032] (7) Anti-SSA / Ro antibodies and anti-SSB / La antibodies: Anti-SSA / Ro antibodies are more common in Sjögren's syndrome (SS) and SLE, with a positive rate of about 40% in the serum of SLE patients. Anti-SSB antibodies only appear in a small number of SLE patients, with a positive rate of 10% to 15%. They are associated with cheek erythema, alopecia, and serositis, but their mechanism is unclear. Anti-SSA and anti-SSB are highly sensitive to SLE but not specific.

[0033] Therefore, in view of the fact that existing biomarkers cannot take into account both sensitivity and specificity, the present invention provides a peripheral blood marker with high sensitivity and specificity, which has application prospects in the clinical diagnosis, disease monitoring and prognosis evaluation of systemic lupus erythematosus. The discovery and verification of the biomarker of the present invention mainly includes the following steps:

[0034] (1) Discovery:

[0035] The transcriptome database was used to analyze the differences in the transcriptional levels of calpain in peripheral blood mononuclear cells between SLE patients and healthy subjects. Peripheral blood was collected from SLE patients and healthy subjects, and mononuclear cells in peripheral blood were separated using lymphocyte separation solution. The cells were lysed with TRIzol and total RNA was extracted and reverse transcribed. Fluorescence quantitative PCR was used to detect the mRNA levels of calpain-1 and calpain-2; Western blot was used to detect the protein level of calpain-1; the substrate method was used to detect the activity of calpain; and ELISA was used to detect the content of SBDP145 in peripheral blood mononuclear cells.

[0036] (2) Verification:

[0037] ① Collect serum from SLE patients and healthy subjects, detect the content of SBDP145 in serum by ELISA and the activity of calpain in serum by substrate method. Analyze the content of SBDP145 and calpain activity in the serum of SLE patients with SLEDAI-2K scores >6 and <6.

[0038] ②Analyze the correlation between serum SBDP145 and calpain activities and relevant clinical detection parameters of SLE patients.

[0039] ③ROC curves were used to evaluate the sensitivity and specificity of SBDP145 and calpain activity as diagnostic markers for SLE.

[0040] ④Analyze the correlation between serum SBDP145 and calpain activities and the progression of complications in SLE patients.

[0041] (3) Results:

[0042] ①The serum SBDP145 level (p<0.001) and calpain activity (p<0.001) of SLE patients were significantly higher than those of the healthy control group.

[0043] ②SBDP145 was positively correlated with anti-dsDNA antibody titer, ESR, high-sensitivity CRP, anti-nucleosome antibody, anti-dsDNA antibody, anti-histone antibody, anti-SSA antibody, and anti-SSB antibody in SLE patients, and negatively correlated with C3 and C4. Calpain activity was positively correlated with anti-double-stranded DNA antibody titer, ESR, anti-nucleosome antibody, anti-dsDNA antibody, anti-histone antibody, and anti-SSA antibody in SLE patients, and negatively correlated with C3 and C4.

[0044] ③The areas under the curve of SBDP145 and Calpain activity were 0.891 and 0.762, respectively. SBDP145, 95% confidence interval was 0.845-0.936, threshold was 2.377ng / ml, sensitivity was 88.7%, and specificity was 80.0%; Calpain activity, 95% confidence interval was 0.697-0.827, threshold was 13.75RFU, sensitivity was 63.1%, and specificity was 81.1%. The AUC of the combined detection of the two indicators was 0.919, and the sensitivity and specificity were 86.3% and 88%, respectively. The diagnostic efficiency of the combined anti-dsDNA titer was as high as 96.0%, which was higher than the diagnostic efficiency of the anti-dsDNA titer alone of 84.0%.

[0045] ④The results of correlation analysis showed that SBDP145 level and calpain activity were correlated with rash (r=0.213, P=0.018; r=0.333, P<0.001) and anemia (r=0.222, P=0.013; r=0.199, P=0.027). Elevated calpain activity was positively correlated with pulmonary interstitialization (r=0.343, P<0.001), arthritis (r=0.255, p=0.004) and renal damage (r=0.303, P<0.001).

[0046] The present invention involves two biomarkers: Calpain (including at least calpain-1 and calpain-2) and SBDP145. Among them:

[0047] Calpain-1, calpain 1, also known as μ-calpain, is a calcium-dependent cysteine ​​protease; Calpain-2, calpain 2, also known as m-Calpain, is also a calcium-dependent cysteine ​​protease. Calpain-1 and Calpain-2 are widely present in a variety of organisms. They play an important role in cells and participate in regulating a variety of biological processes, including cell signaling, cytoskeleton reorganization, cell apoptosis, and protein degradation. At present, some reports have mentioned that Calpain-1 and Calpain-2 play a role in a variety of physiological and pathological processes, but there have been no reports indicating their use in the diagnosis of systemic lupus erythematosus.

[0048] SBDP145, the full name of αII-spectrin breakdown product 145, is one of the fragments of 150kDa and 145kDa produced by activated calpain cleaving its substrate αII-spectrin. It is reported that in cases of brain injury, such as traumatic brain injury (TBI) or cerebral hemorrhage, calpain is activated, the substrate is cleaved to produce the fragment SBDP145, and it can be detected in serum. Therefore, the level of SBDP145 in serum can be used as an indicator of the severity and prognosis of brain injury, but there have been no reports indicating its use in the diagnosis of systemic lupus erythematosus.

[0049] Therefore, the present invention innovatively provides a use of calpain and / or SBDP145 in the preparation of a systemic lupus erythematosus diagnostic product.

[0050] Preferably, the calpain includes at least one of calpain-1 and calpain-2.

[0051] Preferably, the diagnostic product is used to detect the activity of calpain and / or the content of SBDP145 in a sample.

[0052] Preferably, the test samples include but are not limited to serum samples.

[0053] The second object of the present invention is to provide a product for detecting systemic lupus erythematosus, wherein the product contains a first reagent and / or a second reagent, wherein the first reagent is used to detect the activity of calpain, and the second reagent is used to detect the content of SBDP145.

[0054] Preferably, the product is used for clinical diagnosis, disease detection or prognosis assessment of systemic lupus erythematosus.

[0055] Preferably, the product can be used to differentiate between high and low activity of systemic lupus erythematosus.

[0056] Preferably, the first reagent is used to detect the activities of calpain-1 and calpain-2 separately or simultaneously.

[0057] Preferably, containing both the first reagent and the second reagent helps to improve the sensitivity and specificity of the detection.

[0058] Preferably, the product further contains a third reagent, which is used for the detection of anti-double-stranded DNA antibodies, and the combination of the three can further improve the detection effect. Of course, those skilled in the art know that there are many detection methods, including but not limited to enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IIF), radioimmunoassay (RIA), chemiluminescent immunoassay (CLIA), etc.

[0059] Example 1: Analysis of differences in calpain family subtypes in transcriptome sequencing of SLE patients and healthy controls

[0060] By analyzing the GEO database, we obtained the differences in mRNA levels of each subtype of the calpain family in peripheral blood mononuclear cells of SLE patients and healthy controls ( Figure 1 A). Increased calpain activity in peripheral blood mononuclear cells of SLE patients ( Figure 1 B), and the product of calpain cleavage substrate SBDP145 is upregulated ( Figure 1 C).

[0061] Therefore, it was preliminarily determined that calpain and SBDP145 could be used for the detection of SLE, and clinical samples were subsequently used for verification. The clinical samples included 75 healthy controls and 124 SLE patients, all of which were from the First Affiliated Hospital of Soochow University. Among them, 124 SLE patients were diagnosed according to the SLE classification criteria revised by ACR in 1997.

[0062] Example 2: Analysis of SBDP145 content and calpain activity in serum of SLE patients and healthy controls and their relationship with SLE activity

[0063] To evaluate the content of SBDP145 and calpain activity in peripheral blood, we used an ELISA kit to detect the level of serum αII-spectrin cleavage product SBDP145. Figure 2 As shown in A, the SBDP145 level in the serum of SLE patients was significantly higher than that in the healthy control group (p<0.001). At the same time, the calpain activity test showed that the calpain activity level in the serum of SLE patients was significantly increased compared with that in the healthy control group and was statistically significant ( Figure 2 B).

[0064] The results of the analysis showed that serum calpain activity in SLE patients was correlated with SLE disease activity measured by SLEDAI-2K (r=0.349, P<0.001) ( Figure 2 C), and we found that calpain activity was significantly different between low and high disease activity (p<0.001) ( Figure 2D). There was no significant correlation between serum SBDP145 levels and SLEDAI-2K scores (r = 0.047, p = 0.601), and there was no significant difference between patients with high and low disease activity ( Figure 2 EF). Correlation analysis showed that the serum SBDP145 content was positively correlated with calpain activity (r=0.416, P<0.0001) ( Figure 2 G).

[0065] Example 3: Analysis of the correlation between serum SBDP145 content and calpain activity and clinical parameters of SLE patients

[0066] To study the correlation between SBDP145 and Calpain activity and various clinical parameters in SLE patients. SBDP145 was positively correlated with anti-dsDNA antibody titer (r=0.416, p<0.001), ESR (r=0.427, p<0.001), high-sensitivity CRP (r=0.301, p<0.001), anti-nucleosome antibody (r=0.359, p<0.001), anti-dsDNA antibody (r=0.282, p<0.001), anti-histone antibody (r=0.400, p<0.001), anti-SSA antibody (r=0.401, p<0.001), and anti-SSB antibody (r=0.190, p=0.007), and negatively correlated with C3 (r=-0.351, p<0.001) and C4 (r=-0.256, p<0.001), as shown in the figure ( Figure 3 A-3E; Table 1). Calpain activity was positively correlated with anti-double-stranded DNA antibody titer (r=0.305, P<0.001), ESR (r=0.263, P<0.001), anti-nucleosome antibody (r=0.255, P<0.001), anti-dsDNA antibody (r=0.246, P<0.001), anti-histone antibody (r=0.325, P<0.001), and anti-SSA antibody (r=0.310, P<0.001), and negatively correlated with C3 (r=-0.306, P<0.001) and C4 (r=-0.241, P<0.001). Figure 3 F-3J; Table 1).

[0067] Table 1 Correlation between SBDP145 and calpain activities and clinical parameters of SLE

[0068]

[0069]

[0070] Abbreviations: Hb, hemoglobin; WBC, white blood cell; LY, lymphocyte; NEUT, neutrophil; PLT, platelet; hsCRP, hypersensitive C-reactive protein; ESR, Erythrocyte sedimentation rate; phosphatase; LDH, lactate dehydrogenase; C3, complement 3; C4, complement 4.

[0071] Example 4: Univariate and multivariate logistic regression analysis of SLE diagnosis

[0072] In order to evaluate the influence of different factors on distinguishing SLE patients from healthy people, we used logistic regression analysis. The variables with statistical significance in univariate analysis were: sex, hsCRP, LYM, ESR, SBDP145, calpain activity, anti-dsDNA antibody titer, C3, C4 ( Figure 4 A). Variables that showed statistical significance in univariate analysis and were considered to be clinically relevant were included in multivariate logistic regression for further evaluation. In multivariate logistic regression analysis, serum calpain activity was significantly positively correlated with the diagnosis of systemic lupus erythematosus (SLE) (OR: 1.164, 95% CI: 1.016-1.334, P = 0.029) ( Figure 4 B). In addition, in multivariate analysis, SBDP145 levels were statistically significant in distinguishing SLE patients from healthy controls (OR 3.822, 95% CI: 1.928-7.574, P < 0.001) ( Figure 4 B) These results indicate that serum calpain activity and SBDP145 levels are significantly correlated with the diagnosis of SLE.

[0073] Example 5: Diagnostic efficacy of serum SBDP145 and calpain activity in SLE patients

[0074] To further evaluate the sensitivity and specificity of SBDP145 and calpain as diagnostic markers for SLE, we performed ROC curve analysis ( Figure 5 A, C). The areas under the curve of SBDP145 and calpain activity were 0.891 and 0.762, respectively. For SBDP145, the 95% confidence interval was 0.845-0.936, the threshold was 2.377ng / ml, the sensitivity was 88.7%, and the specificity was 80.0%; for calpain activity, the 95% confidence interval was 0.697-0.827, the threshold was 13.75RFU, the sensitivity was 63.1%, and the specificity was 81.1%. The AUC of the combined detection of the two indicators was 0.919, and the sensitivity and specificity were 86.3% and 88.0%, respectively ( Figure 5 BC). Interestingly, serum calpain activity and SBDPs levels increased the diagnostic efficacy of established SLE biomarkers, and the combined diagnostic efficiency of the two and anti-dsDNA titers was as high as 96.0%, which was better than the diagnostic efficiency of anti-dsDNA titers alone, which was 83.2% ( Figure 5 BC).

[0075] Example 6: Correlation between serum SBDP145 and calpain activities and SLE affected organs

[0076] Correlation analysis showed that SBDP145 level and calpain activity were correlated with rash (r=0.213, P=0.018; r=0.333, P<0.001) and anemia (r=0.222, P=0.013; r=0.199, P=0.027). Increased calpain activity was positively correlated with pulmonary interstitialization (r=0.343, P<0.001), arthritis (r=0.255, p=0.004) and renal damage (r=0.303, P<0.001) (Table 2). Currently, the SDI (SLICC / ACR) damage index is an effective means to evaluate the damage of various organs in patients. The correlation analysis of SBDP145, calpain and SDI index of patients was consistent (r=0.250, p=0.005; r=0.342, p<0.001). Currently, the SDI (SLICC / ACR) damage index is an effective means to assess the damage of each organ in patients. We divided patients into groups with or without organ damage by SDI scores and compared them with the levels of calpain activity or SBDP145 in serum. The results showed that the serum calpain activity or SBDP145 in SLE patients with at least one organ damage (SDI-1) was significantly different from that in SLE patients without organ involvement ( Figure 6 ).

[0077] Table 2 Correlation between SBDP145 and calpain activities and organ involvement in SLE

[0078]

[0079] Abbreviations:SDI,SLICC / ACR Damage Index.

[0080] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Use of Calpain and / or SBDP145 in the preparation of diagnostic products for systemic lupus erythematosus.

2. The use according to claim 1, characterized in that: The calpain includes at least one of calpain-1 and calpain-2.

3. The use according to claim 1, characterized in that: The diagnostic product is used to detect the activity of calpain and / or the content of SBDP145 in a sample.

4. The use according to claim 3, characterized in that: The test samples include but are not limited to serum samples.

5. A product for detecting systemic lupus erythematosus, characterized in that: The product contains a first reagent and / or a second reagent, wherein the first reagent is used to detect the activity of calpain, and the second reagent is used to detect the content of SBDP145.

6. The product according to claim 5, characterized in that The product is used for the clinical diagnosis of systemic lupus erythematosus.

7. The product according to claim 5, characterized in that The product is used for monitoring the condition of systemic lupus erythematosus.

8. The product according to claim 5, characterized in that The product is used for the prognosis assessment of systemic lupus erythematosus.

9. The product according to claim 5, characterized in that The product is used to differentiate between high and low activity levels of systemic lupus erythematosus.

10. The product according to claim 5, characterized in that The product also contains a third reagent; the third reagent is used for detecting anti-double-stranded DNA antibodies.

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