Use of a reagent for detecting pyroglutamyl aminopeptidase in the preparation of a diagnostic product for rheumatoid arthritis

Abstract

The present invention relates to the field of biological detection, and discloses the application of a reagent for detecting pyroglutamate aminopeptidase in the preparation of a diagnostic product for rheumatoid arthritis. The reagent can assist in identifying RA patients by detecting the expression level of pyroglutamate aminopeptidase in a subject's sample, and can be used for treatment response evaluation and prognosis judgment, and can also effectively distinguish RA from osteoarthritis. Experiments have proved that the expression of pyroglutamate aminopeptidase is significantly increased in the sera of RA patients, and it also shows a high expression level in RA patients who are negative for rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (CCP). In addition, the expression level of pyroglutamate aminopeptidase is positively correlated with the RA score, and gradually decreases with the improvement of the condition after treatment. The reagent of the present invention has high sensitivity and good specificity, and can be used for the early diagnosis and dynamic monitoring of the condition of RA.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and particularly relates to the application of a reagent for detecting pyroglutamate aminopeptidase in the preparation of a diagnostic product for rheumatoid arthritis. Background Art

[0002] Rheumatoid Arthritis (RA) is a common chronic, systemic, autoimmune disease. The characteristics of RA are synovial inflammation and progressive destruction of joint structure, which may lead to joint deformity and loss of function. The pathogenesis of RA is complex and is closely related to genetic susceptibility, environmental factors, and abnormal immune regulation. The main pathological features of this disease are synovial tissue hyperplasia, neovascularization, and massive infiltration of immune cells, accompanied by excessive release of inflammatory factors. Early diagnosis and intervention are of great significance for delaying the progression of the disease and improving the quality of life of patients. However, due to the diverse clinical manifestations of RA and the lack of a single specific diagnostic criterion, the diagnosis and progression assessment of the disease still face great challenges.

[0003] Currently, the clinical diagnosis of RA mainly relies on comprehensive medical history, imaging examinations, and laboratory tests. Common detection methods are as follows. ① Serological markers: Anti-cyclic citrullinated peptide antibody (CCP): It has high specificity and sensitivity, but there is a problem that some patients are negative. ② Rheumatoid factor (RF): It is the earliest marker applied to the diagnosis of RA, but its specificity is low and it is easily interfered by other diseases or physiological states. ③ Inflammatory indicators: C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) have certain value in reflecting inflammatory activity, but they lack specificity for RA. ④ Imaging examinations: Detect joint destruction through X-ray, ultrasound, or MRI, but specific changes are often difficult to detect in the early stage of the disease. Although the above methods play an important role in the diagnosis and monitoring of RA, there are still many problems, including: ① Existing indicators cannot evaluate the progression degree and prognosis of RA and cannot guide medication; ② Approximately 20%-30% of RA patients are seronegative (that is, both RF and anti-CCP antibody are negative at the time of diagnosis, but they still belong to rheumatoid arthritis, which is defined as seronegative rheumatoid arthritis in medicine). Therefore, it is urgent to discover and develop new diagnostic markers to further improve the diagnostic accuracy of RA and guide the grading of RA conditions.

[0004] Rheumatoid arthritis (RA) and osteoarthritis (OA) are two common joint diseases, but there are significant differences in their pathological mechanisms and treatment strategies. RA is an autoimmune disease characterized by synovial inflammation, infiltration of immune cells, and joint destruction; while OA is a degenerative disease mainly characterized by degradation of articular cartilage and osteophyte formation. Clinically, the symptoms of RA and OA may overlap in the early stage of the disease, such as joint pain, stiffness, and dysfunction. When osteoarthritis appears in the fingers, it may be misdiagnosed as rheumatoid arthritis, which poses a challenge to accurate diagnosis. Therefore, finding a method that can effectively distinguish and diagnose rheumatoid arthritis and osteoarthritis is an urgent problem to be solved. Summary of the Invention

[0005] An object of the present invention is to overcome at least one deficiency of the prior art and provide the use of a reagent for detecting pyroglutamate aminopeptidase in the preparation of a rheumatoid arthritis diagnostic product.

[0006] The technical solution adopted by this application is as follows:

[0007] The first aspect of the present invention provides the use of a reagent for quantitatively detecting pyroglutamate aminopeptidase in the preparation of a rheumatoid arthritis diagnostic product or a rheumatoid arthritis prognosis assessment product.

[0008] Preferably, the reagent for quantitatively detecting pyroglutamate aminopeptidase includes a reagent for quantitatively detecting the mRNA of the pyroglutamate aminopeptidase gene, and / or a reagent for quantitatively detecting the pyroglutamate aminopeptidase protein.

[0009] Preferably, the rheumatoid arthritis includes at least one of serum rheumatoid factor (RF)-positive rheumatoid arthritis, serum anti-cyclic citrullinated peptide antibody (anti-CCP antibody)-positive rheumatoid arthritis, and serum RF and anti-CCP antibody-negative rheumatoid arthritis.

[0010] Preferably, the above detection is achieved by the following method, which includes at least one of enzyme-linked immunosorbent assay, Western blot, immunohistochemistry, fluorescence quantitative PCR, or mass spectrometry analysis.

[0011] Preferably, the above products include at least one of a kit, a chip, a test strip, or a high-throughput sequencing platform.

[0012] Preferably, the rheumatoid arthritis diagnostic product is used to diagnose and determine rheumatoid arthritis in chronic joint diseases including rheumatoid arthritis and osteoarthritis; and / or, the rheumatoid arthritis diagnostic product is used for grading diagnosis of rheumatoid arthritis.

[0013] The second aspect of the present invention provides a detection system for the diagnosis of rheumatoid arthritis, comprising:

[0014] (1) A sample collection module: for collecting biological samples of a subject;

[0015] (2) A sample detection module: for quantitatively detecting the expression level of pyroglutamate aminopeptidase in vitro;

[0016] (3) A data analysis module: for comparing the detection results with a diagnostic threshold and generating a diagnostic report.

[0017] Preferably, the sample includes at least one of whole blood, serum, plasma, and tissue fluid.

[0018] Preferably, the diagnostic threshold is the cut off value; in some embodiments, the cut off value is 57.28 ng / mL, that is, when the expression level of pyroglutamate aminopeptidase in the detection result is greater than or equal to 57.28 ng / mL, it can be preliminarily determined that the patient has RA.

[0019] The third aspect of the present invention provides a rheumatoid arthritis diagnosis product or a rheumatoid arthritis prognosis assessment product, and the product contains a reagent for detecting the expression level of pyroglutamate aminopeptidase in a sample.

[0020] Preferably, the product includes at least one of an ELISA detection kit, a Western Blot detection kit, an IHC staining kit, and an immunochromatographic test strip.

[0021] In some embodiments, the data analysis module further combines the detection results of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and anti-cyclic citrullinated peptide antibody (CCP) to generate a comprehensive diagnostic conclusion.

[0022] A method for evaluating the disease activity of rheumatoid arthritis, by detecting the change trend of the expression level of pyroglutamate aminopeptidase before and after treatment of a patient to judge the disease activity.

[0023] In some embodiments, the disease severity is further evaluated by combining the patient's inflammatory indicators (such as CRP, ESR, etc.) and imaging data.

[0024] A method for formulating an individualized treatment plan for rheumatoid arthritis, according to the dynamic change of the expression level of pyroglutamate aminopeptidase of a subject, selecting a suitable drug, dosage, and treatment cycle.

[0025] In some embodiments, the treatment strategy is optimized by combining the detection result of pyroglutamate aminopeptidase and the patient's immune indicators (such as IL-6, TNF-α levels, etc.).

[0026] Application of reagent for detecting pyroglutamate aminopeptidase in preparing product for differentiating rheumatoid arthritis and osteoarthritis.

[0027] The beneficial effects of this application are as follows:

[0028] (1) It is found in this application research that the content of glutamate aminopeptidase can be used for the diagnosis of seronegative rheumatoid arthritis, especially for the diagnosis of rheumatoid arthritis patients with negative RF and CCP. Using pyroglutamate aminopeptidase for the diagnosis of RA can make up for the deficiencies of existing detection indicators and fill the blank in the current field of RA diagnosis.

[0029] (2) It is found in this application research that the content of pyroglutamate aminopeptidase is linearly correlated with the progression degree of RA. Through the monitoring of pyroglutamate aminopeptidase level, the dynamic evaluation of RA disease activity and treatment response can be realized, providing a scientific basis for formulating individualized treatment plans.

[0030] (3) It is found in this application research that the content of pyroglutamate aminopeptidase can effectively distinguish RA and osteoarthritis (OA) with similar clinical symptoms, improving the diagnostic accuracy of RA.

[0031] In summary, using pyroglutamate aminopeptidase for the diagnosis of RA can not only make up for the deficiencies of existing detection indicators, but also dynamically evaluate RA disease activity and treatment response. At the same time, it helps to distinguish RA and OA, providing important support for the diagnosis and precision medicine of RA, and having significant clinical and industrial value. Description of Drawings

[0032] Figure 1 ROC curve of serum pyroglutamate aminopeptidase for RA patients.

[0033] Figure 2 Statistical chart for detecting differential expression of pyroglutamate aminopeptidase at protein level (***P<0.001).

[0034] Figure 3 Expression level of pyroglutamate aminopeptidase in seronegative rheumatoid arthritis patients (***P<0.001).

[0035] Figure 4 Pyroglutamate aminopeptidase level is positively correlated with clinical score of rheumatoid arthritis. Detailed Implementation Modes

[0036] Term Explanation:

[0037] Pyroglutamate aminopeptidase (EC: 3.4.19.3) can catalyze the removal of L-pyroglutamate from the N-terminus of certain peptides and proteins. Pyroglutamate aminopeptidase has been found in almost all life domains, including mammals, birds, fish, plants, protists, and fungi. In addition, pyroglutamate aminopeptidase is highly conserved and ubiquitously distributed in human tissues, and its new physiological functions have been continuously proposed in recent decades. In previous studies, since pyroglutamate aminopeptidase is present in the small intestine and duodenum, it was considered to be involved in the absorption of peptides and proteins in the mammalian digestive tract. In addition, based on the widespread distribution of pyroglutamate aminopeptidase in tissues with different functions, researchers have proposed that pyroglutamate aminopeptidase may be involved in the intracellular degradation and resynthesis of peptides. Pyroglutamate aminopeptidase is also thought to affect certain disease states (e.g., memory disorders, etc.) by regulating the level of free L-pyroglutamate.

[0038] Although pyroglutamate aminopeptidase may be involved in the regulation of certain diseases, its expression in RA is not clear. More importantly, there is still a lack of clear research evidence in the prior art on whether pyroglutamate aminopeptidase can be used as a diagnostic indicator for RA.

[0039] In the research of this application, it was found that pyroglutamate aminopeptidase is linearly correlated with the progression degree of rheumatoid arthritis, which indicates that the disease progression of rheumatoid arthritis can be judged and the prognosis can be evaluated through pyroglutamate aminopeptidase, which cannot be achieved by existing objective detection indicators (such as RF, CCP). In addition, pyroglutamate aminopeptidase can also be used for the diagnosis of seronegative rheumatoid arthritis, which is a supplement to existing diagnostic indicators (such as RF, CCP) and can make up for the deficiencies of existing methods.

[0040] To better understand the present invention, the following embodiments are used to further specifically illustrate the present invention, but it should not be construed as a limitation of the present invention. For those skilled in the art, some non-essential improvements and adjustments made based on the above-mentioned invention content are also considered to fall within the protection scope of the present invention.

[0041] In the embodiments, unless otherwise specified, the test methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0042] Examples 1 - 3 are the data analysis of patient samples. The diagnosis of all cases of RA conforms to the RA diagnostic criteria revised by the American College of Rheumatology in 1987, that is, the detection indicators include: "morning stiffness (lasting more than 1 hour)", "joint symptoms (swelling of at least 3 joints)", "rheumatoid nodules (subcutaneous nodules commonly found in areas prone to friction)", "RF (elevated serum level)", "X-ray (visible bone erosion changes)". All cases of osteoarthritis patients conform to the diagnostic criteria for OA proposed by Altam, that is, the detection includes: hypertrophy of at least 2 finger joint tissues, bony hypertrophy, bone friction sensation, morning stiffness, etc. All clinical samples used in the research of this application are provided by the Affiliated Hospital of Xi'an Jiaotong University. Informed consent has been obtained from all patients, and it has been approved by the ethics committee.

[0043] Example 4 is the data analysis of the RA mouse model.

[0044] The detection of the pyroglutamyl aminopeptidase protein content in the sample serum in Examples 1 - 3 uses the Human PGPEP1 (Pyroglutamyl-peptidase 1) ELISA Kit. The detection of the pyroglutamyl aminopeptidase protein content in the sample serum in Example 4 uses the Mouse Pyroglutamyl-peptidase 1 (PGPEP1) ELISA Kit. All sera used in the Elisa detection are prepared according to the standard protocol. The sera are diluted, and the detection procedure strictly follows the protocol of the commercial Elisa kit. Example 1

[0045] The pyroglutamyl aminopeptidase protein content in the sera of 10 healthy volunteers and 10 RA patients (patients diagnosed with RA, without considering the distinction between positive and negative RF and CCP) was detected, as shown in Table 1 below. ROC curve analysis was performed to expect to find an index with a relatively high area under the curve (AUC), sensitivity, and specificity to determine the optimal screening positive cut-off value (cut-off value). The closer the AUC is to 1, the better the diagnostic effect.

[0046] Table 1. Pyroglutamyl aminopeptidase content in the sera of RA patients and normal people (venous blood from the upper arm)

[0047]

[0048] As Figure 1As shown, the results were AUC = 0.986, p < 0.0001, indicating that the pyroglutamate aminopeptidase content can be used to clearly distinguish between RA patients and healthy individuals. When the cut-off value = 57.28, the specificity for judging RA was 100% and the sensitivity was 91.67%, that is, when this cut-off value was taken, patients without RA could be identified 100% without false positives; at the same time, the detection rate (or true positive rate) was 91.67%, and the high sensitivity could effectively identify RA patients and reduce the missed diagnosis rate. In other words, when ≥ 57.28 ng / mL, it can be preliminarily determined that the patient has RA.

[0049] Example 2

[0050] In Example 2, 4 RA patients and 4 osteoarthritis patients showed similar external symptoms, namely morning stiffness, hypertrophy of finger joint tissues, bone friction sensation, etc. It was difficult to effectively distinguish only through external joint manifestations. In this example, by detecting the pyroglutamate aminopeptidase content in the venous blood of patients (the detection method was the same as that in Example 1) and using the cut-off value (57.28 ng / mL) in Example 1, RA patients and osteoarthritis patients could be effectively distinguished.

[0051] The results were as Figure 2 shown. The pyroglutamate aminopeptidase content in the serum of osteoarthritis patients was 46.6 ng / mL, lower than the cut-off value (57.28 ng / mL), while the pyroglutamate aminopeptidase content in the serum of RA patients was 86.9 ng / mL, far higher than the cut-off value. This data indicates that pyroglutamate aminopeptidase can effectively distinguish rheumatoid arthritis and osteoarthritis, P < 0.001. Example 3

[0052] In Example 3, the blood samples of 3 RA patients with seronegative rheumatoid arthritis (RF negative, CCP negative) were from RA patients in the hospital, including 2 females and 1 male, with an average age of (55 ± 8) years and an average disease course of (2 ± 1) years. 3 normal blood samples were from volunteers. The detection method was the same as that in Example 1, and the results are shown in Figure 3 .

[0053] As Figure 3 shown, in patients with seronegative rheumatoid arthritis, pyroglutamate aminopeptidase was still highly expressed, at 96 ± 5 ng / mL, far higher than the cut-off value (57.28 ng / mL), P < 0.001, indicating that pyroglutamate aminopeptidase can be used for the diagnosis of seronegative rheumatoid arthritis. Example 4

[0054] Establishing an RA mouse model: 12 male DBA / 1 mice. Lipopolysaccharide was completely emulsified with Freund's adjuvant, and 0.2 mg of the emulsified lipopolysaccharide and Freund's adjuvant was injected intradermally into the mice on day 0 and day 7. Usually, an inflammatory reaction in the joints occurs 10 to 13 days after receiving the stimulation. The thickness of the footpad was measured using a vernier caliper with a precision of 0.01 mm and evaluated according to clinical scoring. The scoring criteria are divided into two aspects, one is the inflammatory score of the mouse toes, and the other is the swelling score of the mouse claws. See Table 2 for details.

[0055] Table 2. Inflammatory response of the footpad joints in mice

[0056]

[0057] The score of each mouse is the sum of the inflammatory score of the toes and the swelling score of the claws (the maximum is 14 points). A score of 1.5 or above can be regarded as a successful model construction. These mice with successful RA model construction were randomly re-grouped to continue the following drug administration experiment.

[0058] Ten mice with successful model establishment (all with a score of 3) were selected to continue the experiment. The experiment was divided into a model control group and a drug administration group, with 5 mice in each group. The mice in the model control group were not treated with anything, and the mice in the drug administration group were orally administered compound methotrexate (dose: 14 mg / kg / week) by gavage once a day for three consecutive weeks. Methotrexate is a cornerstone drug for the treatment of RA. The "2018 Chinese Guidelines for the Diagnosis and Treatment of Rheumatoid Arthritis" recommends the use of methotrexate alone and as the "cornerstone drug" for combination therapy. After the treatment began, the mice were scored for CIA every two weeks, and blood was collected from the tail vein to measure the content of pyroglutamyl aminopeptidase in the blood (detected using Mouse Pyroglutamyl-peptidase 1 (PGPEP1) ELISA Kit). The results showed that the CIA score and the content of pyroglutamyl aminopeptidase in the model control group remained basically unchanged (average CIA score was about 3.0, and average pyroglutamyl aminopeptidase content was about 90 ng / mL). In addition, with the CIA score of the drug administration group gradually decreasing, the content of pyroglutamyl aminopeptidase also gradually decreased, and the regression equation was Y = 23.02X + 20.52, with a goodness of fit R² = 0.9888, as shown in Figure 4 Figure [Figure number not provided in the original]. As the CIA score of the drug administration group gradually decreased, the content of pyroglutamyl aminopeptidase also gradually decreased, and the regression equation was Y = 23.02X + 20.52, with a goodness of fit R² = 0.9888.

[0059] After the RA model mice were treated with methotrexate, the concentration of PGP-1 in their serum gradually decreased with the treatment time, and the decreasing trend was consistent with the improvement trend of the inflammatory score, indicating that PGP-1 can be used to reflect the disease progression during the treatment process and has potential prognostic evaluation value.

[0060] In summary, the above experimental data indicate that the pyroglutamate aminopeptidase level is positively correlated with the progression of RA, suggesting that the serum pyroglutamate aminopeptidase level can be used for the grading of RA and the evaluation of prognosis.

[0061] The above is a further detailed description of the present invention and should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, any simple deduction or substitution without departing from the concept of the present invention falls within the protection scope of the present invention.

Claims

1. Use of a reagent for quantitatively detecting pyroglutamate aminopeptidase in the preparation of a rheumatoid arthritis diagnostic product or a rheumatoid arthritis prognosis assessment product.

2. The application according to claim 1, characterized in that, The rheumatoid arthritis includes at least one of serum rheumatoid factor-positive rheumatoid arthritis, serum anti-cyclic citrullinated peptide antibody-positive rheumatoid arthritis, and serum rheumatoid factor- and serum anti-cyclic citrullinated peptide antibody-negative rheumatoid arthritis.

3. The application according to claim 1, wherein The reagent for quantitatively detecting pyroglutamate aminopeptidase includes a reagent for quantitatively detecting the mRNA of the pyroglutamate aminopeptidase gene, and / or a reagent for quantitatively detecting the pyroglutamate aminopeptidase protein.

4. The application according to claim 1 or 3, characterized in that The detection is achieved by the following methods, which include at least one of enzyme-linked immunosorbent assay, Western blot, immunohistochemistry, fluorescence quantitative polymerase chain reaction, or mass spectrometry.

5. The application according to claim 1, wherein The product includes at least one of a kit, a chip, a test strip, or a high-throughput sequencing platform.

6. The application according to claim 1, characterized in that, The rheumatoid arthritis diagnostic product is used for diagnosing rheumatoid arthritis in chronic joint diseases including rheumatoid arthritis and osteoarthritis; and / or, the rheumatoid arthritis diagnostic product is used for grading diagnosis of rheumatoid arthritis.

7. A detection system for diagnosing rheumatoid arthritis, comprising: (1) A sample collection module: for collecting a biological sample of a subject; (2) A sample detection module: for quantitatively detecting the expression level of pyroglutamate aminopeptidase in vitro; (3) A data analysis module: for comparing the detection result with a diagnostic threshold and generating a diagnostic report.

8. The detection system according to claim 7, wherein, The biological sample includes at least one of whole blood, serum, plasma, and tissue fluid.

9. A rheumatoid arthritis diagnosis product or a rheumatoid arthritis prognosis assessment product, characterized in that The product contains a reagent for detecting the expression level of pyroglutamate aminopeptidase in a sample.

10. The rheumatoid arthritis diagnosis product or rheumatoid arthritis prognosis assessment product according to claim 9, characterized in that, The product includes at least one of an ELISA detection kit, a Western Blot detection kit, an IHC staining kit, and an immunochromatographic test strip.

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