Biomarker for diagnosing idiopathic inflammatory myopathy and application thereof

Abstract

The invention relates to the technical field of identification of idiopathic inflammatory myopathy, in particular to a biomarker for diagnosing idiopathic inflammatory myopathy and application thereof, and the biomarker comprises the following serum markers: cold-induced RNA binding protein, myeloid cell trigger receptor-1 and Toll-like receptor 4. In the biomarker provided by the invention, the CIRP can play a pathological regulation role through a transmembrane receptor. The TLR4 serves as an important bridge for linking innate immunity and adaptive immunity, and the inflammation driving effect of CIRP is mediated in systemic inflammation. TREM-1 is a core action target of CIRP, and tissue damage is amplified by activating inflammatory response of macrophages and neutrophils in various inflammatory diseases.

Description

Technical Field

[0001] This invention relates to the field of idiopathic inflammatory myopathy identification technology, and particularly to a biomarker for diagnosing idiopathic inflammatory myopathy and its application. Background Technology

[0002] Idiopathic inflammatory myopathy (IIM) is a group of highly heterogeneous autoimmune diseases characterized by involvement of the skin and skeletal muscles of the extremities, often accompanied by multi-system involvement, including lesions in organs such as the joints, lungs, and heart. Based on clinical manifestations and myositis-specific antibodies, IIM can be classified into subtypes such as dermatomyositis (DM), anti-synthesizer syndrome (ASS), immune-mediated necrotizing myopathy (IMNM), polymyositis (PM), and inclusion body myositis (IBM). Epidemiological surveys indicate that the incidence of IIM is approximately 0.2–2 per 100,000 person-years, with a prevalence of 2–25 per 100,000 people. Compared to the general population, the overall mortality risk ratio for IIM patients is 3.7, with the highest mortality rate occurring in the first year after diagnosis.

[0003] Currently, the diagnosis of IIM is mainly based on clinical manifestations, laboratory tests, imaging examinations, and muscle pathology results, including symmetrical proximal muscle weakness, typical skin lesions, muscle biopsy and electromyography characteristics, and serum muscle enzyme levels. However, existing diagnostic methods have limitations: muscle biopsy is an invasive procedure with low patient acceptance; muscle enzyme levels, such as creatine kinase (CK), may be normal in some IIM subtypes; and some patients present with atypical clinical manifestations, making it easy to miss or misdiagnose. Therefore, developing serum biomarkers with early diagnostic value and innovative treatment strategies is of great clinical significance for effectively reducing the risk of death from this disease and optimizing long-term clinical outcomes for patients. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a biomarker for the diagnosis of idiopathic inflammatory myopathy and its application, which can effectively reduce the risk of death from the disease and optimize the long-term clinical outcomes of patients.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: According to a first aspect of the present invention, a biomarker is provided, comprising the following serum biomarkers: Cold-induced RNA-binding protein (CIRP), myeloid trigger receptor-1 (TREM-1), and Toll-like receptor 4 (TLR4).

[0006] According to a second aspect of the invention, there is provided the use of a combination of biomarkers or a detection reagent thereof in the preparation of a product for diagnosing or identifying idiopathic inflammatory myopathy, the detection reagent being used to detect the level of the combination of biomarkers in a sample to be tested.

[0007] According to a third aspect of the present invention, a diagnostic kit for diagnosing or identifying idiopathic inflammatory myopathy is provided, the diagnostic kit comprising reagents for detecting the levels of a combination of biomarkers in a sample to be tested.

[0008] According to a fourth aspect of the invention, there is provided an application of a combination of biomarkers or a detection reagent thereof in the preparation of a product for assessing disease activity or treatment efficacy in idiopathic inflammatory myopathy, the detection reagent being used to detect the level of the combination of biomarkers in a sample to be tested.

[0009] According to a fifth aspect of the present invention, a diagnostic kit is provided for assessing disease activity or treatment efficacy in idiopathic inflammatory myopathy, the diagnostic kit comprising reagents for detecting the levels of a combination of biomarkers in a test sample.

[0010] According to a sixth aspect of the present invention, an evaluation system for monitoring idiopathic inflammatory myopathy (IIM) is provided, comprising a detection module, a data analysis module, and an evaluation module: The detection module is used to detect the levels of biomarker combinations in the sample to be tested and obtain the detection results. The data analysis module is used to analyze and calculate the detection results of the detection module to obtain the idiopathic inflammatory myopathy score; The assessment module is used to identify idiopathic inflammatory myopathy based on the idiopathic inflammatory myopathy score from the data analysis module.

[0011] According to a seventh aspect of the present invention, an evaluation system for monitoring the activity or treatment efficacy of idiopathic inflammatory myopathy is provided, comprising a detection module, a data analysis module, and an evaluation module: The detection module is used to detect the level of biomarker combination in the first sample to be tested and obtain the first detection result; The detection module is used to detect the level of biomarker combination in the second sample to be tested, and to obtain a second detection result; The data analysis module is used to analyze and calculate the changes in the first and second detection results of the detection module to obtain a score for the activity or treatment effect of idiopathic inflammatory myopathy. The assessment module is used to evaluate the activity or treatment effect of idiopathic inflammatory myopathy based on the activity or treatment effect score of idiopathic inflammatory myopathy from the data analysis module.

[0012] According to an eighth aspect of the present invention, a combination of biomarkers or their detection reagents are provided for use in the preparation of products for screening high-risk individuals for idiopathic inflammatory myopathy, wherein the expression levels of cold-induced RNA-binding protein (CIRP), myeloid trigger receptor-1 (TREM-1), and Toll-like receptor 4 (TLR4) in the serum of the test subject are detected and compared with preset thresholds.

[0013] According to a ninth aspect of the present invention, a test kit for screening high-risk individuals for idiopathic inflammatory myopathy is provided, the test kit comprising reagents for detecting the levels of a combination of biomarkers in a sample to be tested.

[0014] Among the biomarkers provided in this invention, CIRP can exert pathological regulatory effects by targeting transmembrane receptors such as Toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells-1 (TREM-1). TLR4, as an important bridge connecting innate and adaptive immunity, mediates the inflammatory-driven effect of CIRP in systemic inflammation. TREM-1 is the core target of CIRP, amplifying tissue damage in various inflammatory diseases by activating the inflammatory responses of macrophages and neutrophils. Attached Figure Description

[0015] The present invention includes the following figures: Figure 1 This is the CIRP standard concentration curve in Example 1 of the present invention; Figure 2 This is the TREM-1 standard concentration curve in Example 1 of the present invention; Figure 3 This is the TLR4 standard concentration curve in Example 1 of the present invention; Figure 4 These are the experimental results of serum CIRP, TREM-1, and TLR4 levels in the IIM group and the healthy control group in Example 2 of this invention; Figure 5 MDA5 in Embodiment 2 of the present invention + DM group, MDA5 - Experimental results of serum CIRP, TREM-1, and TLR4 levels in the DM group, ASS group, myositis group, and healthy control group; Figure 6 These are the experimental results of serum CIRP, TREM-1, and TLR4 levels in different types of myositis-specific antibody groups and healthy control groups in Example 2 of this invention; Figure 7The results show the correlation between serum CIRP and AST, LDH, CK, CKMB, ESR, and creatinine in the IIM group in Example 3 of this invention. Figure 8 This is the experimental result of the correlation between serum TREM-1 and CKMB and creatinine in the IIM group in Example 3 of the present invention; Figure 9 The results show the correlation between serum TLR4 and AST, LDH, CK, CKMB, and creatinine in the IIM group in Example 3 of this invention. Figure 10 These are the experimental results of serum CIRP, TREM-1, and TLR4 levels before and after treatment in the IIM group in Example 4 of this invention; Figure 11 This is the experimental result of the predictive value of serum CIRP, TREM-1, and TLR4 expression levels for the diagnosis of IIM in Example 5 of the present invention; Figure 12 These are the experimental results of serum CIRP, TREM-1, and TLR4 levels in the IIM2 group and the healthy control group in Example 6 of this invention; Figure 13 The ASS group and MDA5 in Embodiment 6 of the present invention + DM group, MDA5 - Experimental results of serum CIRP, TREM-1, and TLR4 levels in the DM group, myositis group, and healthy control group; Figure 14 This is the experimental result of the predictive value of serum CIRP, TREM-1, and TLR4 expression levels for the diagnosis of IIM in Example 6 of the present invention; Figure 15 These are the experimental results of serum CIRP, TREM-1, and TLR4 levels before and after treatment in the IIM group in Example 6 of this invention; Figure 16 The experimental results of CIRBP levels in the IIM group and healthy control group in GSE142807 and GSE128470 in Example 6 of this invention; Figure 17 The results show the experimental results of TLR4 levels in the IIM group and the healthy control group in GSE142807 and GSE128314 in Example 6 of this invention. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0017] Example 1: Screening of biomarkers and construction of evaluation models 1. Subject screening: The subjects were 95 newly diagnosed and relapsed IIM patients who were hospitalized at Tianjin Medical University General Hospital. Based on their clinical manifestations and myositis-specific antibodies, they were divided into 25 ASS patients and 13 MDA5 patients. + DM patients, 36 MDA5 cases - The study included DM patients and 21 myositis patients, including PM, IMNM, and IBM patients.

[0018] Inclusion criteria: (1) All IIM patients met the classification criteria for idiopathic inflammatory myopathy published by the European League of Rheumatology (EULAR) and the American College of Rheumatology (ACR); (2) The Myositis Disease Activity Assessment Tool (MDAAT) was used to score the disease activity of patients based on their clinical data. The assessment included eight parts: general condition, skin and mucous membranes, bones and joints, gastrointestinal tract, lungs, heart, muscles, and total disease activity, with a total of 26 items. Each item was recorded as none, improved, unchanged, worsened, and new, with corresponding scores of 0, 1, 2, 3, and 4. The Visual Analogue Scale (VAS) was used to assess the total activity of each system, ranging from 0 to 10, where 0 represents "no activity" and 10 represents "high activity". Patients with IIM who scored ≥2 were included.

[0019] Healthy control group: 49 healthy individuals were included in the study, including 40 females (81.63%) and 9 males (18.37%), with an age range of 28-82 years and a mean age of (54.94±13.91) years. Statistical analysis showed no significant differences between the case group and the healthy control group in baseline characteristics such as gender distribution and age range. p >0.05), which meets the requirements for research comparability.

[0020] This protocol has passed the ethical review of the Medical Ethics Committee of Tianjin Medical University General Hospital (No.: IRB2024-YX-496-01) before implementation. Table 1 shows the demographic and clinical characteristics of the subjects.

[0021] Table 1. Demographic and clinical characteristics of the subjects 2. CIRP, TREM-1, and TLR4 assays in subjects The main reagents include: CIRP ELISA Kit (Tianjin Meisheng Technology Co., Ltd., China) TREM-1 ELISA Kit (Tianjin Meisheng Technology Co., Ltd., China) TLR4 ELISA Kit (Tianjin Meisheng Technology Co., Ltd., China) This embodiment strictly adheres to the standardized collection process: All subjects underwent 4 ml of antecubital venous blood collection via standard venipuncture technique in the morning on an empty stomach, and the samples were then placed in sterile vacuum blood collection tubes. After the blood samples were allowed to stand at room temperature for 60 minutes to promote coagulation, serum separation was performed using a low-speed centrifuge (centrifugation parameters set at 3000 rpm, room temperature for 5 minutes). The clarified serum layer was precisely aspirated using a micropipette, aliquoted into pre-frozen storage tubes, and immediately labeled with a unique code and collection time. Finally, the tubes were transferred to ultra-low temperature storage equipment (-80℃) for a single-use cryopreservation strategy. The entire process strictly followed biobank management guidelines to avoid the impact of repeated freeze-thaw cycles on protein stability. All subjects' antecubital venous blood samples were subjected to CIRP, TREM-1, and TLR4 tests, as detailed below: (1) CIRP detection Dilution of Standards: This kit provides one vial of original standard, which users can dilute in small test tubes according to the following chart 2; Table 2 Dilution of Standards Sample addition: Set up blank wells (blank control wells do not contain sample or enzyme-labeled reagent, all other steps are the same), standard wells, and sample wells. Accurately add 50 μl of standard to the enzyme-labeled plate. Add 40 μl of sample diluent to the sample wells, then add 10 μl of the sample to be tested (final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix. Incubation: After sealing the plate with sealing film, incubate at 37°C for 30 minutes; Solution preparation: Dilute the 30-fold concentrated washing solution with distilled water 30 times and set aside; Washing: Carefully peel off the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 30 seconds and then discard, repeat this 5 times, then pat dry; Add enzyme: Add 50 μl of enzyme-labeled reagent to each well, except for the blank wells; After incubation and washing, develop the color: Add 50 μl of color developer A to each well, then add 50 μl of color developer B, gently shake to mix, and develop the color at 37°C in the dark for 10 minutes. Termination: Add 50 μl of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). Measurement: Zero the instrument using the blank well, and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. Measurements should be performed within 15 minutes of adding the stop solution. A CIRP standard concentration curve was plotted based on experimental data. Figure 1 The CIRP concentration (ng / L) of each well was calculated based on the concentration curve.

[0022] (2) TREM-1 detection: The ELISA detection method is the same as the CIRP detection method, and the results are as follows: Figure 2 As shown, Figure 2 This is a curve showing the standard concentration of TREM-1.

[0023] (3) TLR4 detection: The ELISA detection method is the same as the CIRP detection method, and the results are as follows: Figure 3 As shown, Figure 3 This is a curve showing the standard concentration of TLR4.

[0024] Example 2: Serum CIRP, TREM-1, and TLR4 expression levels in the IIM group and healthy control group 1. Serum CIRP, TREM-1, and TLR4 expression levels in the IIM group and healthy control group Mann-Whitney U test analysis showed significant differences in serum CIRP, TREM-1, and TLR4 expression levels between the IIM group (n=95) and the healthy control group (n=49). The results indicated: (1) Serum CIRP level: The median concentration in the IIM group was 141.5 ng / L (IQR=116.5), which was significantly higher than that in the healthy control group [115.0 ng / L (IQR=57.23)]. p =0.0029). (Table 3, Figure 4 ) (2) Serum TREM-1 level: The median level in the IIM group was 430.7 ng / L (IQR=230.3), which was significantly higher than that in the healthy control group [267.8 ng / L (IQR=145.4)]. p <0.0001). (Table 3, Figure 4 ) (3) Serum TLR4 level: The median expression in the IIM group was 13.78 ng / ml (IQR=11.4), which was significantly higher than that in the healthy control group [9.386 ng / ml (IQR=4.199)]. p <0.0001). (Table 3, Figure 4 ) The results show that CIRP, TREM-1, and TLR4 are all highly expressed in the serum of IIM patients, and may be involved in the pathogenesis of the disease.

[0025] Table 3. Serum CIRP, TREM-1, and TLR4 expression levels in the IIM group and the healthy control group. Note:*: p <0.05; **: p<0.01; ***: p <0.001; ****: p <0.0001 2. Comparison of serum CIRP, TREM-1, and TLR4 levels between each subgroup and the healthy control group MDA5 + DM group, MDA5 - Comparison of serum CIRP, TREM-1, and TLR4 levels in the DM group, ASS group, myositis group, and healthy control group The Kruskal-Wallis H test was used to differentiate between different IIM subtypes (MDA5). + DM group 13 cases, MDA5 - Serum CIRP, TREM-1, and TLR4 levels were compared among the groups (36 cases in the DM group, 25 cases in the ASS group, 21 cases in the myositis group, and 49 healthy controls). The results showed that: (1) Compared with the healthy control group, MDA5 - Serum CIRP levels were elevated in the DM, ASS, and myositis groups, with a statistically significant difference in the myositis group (p=0.0001); compared with MDA5 + Compared with the DM group, the serum CIRP level in the myositis group was significantly higher, and the difference was statistically significant (p<0.05). (Table 4) Figure 5 ) (2) Compared with the healthy control group, MDA5 + DM, MDA5 - Serum TREM-1 levels were elevated in the DM group, ASS group, and myositis group, and the differences were statistically significant (p<0.05), while there were no statistically significant differences among the subgroups. (Table 4) Figure 5 ) (3) Compared with the healthy control group, MDA5 + DM, MDA5 - Serum TLR4 levels were elevated in the DM group, ASS group, and myositis group, and were also elevated in MDA5. - The differences between the DM and myositis groups were statistically significant (p<0.0001). Compared with MDA5 + Compared with the ASS group, the serum TLR4 level in the myositis group was significantly increased, and the difference was statistically significant (p < 0.05). (Table 4) Figure 5 ) Table 4 MDA5 + DM group, MDA5 - Serum CIRP, TREM-1, and TLR4 levels in the DM group, ASS group, myositis group, and healthy control group Note:*: p<0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; 'a' indicates a comparison with the control group. p <0.05; b indicates the difference between MDA5 and MDA5. + DM comparison, p <0.05; d indicates comparison with ASS, p <0.05 3. Comparison of serum CIRP, TREM-1, and TLR4 levels between different types of myositis-specific antibody groups and healthy controls. The Kruskal-Wallis H test was used to compare serum CIRP, TREM-1, and TLR4 levels of different types of myositis-specific antibodies (18 cases positive for anti-JO-1 antibody, 13 cases positive for anti-MDA5 antibody, 11 cases positive for anti-Mi-2 antibody, 4 cases positive for anti-HMGCR antibody, 48 cases positive for anti-RO-52 antibody, and 20 cases negative for antibodies) with a healthy control group (49 cases). The results showed: (1) Compared with the healthy control group, serum CIRP levels were elevated in the groups with positive anti-JO-1 antibody, positive anti-Mi-2 antibody, positive anti-HMGCR antibody, positive anti-RO-52 antibody, and negative antibody, but the differences were not statistically significant. (Table 5) Figure 6 ) (2) Compared with healthy controls, serum TREM-1 and TLR4 levels were significantly increased in the groups with positive anti-JO-1 antibody, positive anti-Mi-2 antibody, positive anti-HMGCR antibody, positive anti-RO-52 antibody, and negative antibody, with statistically significant differences. However, no significant differences were observed among the antibody groups. (Table 5) Figure 6 ) Table 5. Serum CIRP, TREM-1, and TLR4 levels in different types of myositis-specific antibody groups and healthy controls. Note:*: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; 'a' indicates a comparison with the control group. p <0.05.

[0026] Example 3: Correlation between serum CIRP, TREM-1, TLR4 levels and laboratory test indicators in IIM patients Spearman rank correlation analysis was used to assess the relationship between serum CIRP, TREM-1, TLR4 levels and laboratory indicators in patients with intra-infectious disease (IIM). The results showed... (1) Serum CIRP levels were positively correlated with aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CKMB), and erythrocyte sedimentation rate (ESR), and negatively correlated with creatinine. (Table 6, Figure 7 ) (2) Serum TREM-1 levels were positively correlated with creatine kinase isoenzymes and negatively correlated with creatinine. (Table 6) Figure 8 ) (3) Serum TLR4 levels were positively correlated with aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase, and creatine kinase isoenzymes, and negatively correlated with creatinine. (Table 6) Figure 9 ) Table 6. Correlation between serum CIRP, TREM-1, and TLR4 levels and laboratory test indicators in IIM patients Note:*: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001 Example 4: Changes in serum CIRP, TREM-1, and TLR4 levels before and after IIM treatment in the group To investigate the changes in CIRP, TREM-1, and TLR4 levels before and after treatment, we included 5 patients with intraepithelial neoplasia (IIM). These 5 newly diagnosed IIM patients were followed up after treatment with a combination of hormones and immunosuppressants, and their condition was assessed as stable. Samples were then resampled for testing. Results showed that serum CIRP, TREM-1, and TLR4 levels in IIM patients significantly decreased after treatment. Figure 10 ) Example 5: Predictive value of serum CIRP, TREM-1, and TLR4 expression levels for the diagnosis of IIM The independent and combined predictive abilities of serum CIRP, TREM-1, and TLR4 for IIM were evaluated by constructing a binary logistic regression model, and their diagnostic efficacy was quantified by ROC curves. The results showed: (1) When the serum CIRP cutoff value was 99.08 ng / L, its sensitivity was 93.68%, specificity was 36.73%, and AUC was 0.6509. (Table 7) Figure 11 ) (2) When the serum TREM-1 cutoff value was 333.6 ng / L, its sensitivity was 82.11%, its specificity was 69.39%, and its AUC was 0.7955. (Table 7) Figure 11 ) (3) When the serum TLR4 cutoff value was 10.09 ng / ml, the sensitivity was 84.21%, the specificity was 65.31%, and the AUC was 0.7893. (Table 7) Figure 11 ) (4) When serum CIRP was combined with serum TREM-1, the sensitivity was 80.0%, the specificity was 73.5%, and the AUC was 0.815. (Table 7) Figure 11 ) (5) When serum CIRP was combined with serum TLR4, the sensitivity was 84.2%, the specificity was 67.3%, and the AUC was 0.813. (Table 7) Figure 11 ) (6) When serum TREM-1 was combined with serum TLR4, the sensitivity was 86.3%, the specificity was 69.4%, and the AUC was 0.812. (Table 7) Figure 11 ) (7) When serum CIRP, serum TREM-1, and serum TLR4 were combined, the sensitivity was 89.5%, the specificity was 67.3%, and the AUC was 0.842. (Table 7) Figure 11 ) Table 7. Predictive value of serum CIRP, TREM-1, and TLR4 expression levels for the diagnosis of IIM This indicates that serum CIRP levels in IIM patients were significantly higher than in healthy controls, and serum TREM-1 and TLR4 levels were also significantly higher in IIM patients, with statistically significant differences. Therefore, it can be inferred that CIRP acts as a DAMP in IIM patients, driving inflammatory responses through the CIRP-TREM-1 and CIRP-TLR4 axes and participating in the pathogenesis of IIM. Currently, the diagnosis of IIM is challenging, mainly based on clinical manifestations, laboratory tests, imaging examinations, and pathological results, including the presence or absence of symmetrical proximal muscle weakness and typical skin lesions, muscle biopsy and electromyography characteristics, serological assessment, and serum muscle enzyme levels. This invention found that serum CIRP levels are related to AST (r = 0.2019, p = 0.0498), LDH (r= 0.2113, p =0.0398), CK (r= 0.2467, p = 0.016), CKMB (r = 0.2971, p = 0.0035), ESR (r= 0.23, p The correlation between CIRP and inflammatory response (=0.025) is positive, suggesting that CIRP has some value in assessing the degree of muscle damage, the level of inflammatory response, and early diagnosis of IIM.

[0027] Creatinine is a compound produced during muscle metabolism, primarily from the metabolism of phosphocreatine in muscles. After entering the bloodstream, it is filtered by the glomeruli of the kidneys and ultimately excreted in urine. Serum creatinine is a rough estimate of muscle mass and is positively correlated with it. When muscle damage worsens in IIM patients, serum creatinine levels decrease. This indicates that CIRP can, to some extent, reflect the degree of muscle damage in IIM patients, and its level changes can reflect muscle damage and repair in IIM patients.

[0028] In different subgroup analyses, this invention found that serum CIRP levels in the myositis group were significantly higher than those in the healthy control group. p <0.05, suggesting that serum CIRP may better reflect the association with muscle injury-related inflammation, a finding we also observed in other subgroup analyses. In both the rash and non-rash groups, serum CIRP in the non-rash group was significantly higher than in the healthy control group ( p <0.05. Serum CIRP levels were higher in both the ILD group and the non-ILD group than in the healthy control group ( p <0.05), but there was no statistically significant difference between the two groups. In the correlation analysis between CIRP and laboratory indicators, CIRP was positively correlated with muscle injury markers such as AST, LDH, CK, and CKMB, but not with KL-6. This suggests its mediating role in muscle injury.

[0029] This invention found that the levels of TREM-1 and TLR4 were significantly increased in the anti-RO-52 antibody positive group ( p <0.05), suggesting that the antibody may exacerbate systemic inflammation through TREM-1 and TLR4. We can reasonably speculate that the anti-RO-52 antibody may participate in the pathogenesis of IIM through interaction with TREM-1 and / or TLR4, thereby exhibiting more severe IIM-related complications.

[0030] Example 6: Validation of the diagnostic value of serum CIRP, TREM-1, and TLR4 expression levels for IIM 1. Subject screening: The subjects were 47 newly diagnosed and relapsed IIM patients who were hospitalized at Tianjin Medical University General Hospital from February 2023 to February 2025. These patients were assigned to the IIM2 group and further divided into 12 ASS patients and 7 MDA5 patients based on their clinical manifestations and myositis-specific antibodies. + DM patients, 18 MDA5 - The study included DM patients and 10 myositis patients, including PM, IMNM, and IBM patients. The inclusion criteria were the same as in Example 1.

[0031] 2. The main reagents and experimental methods are the same as in Example 1. 3. Experimental Results This invention systematically collects clinical data and laboratory test results from patients with intraepithelial neoplasia (IIM), specifically covering medical records, ALT, AST, LDH, CK, CKMB, ESR, CRP, etc. Rheumatologists performed standardized physical assessments on enrolled patients, focusing on the presence of characteristic skin rashes, including sunspots, Gottron's sign, V-sign, and shawl sign. All laboratory test results were obtained from our hospital's laboratory and rheumatology / immunology department.

[0032] (1) The expression levels of serum CIRP, TREM-1, and TLR4 in the IIM2 group and the healthy control group were determined using the same experimental methods as in Example 2. The results are shown in Table 8. Figure 12 As shown.

[0033] Table 8. Serum CIRP, TREM-1, and TLR4 expression levels in the IIM2 group and the healthy control group. Note: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001 (2) Comparison of serum CIRP, TREM-1, and TLR4 levels between each subgroup of IIM2 group and the healthy control group. The experimental method was the same as in Example 2, and the results are shown in Table 9. Figure 13 As shown.

[0034] Table 9 ASS Group, MDA5 + DM group, MDA5 - Serum CIRP, TREM-1, and TLR4 levels in the DM group, myositis group, and healthy control group Note: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; a indicates comparison with the control group, p<0.05; b indicates comparison with ASS, p<0.05; c indicates comparison with MDA5 + For comparison with DM, p < 0.05; d indicates comparison with MDA5-DM, p < 0.05. (3) The predictive value of serum CIRP, TREM-1, and TLR4 expression levels for the diagnosis of IIM was determined using the same experimental methods as in Example 5, and the results are shown in Table 10. Figure 14 As shown.

[0035] Table 10. Predictive value of serum CIRP, TREM-1, and TLR4 expression levels for the diagnosis of IIM. The results show that CIRP, TREM-1, and TLR4 are all highly expressed in the serum of IIM patients, and may be involved in the pathogenesis of the disease.

[0036] (4) Changes in serum CIRP, TREM-1, and TLR4 levels before and after IIM treatment in the IIM group To investigate the changes in CIRP, TREM-1, and TLR4 levels before and after treatment, we included 5 patients with intraepithelial neoplasia (IIM). These 5 newly diagnosed IIM patients were followed up after treatment with a combination of hormones and immunosuppressants, and their condition was assessed as stable. Samples were then resampled for testing. Results showed that serum CIRP, TREM-1, and TLR4 levels in IIM patients significantly decreased after treatment. (See attached figures.) Figure 15 As shown.

[0037] (5) Compare the experimental results with those in the publicly available database. Significantly elevated CIRBP was found in IIM patients in GSE142807 and GSE128470. Figure 16 ); Significantly elevated TLR4 levels were found in IIM patients in GSE142807 and GSE128314. Figure 17 ) Data comparison and verification have shown that IIM patients have higher levels of CIRP, TREM-1, and TLR4 compared to healthy controls, and these levels decrease after treatment. This has also been confirmed in publicly available databases, providing preliminary evidence that these levels are involved in the pathogenesis of IIM. Furthermore, serum CIRP, TREM-1, and TLR4 have certain diagnostic and predictive value in IIM.

[0038] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those skilled in the art can make various changes and modifications without departing from the essence and scope of the present invention. Therefore, all equivalent technical solutions also fall within the protection scope of the present invention.

[0039] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. A combination of biomarkers for idiopathic inflammatory myopathy, characterized in that, Including the following serum biomarkers: Cold-induced RNA-binding protein, myeloid trigger receptor-1, and Toll-like receptor 4.

2. The use of the biomarker combination or its detection reagent according to claim 1 in the preparation of products for diagnosing or identifying idiopathic inflammatory myopathy, characterized in that, The detection reagent is used to detect the level of the combination of biomarkers in the sample to be tested.

3. A diagnostic kit for diagnosing or differentiating idiopathic inflammatory myopathy, characterized in that, The test kit includes reagents for detecting the levels of the combination of biomarkers as described in claim 1 in a sample to be tested.

4. The use of the biomarker combination or its detection reagent according to claim 1 in the preparation of products for evaluating the activity or treatment efficacy of idiopathic inflammatory myopathy, characterized in that, The detection reagent is used to detect the level of the combination of biomarkers in the sample to be tested.

5. A diagnostic kit for assessing disease activity or treatment efficacy in idiopathic inflammatory myopathy, characterized in that, The test kit includes reagents for detecting the levels of the combination of biomarkers as described in claim 1 in a sample to be tested.

6. An evaluation system for monitoring idiopathic inflammatory myopathy, characterized in that, It includes a detection module, a data analysis module, and an evaluation module: The detection module is used to detect the level of the combination of biomarkers as described in claim 1 in the sample to be tested, and to obtain the detection results; The data analysis module is used to analyze and calculate the detection results of the detection module to obtain the idiopathic inflammatory myopathy score; The assessment module is used to identify idiopathic inflammatory myopathy based on the idiopathic inflammatory myopathy score from the data analysis module.

7. An evaluation system for monitoring disease activity or treatment efficacy in idiopathic inflammatory myopathy, characterized in that, It includes a detection module, a data analysis module, and an evaluation module: The detection module is used to detect the level of the combination of biomarkers as described in claim 1 in the first sample to be tested, and to obtain a first detection result; The detection module is used to detect the level of the combination of biomarkers as described in claim 1 in the second sample to be tested, and to obtain a second detection result; The data analysis module is used to analyze and calculate the changes in the first and second detection results of the detection module to obtain the activity or treatment effect score of idiopathic inflammatory myopathy. The assessment module is used to evaluate the activity or treatment effect of idiopathic inflammatory myopathy based on the activity or treatment effect score of idiopathic inflammatory myopathy from the data analysis module.

8. The use of the biomarker combination or its detection reagent as described in claim 1 in the preparation of products for screening high-risk populations for idiopathic inflammatory myopathy, characterized in that: The expression levels of cold-induced RNA binding protein, myeloid trigger receptor-1, and Toll-like receptor 4 in the serum of the subjects were detected and compared with preset thresholds.

9. A diagnostic kit for screening high-risk individuals for idiopathic inflammatory myopathy, characterized in that, The test kit includes reagents for detecting the levels of the combination of biomarkers as described in claim 1 in a sample to be tested.

Images