Application of VSIG4 as a biomarker in assessing the risk of progression of pulmonary fibrosis

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of VSIG4 as a biomarker in evaluation of risk of lung fibrosis progression. The application first discovers and verifies that VSIG4 is significantly highly expressed in ILD patients with active inflammatory response, and the high expression is closely related to a signal of continuous progression of the disease by integrating peripheral blood exosome proteomics data and lung tissue single-cell transcriptome data. Based on the discovery, the application provides a kit and a detection method for detecting a VSIG4 protein level in peripheral blood. Data show that the PPF patients can be effectively identified by detecting the content or level of the exosome protein VSIG4 in peripheral blood, and the method has high sensitivity and specificity. The application provides a new non-invasive solution for early risk prediction and disease progression monitoring of lung fibrosis, and has important clinical value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of VSIG4 as a biomarker in assessing the risk of pulmonary fibrosis progression. Background Technology

[0002] Interstitial lung disease (ILD) is a group of diffuse lung diseases characterized by inflammation and interstitial fibrosis of the alveolar units. Due to varying etiologies, the pathological manifestations, treatment strategies, and prognoses of ILD differ. Numerous clinical studies have found that some ILD patients eventually develop pulmonary fibrosis during the disease process, and even after treatment with the underlying cause and conventional therapies (such as glucocorticoids and immunosuppressants), the pulmonary fibrosis continues to progress. These diseases are termed progressive fibrosing ILD (PF-ILD). Currently, idiopathic pulmonary fibrosis (IPF) is generally recognized as a typical chronic, continuously progressive fibrotic ILD phenotype, while the progressive worsening of fibrosis in ILD other than IPF is termed progressive pulmonary fibrosis (PFF).

[0003] Early identification of ILD patients at high risk of fibrosis progression is a core challenge in clinical management. The natural course of idiopathic pulmonary fibrosis and other progressive fibrotic ILDs exhibits significant heterogeneity; some patients have stable conditions, while others experience rapid progression, leading to irreversible loss of lung function or even death. Current evidence suggests that early intervention may slow disease progression, but the key lies in identifying high-risk individuals before significant, irreversible damage to the lung parenchyma occurs. Currently, clinical practice primarily relies on high-resolution CT (HRCT) and pulmonary function tests (PFT), such as forced vital capacity (FVC) and carbon monoxide diffusing capacity (DLCO). However, these traditional methods have significant limitations, restricting their value in early warning. While HRCT can visually visualize lung structural changes, it suffers from radiation exposure, high cost, and limited equipment accessibility. More importantly, HRCT's sensitivity is insufficient to capture early, microscopic pathophysiological changes, often only detecting clear abnormalities when significant fibrosis has already occurred in the lung parenchyma. While pulmonary function tests such as FVC and DLCO are non-invasive, their sensitivity is insufficient; significant declines in these tests often lag behind pathological changes and are easily affected by patient compliance and comorbidities (such as emphysema). Furthermore, normal pulmonary function test results do not rule out the presence of ILD. In summary, existing physiological and imaging measurements have inherent limitations in assessing disease progression and severity, necessitating the development of more sensitive early biomarkers.

[0004] Some researchers have conducted studies on biomarkers for ILD. For example, CN118064575A discloses a microRNA molecular marker, hsa-miR-221-5p, which has been found to serve as a molecular marker for the early diagnosis of rheumatoid arthritis complicated with interstitial lung disease, and can be applied to assess the severity and prognosis of rheumatoid arthritis complicated with interstitial lung disease. Another example is patent CN118501453A, which discloses that exosomal calpain CAPN2 can be used as a biomarker for the auxiliary diagnosis of interstitial lung disease, and that the combination of exosomal KL-6, SP-B, and CAPN2 protein detection can be used for the auxiliary diagnosis of interstitial lung disease.

[0005] Therefore, finding more sensitive and specific biomarkers is of great significance for the early diagnosis and treatment of interstitial lung disease. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide the use of V-set immunoglobulin domain-containing protein 4 (VSIG4) as a biomarker in the preparation of products for diagnosing interstitial lung disease and / or assessing the risk of pulmonary fibrosis progression in patients with interstitial lung disease.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] Application of VSIG4 as a biomarker in the development of products for diagnosing interstitial lung disease and / or assessing the risk of pulmonary fibrosis progression in patients with interstitial lung disease.

[0009] Preferably, the VSIG4 has the ID number Q9Y279 in the UniProt database.

[0010] Preferably, the interstitial lung disease includes progressive pulmonary fibrosis.

[0011] Preferably, the test sample for the product is a peripheral blood sample, which includes one or more of serum, plasma, and exosomes isolated from serum or plasma.

[0012] Preferably, the product includes one or more of the following: reagent kit, chip, antibody, probe, and primer.

[0013] Preferably, the antibody includes one or more of monoclonal antibodies, polyclonal antibodies, and nanobodies.

[0014] Preferably, the diagnosis includes differentiating ILD patients from healthy subjects; the assessment includes differentiating patients with interstitial lung disease who are at high risk of pulmonary fibrosis progression from low-risk patients.

[0015] A second objective of this invention is to provide a kit for diagnosing interstitial lung disease and / or assessing the risk of pulmonary fibrosis progression in patients with interstitial lung disease.

[0016] To achieve the above objectives, the present invention adopts the following technical solution:

[0017] A kit for diagnosing interstitial lung disease and / or assessing the risk of pulmonary fibrosis progression in patients with interstitial lung disease, the kit comprising a reagent for detecting VSIG4 protein levels in peripheral blood samples.

[0018] This kit diagnoses interstitial lung disease or assesses the risk of pulmonary fibrosis progression in patients with interstitial lung disease (ILD) by detecting VSIG4 protein levels in peripheral blood (including exosomes).

[0019] Preferably, the kit includes a VSIG4 capture antibody and a VSIG4 detection antibody.

[0020] Preferably, the kit includes a sample processing solution, a solid-phase carrier coated with VSIG4 capture antibody, a VSIG4 detection antibody labeled with a signal-generating molecule, and / or a VSIG4 recombinant protein standard.

[0021] Preferably, the signal-generating molecule includes one or more of enzymes and chemiluminescent substances.

[0022] Preferably, the signal-generating molecule is an acridine ester coupled with NHS.

[0023] Preferably, the solid-phase support includes one or more of magnetic beads and ELISA plates; the sample processing solution includes the following components by weight percentage: TRIS 0.7%~1.1%, EDTA-2Na 0.02%~0.08%, NaCl 0.7%~1.1%, BSA 1%~5%, CHAPS 0.5%~2%, Tween-80 0.02%~0.08%, trehalose 1%~5%, ProClin 300 0.05%~0.2%, with the remainder being water, and the pH being 7~8.

[0024] Preferably, the sample processing solution comprises the following components by weight percentage: TRIS 0.9%, EDTA-2Na 0.05%, NaCl 0.9%, BSA 2%, CHAPS 1%, Tween-80 0.5%, trehalose 2%, ProClin 300 0.1%, with the remainder being water, pH 7.5.

[0025] Preferably, the kit further includes a reaction substrate; more preferably, the reaction substrate includes an AE pre-activation solution and an activation solution.

[0026] The third objective of this invention is to provide a method for detecting VSIG4 content in peripheral blood samples.

[0027] To achieve the above objectives, the present invention adopts the following technical solution:

[0028] A method for detecting VSIG4 levels in peripheral blood samples, wherein the method is not intended for disease diagnosis or treatment, and the method includes the following steps:

[0029] (1) Obtain the peripheral blood sample to be tested;

[0030] (2) Using the aforementioned kit, the content or level of VSIG4 protein in the sample was detected by chemiluminescent immunoassay (CLIA) or enzyme-linked immunosorbent assay (ELISA).

[0031] Preferably, the detection method specifically includes:

[0032] (1) Sample pretreatment: Mix the sample to be tested with the sample treatment solution and react at 37°C for 5 min to obtain the pretreated sample;

[0033] (2) Add the magnetic bead coating working solution to the pretreated sample obtained in step (1), mix well and incubate at 37°C for 5 min; then separate by magnetic separation, wash and remove the supernatant to obtain the magnetic bead-antigen complex.

[0034] (3) Add the label-detection antibody complex to the magnetic bead-antigen complex obtained in step (2), mix well and incubate at 37°C for 5 min; then separate by magnetic separation, wash and remove the supernatant to obtain the magnetic bead-antigen-detection antibody complex.

[0035] (4) Add the reaction substrate to the magnetic bead-antigen-detection antibody complex obtained in step (3), mix thoroughly, and measure the maximum luminescence intensity; fit a standard curve based on the luminescence intensity detected by the standard, and calculate the VSIG4 content in the sample to be tested through the standard curve.

[0036] Preferably, the marker-detection antibody complex is prepared using the following method:

[0037] 1) Add the VSIG4 detection antibody to the labeling buffer, then add the acridine ester conjugated with NHS, and mix well;

[0038] 2) Add a quencher to the reaction solution obtained in step 1). After the reaction is complete, remove unreacted substances by desalting column method to obtain acridinium ester marker for VSIG4 detection antibody;

[0039] 3) Dilute the acridine ester label of the VSIG4 detection antibody with acridine ester label diluent to obtain the working solution of acridine ester label.

[0040] Preferably, in step 1), the labeling buffer is an aqueous solution containing 0.1 mol / L sodium bicarbonate, with a pH of 8.5.

[0041] Preferably, in step 1), the concentration of the acridine ester coupled with NHS is 10 mg / mL.

[0042] Preferably, in step 1), the final concentration of the luminescent material is 0.5 mg / mL.

[0043] Preferably, in step 1), the mixing reaction is carried out for 30-90 minutes, more preferably 60 minutes.

[0044] Preferably, in step 2), the quenching agent is an aqueous solution containing 10% lysine.

[0045] Preferably, in step 2), after adding the quencher, the reaction is carried out at room temperature for 20-60 minutes, preferably 30 minutes.

[0046] Preferably, the luminescent marker obtained in step 2) is diluted to a concentration of 200 μg / mL using a marker preservation solution and stored at 2-8°C.

[0047] Preferably, in step 3), the acridine ester label diluent comprises the following components: MES 1%, NaCl 0.9%, BSA 2%, Tween-20 0.1%, NLS 0.2%, SB3-14 0.2%, bovine γ-globulin 0.05%, Proclin 300 0.1%, and the remainder is water, pH 6.0.

[0048] Preferably, in step 3), the final concentration of the obtained acridine ester labeled working solution is 0.4 μg / mL.

[0049] The fourth objective of this invention is to provide a reagent for detecting VSIG4 levels and / or the aforementioned kit for use in the preparation of products for diagnosing interstitial lung disease and / or assessing the risk of pulmonary fibrosis progression in patients with interstitial lung disease.

[0050] To achieve the above objectives, the present invention adopts the following technical solution:

[0051] The use of reagents for detecting VSIG4 levels and / or the aforementioned kits in the preparation of products for diagnosing interstitial lung disease and / or assessing the risk of progression of pulmonary fibrosis in patients with interstitial lung disease.

[0052] The beneficial effects of this invention are as follows:

[0053] 1. This invention confirms that the level of VSIG4 in peripheral blood has the potential to noninvasively reflect the activity of pro-fibrotic cell subsets in the lungs. By detecting the level of VSIG4 protein in peripheral blood, patients with interstitial lung disease at high risk of fibrosis progression can be identified at an early stage. This helps to identify high-risk individuals before significant and irreversible damage to the lung parenchyma occurs, thereby enabling early intervention and treatment. This invention provides a new non-invasive solution for early risk prediction and disease progression monitoring of pulmonary fibrosis, and has significant clinical value.

[0054] 2. This invention is the first to discover and verify that VSIG4 is significantly overexpressed in patients with interstitial lung disease and has an active inflammatory response, and that its overexpression is closely related to signals of continued disease progression. This discovery provides a new biomarker for the early diagnosis and monitoring of pulmonary fibrosis, and can effectively address the following problems in existing pulmonary fibrosis risk assessment techniques: 1) lack of non-invasive, early, and highly sensitive detection methods; 2) existing blood biomarkers are difficult to accurately reflect the pathological processes of pulmonary heterogeneity (such as inflammation-mediated pro-fibrotic activity).

[0055] 3. This invention provides a kit for assessing the risk of pulmonary fibrosis. The kit can detect the content or level of VSIG4 protein in a sample by chemiluminescent immunoassay or enzyme-linked immunosorbent assay. It is simple to operate, highly sensitive, and suitable for routine testing in clinical laboratories. It has broad applicability and promotional value. Attached Figure Description

[0056] Figures 1-2 This diagram illustrates the process of analyzing differentially expressed genes in SFTPB+SCGB3A2+ alveolar epithelial cells from single-cell sequencing results, along with the GO analysis results of these differentially expressed genes. Figure 1 A flowchart illustrating the process of analyzing differentially expressed genes in SFTPB+SCGB3A2+ alveolar epithelial cells from single-cell sequencing results; Figure 2 The image shows the results of the differential gene GO analysis.

[0057] Figures 3-5 This image shows the results of cell communication analysis between SFTPB+SCGB3A2+ alveolar epithelial cells and other cell types. Figure 3 This shows the overall result; Figure 4 and Figure 5 The figure shows the results of the detection of the TOP15 signaling pathways in which SFTPB+SCGB3A2+ alveolar epithelial cells communicate with macrophages as signal transmitters.

[0058] Figure 6 The figure shows the results of cross-analysis of exosomal proteins highly expressed in peripheral blood exosomes of ILD patients and single-cell sequencing results, highlighting the markers that are specifically highly expressed in alveolar epithelial cells and macrophages of SFTPB+SCGB3A2+.

[0059] Figure 7 This figure shows the results of VSIG4 expression level detection in the ILD group and healthy control (HC) group in the peripheral blood exosome proteomic profile.

[0060] Figure 8 The figure shows the results of using chemiluminescence assay to detect the difference in serum VSIG4 expression between the ILD and HC groups.

[0061] Figure 9 This is a standard curve graph.

[0062] Figure 10 The image shows the difference in VSIG4 expression in peripheral blood between non-PPF and PPF patients and the corresponding ROC curves. In the image, AB represents the measured VSIG4 protein levels in peripheral blood of the non-PPF group and the PPF group (A) and the corresponding ROC curve (B); CD represents the measured KL-6 protein levels in the non-PPF group and the PPF group (C) and the corresponding ROC curve (D). Detailed Implementation

[0063] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0064] Exosomes are nanoscale (30-150 nm) vesicles secreted by cells, carrying information molecules such as proteins, lipids, mRNA, and microRNAs (miRNAs) from their source cells. They play a crucial role in intercellular communication and the regulation of the disease microenvironment. Their stable presence and ease of detection in body fluids (such as blood, sputum, and bronchoalveolar lavage fluid, BALF) provide a novel perspective for non-invasive disease diagnosis. Theoretically, exosome cargo molecules can dynamically reflect specific pathological processes in the lungs (such as epithelial-mesenchymal transition, fibroblast activation, and extracellular matrix deposition), thus providing earlier warnings of fibrosis progression risk than macroscopic imaging or overall lung function tests. In this embodiment of the invention, by detecting peripheral blood exosome VSIG4, interstitial lung disease with a high risk of fibrosis progression can be non-invasively identified.

[0065] Example 1

[0066] 1. A kit for detecting peripheral blood protein VSIG4, mainly comprising the following components:

[0067] (1) VSIG4 antibody-coated magnetic bead suspension: magnetic microspheres labeled with VSIG4 specific monoclonal antibody, diluted with magnetic bead coating diluent to form magnetic bead coating working solution.

[0068] (2) Luminescent labeling antibody reagent: Acridine ester (AE) labeled VSIG4 detection antibody is diluted with acridine ester labeling diluent to form acridine ester labeling working solution.

[0069] (3) Standards: Solutions of VSIG4 recombinant protein of known concentration (purchased from Huamei Biotechnology, Cat: CSB-MP896869HU) were prepared by gradient dilution and used to establish a standard curve.

[0070] 2. The method for detecting peripheral blood protein VSIG4 using the aforementioned kit includes the following steps:

[0071] (1) Preparation of magnetic bead coating material

[0072] 1) Place the magnetic beads on the blood mixing device for more than 10 minutes until the magnetic beads are completely and evenly dispersed.

[0073] 2) Prepare one centrifuge tube, transfer 10mg of magnetic beads with a pipette, remove the supernatant by magnetic separation, and add 1mL of magnetic bead activation buffer to the centrifuge tube to make the magnetic bead concentration 10mg / mL.

[0074] 3) Add 25 μL of EDC solution (20 mg / mL) and 25 μL of NHS solution (40 mg / mL) to the above magnetic bead solution in sequence, react at room temperature for 60 min, and then remove the supernatant by magnetic separation.

[0075] 4) Resuspend the magnetic beads in 1 mL of magnetic bead coupling buffer, then add 200 μg of the VSIG4 capture antibody to be coated (purchased from Sinocare, Cat: 12163-R005), and incubate at room temperature for 2 h.

[0076] 5) Add 100 μL of blocking agent (PBS buffer containing 5% BSA) to the above 1 mL magnetic bead coupling buffer and incubate at room temperature for 1 h.

[0077] 6) Place the above solution on a magnetic separator and remove the supernatant by magnetic separation. Repeat this process 3 times.

[0078] 7) Place the above magnetic bead solution on a touch-sensitive ultrasonic cell disruptor at 10% power.

[0079] 8) Place the ultrasonicated magnetic bead solution on a magnetic separator and remove the supernatant by magnetic separation.

[0080] 9) Dilute the capture antibody-coated magnetic beads with magnetic bead coating diluent to obtain the working solution of the magnetic bead coating. The concentration of the working solution after dilution is 0.8 mg / mL. The main components of the magnetic bead coating diluent are: TRIS 0.1%, NaCl 0.9%, BSA 3%, Tween-20 0.1%, sodium azide 0.1%, and the remainder is water.

[0081] (2) Preparation of marker-detection antibody complex

[0082] 1) Add 200 μg of VSIG4 detection antibody (purchased from Sinocare, Cat: 12163-T24) to a pre-prepared 500 μL labeling buffer (an aqueous solution containing 0.1 mol / L sodium bicarbonate, pH 8.5), and then add 25 μL of NHS-conjugated acridine ester (concentration 10 mg / mL) to make the final concentration of the luminescent material 0.5 mg / mL.

[0083] 2) Place the mixed liquid from step 1) on a constant temperature mixer and react for 60 minutes.

[0084] 3) Add 100 μL of quencher (an aqueous solution containing 10% lysine) to the reaction solution obtained in step 2), react at room temperature for 30 min, and then remove unreacted substances by desalting column method; dilute the obtained luminescent label to a concentration of 200 μg / mL with label preservation solution and store at 2-8℃.

[0085] 4) Preparation of acridine ester labeling working solution: The acridine ester label of the VSIG4 detection antibody was diluted with acridine ester labeling diluent (MES 1%, NaCl 0.9%, BSA 2%, Tween-20 0.1%, NLS 0.2%, SB3-14 0.2%, bovine γ-globulin 0.05%, Proclin 300 0.1%, the remainder being water, pH 6.0) to obtain an acridine ester labeling working solution with a concentration of 0.4 μg / mL.

[0086] (3) Sample preprocessing steps

[0087] Sample pretreatment: Mix 10 μL of sample with 40 μL of sample processing solution, and then react at 37 °C for 5 min. The sample processing solution contains the following raw materials by weight percentage: TRIS 0.9%, EDTA-2Na 0.05%, NaCl 0.9%, BSA 2%, CHAPS 1%, Tween-80 0.5%, trehalose 2%, ProClin 300 0.1%, and the remainder is water, pH 7.5.

[0088] (4) Detection and calculation of VSIG4 content in the sample to be tested

[0089] 1) Add 50 μL of the magnetic bead coating working solution obtained in step (1) to the sample after pretreatment in step (3), mix well and incubate at 37°C for 5 min; then perform magnetic separation, wash to remove unbound substances and remove the supernatant to obtain the magnetic bead-antigen complex.

[0090] 2) Add 50 μL of the label-detection antibody complex obtained in step (2) to the magnetic bead-antigen complex obtained in step 1), mix well and incubate at 37°C for 5 min; then perform magnetic separation, wash to remove unbound substances and remove the supernatant to obtain the magnetic bead-antigen-detection antibody complex.

[0091] 3) Add the reaction substrate (100 μL AE pre-excitation solution and 100 μL excitation solution) to the magnetic bead-antigen-detection antibody complex obtained in step 2), mix thoroughly, and measure the maximum luminescence intensity. Based on the luminescence intensity detected by the standard, fit a standard curve, and calculate the VSIG4 content in the sample using the standard curve. The standard curve is shown below. Figure 9 As shown.

[0092] Example 2

[0093] This invention identified SFTPB in 107,847 cells through single-cell RNA sequencing data analysis of healthy controls (n=10) and ILD patients (n=10). + Two subsets with different phenotypes were identified: SFN+KRT19+ and SFTPB+SCGB3A2+. SFTPB+SFN+KRT19+ cells were present in both healthy and ILD samples, with highly expressed genes enriched in repair-related functions such as cell proliferation, migration, and adhesion. SFTPB+SCGB3A2+ cells were only found in ILD samples, with upregulated genes significantly enriched in processes such as immune response regulation, antigen presentation, and MHC class II complex assembly, suggesting that this subset may be involved in the immunopathological process of ILD. (See details...) Figures 1-2 .

[0094] This invention further elucidates the mechanism by which SFTPB+SCGB3A2+ cells interact with myeloid immune cells through multiple signaling pathways using cellphoneDB-based cell communication analysis. Cell communication network analysis shows that SFTPB+SCGB3A2+ cells primarily send signals to macrophages and dendritic cells. The APP-CD74 pathway is highly active in both paracrine and autocrine environments, exhibiting particularly high communication strength in the autocrine circuit, suggesting its potential role in maintaining the activated state of this epithelial cell subset within the pulmonary fibrosis microenvironment. (See details...) Figures 3-5 .

[0095] Through pseudo-time-series trajectory analysis, this invention clarifies the dynamic changes of these signaling pathways during the development of pulmonary fibrosis. In the early stage, the ICAM1 adhesion pathway and the SFTPD-ADGRE5 pathway are dominant; in the middle stage, the CD44-TYROBP and PLAU-PLAUR pathways are enhanced; and in the late stage, most pathways, such as APP-CD74, SAA1-FPR2, complement C3 pathway, and CLU stress signaling, are continuously enhanced and reach peak values. This indicates that in the pathological process of immune inflammation in pulmonary fibrosis, the role of alveolar epithelial cells may gradually shift from early direct immune regulation to later more complex immune activation mediated by continuous and enhanced myeloid cell interactions.

[0096] Based on the above findings, this invention further cross-analyzes peripheral blood exosome proteomics data with lung tissue single-cell transcriptomics data, providing SFTPB+SCGB3A2+ cells and their related signaling pathways as potential new targets for diagnosing and intervening in the progression of pulmonary fibrosis. This lays an important foundation for developing diagnostic strategies and therapeutic methods targeting the immune-inflammatory microenvironment of pulmonary fibrosis. Among the newly discovered biomarkers derived from SFTPB+SCGB3A2+ cells are IVL and SDC1; the significantly different newly discovered biomarkers derived from M2 macrophages include TGOLN2 and VSIG4, etc., for details. Figure 6 In the peripheral blood exosome proteomic analysis, the expression levels of VSIG4 in the ILD group and the healthy control (HC) group were as follows: Figure 7 As shown.

[0097] Example 3

[0098] Based on the experimental results of Example 2, the present invention conducted a preliminary clinical evaluation of VSIG4 (V-set immunoglobulin domain-containing protein 4), which showed the most significant differences. Specifically, serum samples from 10 healthy controls and 36 ILD patients were collected and then detected using a fully automated chemiluminescence immunoassay analyzer (Shine i2910) with the VSIG4 detection kit and method described in Example 1.

[0099] Test results as follows Figure 8 As shown in Table 1, VSIG4 protein was significantly overexpressed in the peripheral blood of ILD patients (p=0.0011). Based on this, a retrospective analysis of the clinical information of nine selected patients with high VSIG4 expression was performed. The results showed that almost all of these patients had specific autoantibodies, and the main diagnoses were IPAF or CTD-ILD. Functional imaging (FAPI-PET) in several of these patients confirmed that fibrosis was in an active phase, rather than a quiescent phase, suggesting that peripheral blood VSIG4 testing may be useful in monitoring the progression of pulmonary fibrosis. Elevated VSIG4 levels indicate an active autoimmune or immune-inflammatory background behind ILD. As an immune checkpoint molecule, VSIG4 upregulation may be a feedback attempt to suppress an overactive autoimmune response. In autoimmune interstitial lung disease, persistent stimulation by autoantigens leads to widespread activation of pulmonary macrophages. These activated macrophages highly express VSIG4, attempting to suppress T cell responses that may exacerbate tissue damage. Therefore, elevated VSIG4 is a key node and biomarker in the combined effect of immune dysregulation and fibrosis progression.

[0100] Table 1. Clinical information and analysis results of patients with high VSIG4 expression in the ILD group.

[0101]

[0102] Example 4. Comparison of detection results of peripheral blood exosome protein VSIG4 in non-PPF and PPF samples.

[0103] Based on the detection results of the aforementioned embodiments, this embodiment assesses the risk of pulmonary fibrosis progression by detecting the VSIG4 protein level in peripheral blood samples (including serum, plasma, or exosomes isolated therefrom) of the subjects. Specifically, serum samples from 15 patients with PPF and serum samples from 13 patients with non-PPF were collected. Exosomes were extracted from these samples using a kit method, and then the extracted exosomes were detected using a fully automated chemiluminescence immunoassay analyzer (Shine i2910) with the VSIG4 detection kit and method described in Example 1. At the same time, commercially available salivary glycan antigen KL-6 reagent (registration certificate number: National Medical Device Registration Certificate 20152400741) was purchased and the above samples were tested simultaneously. The inclusion criteria for PPF were: patients with known or unknown etiology of ILD other than IPF who have radiological evidence of pulmonary fibrosis and meet at least two of the following three criteria in the past year. (1) Worsening of respiratory symptoms. (2) There is physiological evidence of disease progression, including: ① a decrease of ≥5% in the absolute value of FVC%pred; ② a decrease of ≥10% in the absolute value of DLCO%pred after hemoglobin correction.

[0104] The comparison results are as follows Figure 10 As shown, the concentration of peripheral blood exosome protein VSIG4 in PPF patients was significantly higher than that in non-PPF patients (P<0.0001). In contrast, the KL-6 assay kit showed no significant difference in distinguishing between PPF and non-PPF patients. ROC curves were plotted based on the above data. The AUC of the VSIG4 assay was 0.923, with a sensitivity of 92.31% and a specificity of 86.67%. In contrast, the AUC of the KL-6 assay kit was only 0.741, with a sensitivity of only 63.64% and a specificity of 84.62%. These results indicate that the peripheral blood exosome protein VSIG4 assay kit can effectively identify PPF patients with high sensitivity and specificity.

Claims

1. The use of VSIG4 as a biomarker in the preparation of products for the diagnosis of interstitial lung disease and / or the assessment of the risk of pulmonary fibrosis progression in patients with interstitial lung disease.

2. The application according to claim 1, characterized in that, The interstitial lung disease includes progressive pulmonary fibrosis.

3. The application according to claim 1, characterized in that, The test sample for the product is a peripheral blood sample, which includes one or more of serum, plasma, and exosomes isolated from serum or plasma.

4. The application according to claim 1, characterized in that, The products include any one or more of the following: reagent kits, chips, antibodies, probes, and primers.

5. The application according to claim 1, characterized in that, The diagnosis includes differentiating ILD patients from healthy subjects; the assessment includes differentiating patients with interstitial lung disease at high risk of pulmonary fibrosis progression from low-risk patients.

6. A kit for diagnosing interstitial lung disease and / or assessing the risk of pulmonary fibrosis progression in patients with interstitial lung disease, characterized in that, The kit includes reagents for detecting VSIG4 protein levels in peripheral blood samples.

7. The reagent kit according to claim 6, characterized in that, The kit includes a sample processing solution, a solid-phase carrier coated with VSIG4 capture antibody, a VSIG4 detection antibody labeled with a signal-generating molecule, and / or a VSIG4 recombinant protein standard.

8. The reagent kit according to claim 7, characterized in that, The solid-phase support includes one or more of magnetic beads and ELISA plates; the sample processing solution includes the following components by weight percentage: TRIS 0.7%~1.1%, EDTA-2Na 0.02%~0.08%, NaCl 0.7%~1.1%, BSA 1%~5%, CHAPS 0.5%~2%, Tween-80 0.02%~0.08%, trehalose 1%~5%, ProClin 300 0.05%~0.2%, with the remainder being water, and a pH of 7~8.

9. A method for detecting VSIG4 levels in peripheral blood samples, characterized in that, The method is not intended for disease diagnosis or treatment, and includes the following steps: (1) Obtain the peripheral blood sample to be tested; (2) Using the kit described in any one of claims 6 to 8, the content or level of VSIG4 protein in the sample is detected by chemiluminescent immunoassay or enzyme-linked immunosorbent assay.

10. The use of reagents for detecting VSIG4 levels and / or the kits according to any one of claims 6 to 8 in the preparation of products for diagnosing interstitial lung disease and / or assessing the risk of progression of pulmonary fibrosis in patients with interstitial lung disease.

Images