Application of soluble myeloid cell triggered receptor-1 as biomarker and kit

Abstract

The invention relates to in vitro detection of biomarkers associated with drug-induced liver injury; belongs to the technical field of immunokits. The technical scheme provided by the invention is as follows: the soluble myeloid cell triggered receptor-1 is applied to preparation of a product for diagnosing the drug-induced liver injury as a biomarker. The invention discloses and proves that sTREM1 in serum or plasma can be used as a specific biomarker for diagnosing drug-induced liver injury (DILI), especially drug-induced liver fatty degeneration injury for the first time. The serum / plasma sTREM1 can detect abnormal rise in the extremely early stage (such as 7 days after medication) of the drug-induced liver injury, which is about 5-7 days earlier than that of the traditional marker ALT / AST, so that a key early intervention time window is provided for clinicians, and the liver injury can be prevented from further worsening.

Description

Technical Field

[0001] This invention relates to a biomarker for in vitro detection, particularly for the in vitro detection of biomarkers associated with drug-induced liver injury; it belongs to the field of immunoassay kit technology. Background Technology

[0002] Drug-induced liver injury (DILI) is a common and serious adverse drug reaction in clinical practice, and its accurate and timely diagnosis is crucial for patient management and drug safety assessment. Currently, the diagnosis of DILI mainly relies on traditional liver function biochemical indicators, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as imaging examinations and liver tissue biopsy (the gold standard). However, these methods have significant limitations: indicators such as ALT and AST usually only rise significantly when liver injury progresses to a more severe stage, resulting in insufficient sensitivity and making it difficult to provide early warning; while liver tissue biopsy is accurate, it is invasive, complex to perform, expensive, and carries the risk of complications, making it unsuitable as a routine screening or dynamic monitoring method.

[0003] In recent years, although some studies have explored potential novel biomarkers for DILI, such as PPARα and specific miRNAs, these biomarkers are often interfered with by metabolic diseases (such as non-alcoholic fatty liver disease NAFLD) or lack sufficient consistency and reproducibility across different studies, limiting their clinical application value. Therefore, there is an urgent need in this field to develop DILI biomarkers and their in vitro detection products that are highly sensitive, highly specific, non-invasive, and capable of early diagnosis. Summary of the Invention

[0004] This invention aims to address the problems of insufficient sensitivity (difficulty in early detection) and invasiveness of some existing DILI diagnostic technologies, and provides an in vitro detection and disease identification solution based on the biomarker soluble myeloid cell trigger receptor-1 (sTREM1).

[0005] Firstly, the present invention provides an application, the specific technical solution of which is as follows:

[0006] Application of soluble myeloid cell triggering receptor-1 as a biomarker in the preparation of products for diagnosing drug-induced liver injury.

[0007] Furthermore, the application of soluble myeloid triggering receptor-1 as a biomarker in the preparation of products for the early diagnosis of drug-induced liver injury with hepatic steatosis.

[0008] Furthermore, the application of soluble myeloid trigger receptor-1 as a biomarker in the preparation of products for diagnosing drug-induced liver injury induced by triazole antifungals.

[0009] Secondly, the present invention provides a reagent kit, the technical solution of which is as follows:

[0010] A kit for detecting soluble myeloid cell triggering receptor-1, the kit comprising an antibody capable of immunobinding to the soluble myeloid cell triggering receptor-1.

[0011] Furthermore, the kit is selected from one of the following: enzyme-linked immunosorbent assay kit, chemiluminescent immunoassay kit, and lateral flow immunochromatographic assay kit.

[0012] Furthermore, the kit also includes reagents for detecting additional biomarkers selected from at least one of alanine aminotransferase, triglycerides, and interleukin-1β.

[0013] Thirdly, the present invention provides a method for detecting the above-mentioned markers, the technical solution of which is as follows:

[0014] A method for detecting soluble myeloid cell trigger receptor-1 in blood for non-diagnostic purposes includes the following steps:

[0015] a) Obtain serum or plasma samples from the subject;

[0016] b) The level of soluble myeloid cell triggering receptor-1 in the sample was detected using a kit containing an antibody that can bind to the soluble myeloid cell triggering receptor-1.

[0017] Furthermore, the method for detecting the level of soluble myeloid cell trigger receptor-1 in the sample is selected from one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or lateral flow immunochromatography.

[0018] Furthermore, the following steps are also included:

[0019] c) Detect the levels of additional biomarkers in the sample;

[0020] The additional biomarker is selected from at least one of alanine aminotransferase, triglycerides, and interleukin-1β.

[0021] This invention is the first to reveal and demonstrate that serum or plasma sTREM1 can serve as a specific biomarker for diagnosing drug-induced liver injury (DILI), particularly drug-induced steatosis. While the role of sTREM1 (the soluble form of TREM1) in infectious diseases has been studied, this invention, through rigorous clinical cohort studies and animal model experiments, is the first to discover and demonstrate that serum / plasma sTREM1 levels specifically and significantly increase in patients and model animals during drug-induced liver injury (especially drug-induced steatosis), and this increase occurs earlier than significant changes in traditional liver function indicators (such as ALT and AST). Furthermore, serum / plasma sTREM1 levels are significantly positively correlated with the degree of steatosis in liver tissue, the degree of inflammatory infiltration, and the activation status of macrophages.

[0022] Specifically, in a mouse model of drug-induced liver injury induced by voriconazole, the mRNA and protein expression levels of TREM1 in liver tissue were significantly upregulated (see [link to relevant documentation]). Figure 1 Serum sTREM1 levels continued to rise with increasing hepatic steatosis and inflammation (see [link to article]). Figure 1 ).

[0023] At the end of the first week of treatment (day 7), serum sTREM1 levels in mice with liver damage showed an abnormally high increase, while ALT levels had not yet shown a statistically significant change (see...). Figure 2 This indicates that changes in sTREM1 occurred earlier than those in ALT.

[0024] Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) of serum sTREM1 in diagnosing DILI at this stage was 0.85, significantly better than the AUC of ALT (0.57) during the same period (see [link to ROC curve analysis]). Figure 2 These results demonstrate that serum / plasma sTREM1 can serve not only as an early diagnostic marker for DILI, but also has the potential to assess the severity of liver injury.

[0025] Based on the above findings, this invention provides a specific technical solution for preparing diagnostic DILI products using sTREM1 detection. The product contains reagents capable of specifically recognizing and binding to human or animal-derived sTREM1 protein, used for quantitative or qualitative detection of sTREM1 levels in serum or plasma samples of subjects.

[0026] Typical forms of diagnostic products include:

[0027] The ELISA kit contains a solid-phase carrier (e.g., a microplate) coated with anti-sTREM1 capture antibody, a series of concentrations of sTREM1 standards, an anti-sTREM1 detection antibody (or secondary antibody) labeled with a reporter molecule (e.g., horseradish peroxidase HRP), appropriate wash buffer, chromogenic substrate solution, and stop solution. The concentration of sTREM1 in the sample is calculated by measuring the optical density (OD) value after the sample reaction and comparing it to a standard curve.

[0028] Chemiluminescent immunoassay (CLIA) kits or detection devices: These devices use chemiluminescent signals for detection, similar in principle to ELISA, but the antibodies used to detect the chemiluminescent substances (such as acridinium ester, luminol, etc.) have higher sensitivity.

[0029] Lateral flow immunochromatography (LFIA) test strips: Includes a sample pad, a conjugation pad (pre-coated with labeled anti-sTREM1 antibody), a nitrocellulose membrane (coated with the T-line anti-sTREM1 antibody for the test line and the C-line antibody for the control line), and an absorbent pad. Suitable for point-of-care testing (POCT), providing qualitative (positive / negative) or semi-quantitative results.

[0030] In summary, the present invention has the following beneficial effects:

[0031] 1. Early diagnostic advantage: Serum / plasma sTREM1 can be detected abnormally elevated in the very early stages of drug-induced liver injury (such as voriconazole-induced injury) (e.g., 7 days after drug administration), approximately 5-7 days earlier than the traditional biomarkers ALT / AST (see...). Figure 2 This provides clinicians with a crucial window for early intervention, helping to prevent further deterioration of liver damage;

[0032] 2. Non-invasive: The test is based on serum or plasma samples and does not require invasive liver biopsy, which greatly reduces patient suffering, lowers medical risks and costs, and is easier to promote and apply to routine screening and dynamic monitoring.

[0033] 3. High sensitivity and specificity: In the clinical cohort of voriconazole-associated drug-induced liver injury (DILI), serum sTREM1 alone achieved an AUC of 0.877, significantly superior to the AUC of early ALT (0.57), and was closely related to pathological changes in liver tissue (steatodegeneration, inflammation), demonstrating good specificity for drug-induced liver injury (especially hepatic steatosis). (See [link to relevant documentation]) Figure 14 );

[0034] 4. Clear Mechanism, Combining Diagnostic and Mechanism Indication: This invention not only establishes sTREM1 as a diagnostic biomarker, but also elucidates the core role of the TREM1 / sTREM1 pathway in the development and progression of drug-induced hepatic steatosis through functional experiments (knockout, inhibition, and overexpression) (see...). Figures 9-13 This provides a solid theoretical basis for the application of this biomarker. Changes in its level can reflect the degree of intrahepatic inflammation and lipid metabolism disorders.

[0035] 5. Wide range of applications: In clinical applications, it can be used in clinical laboratories of hospitals at all levels as an auxiliary diagnostic, early warning and severity assessment tool for DILI (especially drug-induced fatty liver);

[0036] 6. Drug development and safety evaluation: In the preclinical (animal experiment) and clinical trial stages of new drug development, monitoring changes in sTREM1 levels after administration can serve as a sensitive indicator for assessing the potential hepatotoxicity of candidate drugs. This helps to provide early warning of liver injury risks, optimize the drug safety evaluation process, improve the detection rate of hepatotoxicity, and reduce the risks of new drug development.

[0037] 7. Treatment monitoring: Dynamic monitoring of sTREM1 levels can be used to assess the effectiveness of liver protection treatment and disease recovery.

[0038] 8. Multi-mode detection is possible: Diagnostic products based on sTREM1 can be flexibly developed into highly sensitive laboratory tests (ELISA, CLIA) or convenient point-of-care rapid tests (LFIA) to meet different needs. Attached Figure Description

[0039] Figure 1 Expression characteristics of TREM1 / sTREM1 and its correlation with liver injury markers in a voriconazole-induced liver injury mouse model: Figure 1 A in the figure represents the relative expression level of TREM1 mRNA in liver tissue. Figure 1 B in the figure represents the expression level of TREM1 protein in liver tissue. Figure 1 C in the text represents the dynamic changes in serum sTREM1 concentration. Figure 1 D: Pearson correlation analysis of serum sTREM1 with liver weight, ALT, AST, TG, and TC;

[0040] Figure 2 A comparison of the diagnostic efficacy of sTREM1 and ALT in the early stages of liver injury: Figure 2 A: HE staining pathological images of liver tissue from mice in the control group and liver injury group; Figure 2 B: Statistical analysis of serum ALT in mice on day 7 after drug administration; Figure 2 C: Statistical analysis of serum sTREM1 levels in mice on day 7 after drug administration; Figure 2D in the figure: Analysis of the differential diagnostic efficacy of ALT and sTREM1 for liver injury;

[0041] Figure 3 Transcriptomic-proteomic analysis and pathway enrichment of liver tissue: Figure 3 A: Volcano diagram of differentially expressed TREM1 genes; Figure 3 B: Differential gene enrichment in the KEGG pathway; Figure 3 D: Differential protein enrichment in the KEGG pathway;

[0042] Figure 4 Immunofluorescence staining image of TREM1 in liver tissue (green, CD86; red, TREM1; blue, DAPI).

[0043] Figure 5 Flow cytometry analysis of TREM1+ cell proportion in liver tissue (control group).

[0044] Figure 6 Flow cytometry analysis of TREM1+ cell proportion in liver tissue (experimental group).

[0045] Figure 7 This is a graph showing the statistical results of TREM1+ cells;

[0046] Figure 8 Figure showing the correlation analysis results between the proportion of TREM1+ cells and serum sTREM1 levels;

[0047] Figure 9 Effects of TREM1 knockout on hepatic lipid accumulation: Figure 9 A in the text refers to TREM1 knockdown of TG / TC levels in HepG2 cells cultured in conditioned medium for macrophages; Figure 9 B in the text refers to the process of constructing macrophage-specific TREM1 knockout mice. Figure 9 C in the text refers to Oil Red O staining of liver tissue from knockout mice. Figure 9 D in the text represents the serum sTREM1 level in knockout mice.

[0048] Figure 10 The effect of pretreatment with the TREM1-specific inhibitor LR12 on improving hepatic lipid accumulation: Figure 10 A: TG / TC levels in hepatocytes after LR12 pretreatment of macrophages; Figure 10 Image of Oil Red O staining results of liver tissue from mice pretreated with B:LR12;

[0049] Figure 11 Lipid accumulation in hepatocytes following TREM1 overexpression / activation;

[0050] Figure 12The procedure for constructing macrophage-specific TREM1 overexpression mice and the serum sTREM1 content of macrophage-specific TREM1 overexpression mouse models;

[0051] Figure 13 Image showing Oil Red O staining results of liver tissue from a mouse model with macrophage-specific TREM1 overexpression;

[0052] Figure 14 To validate the diagnostic efficacy of sTREM1 in a clinical cohort: Figure 14 A: Comparison of serum sTREM1 levels between DILI patients and the control group; Figure 14 A heatmap showing the correlation between B: sTREM1 and liver injury markers; Figure 14 ROC curve of C: sTREM1 for diagnosing DILI (AUC=0.877).

[0053] Figure 15 ROC curve of sTREM1 combined with diagnostic model: AUC value of sTREM1 combined with ALT, TG, IL-1β multiparameter model. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0055] Example 1: Construction of a voriconazole-induced liver injury mouse model and validation of sTREM1 diagnostic efficacy

[0056] 1. Model Construction and Grouping:

[0057] 1.1 Animal source and ethics: SPF-grade 4-week-old male C57BL / 6 mice (purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd.) were used. The experiment was approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (batch number: 2024952).

[0058] 1.2 Group Design: Four-week-old male C57BL / 6 mice were selected.

[0059] a) DILI group: Voriconazole (40 mg / kg, dissolved in 100 μL 0.9% saline) was administered by gavage daily for 3 weeks;

[0060] b) Control group: The same volume of physiological saline was administered by gavage (100 μL / animal) daily for 3 weeks.

[0061] 1.3 Sample Collection: Serum and liver tissue were collected at weeks 1, 2, and 3 after drug administration.

[0062] a) Serum collection: Blood was collected from the orbital venous plexus and the serum was separated by centrifugation;

[0063] b) Liver processing: Some tissue was fixed in 4% paraformaldehyde for pathological sections (HE / Oil Red O staining / immunofluorescence staining); some was flash-frozen in liquid nitrogen and stored at -80°C for further testing and analysis.

[0064] 2. Core Indicator Testing

[0065] 2.1 Biochemical indicators of liver injury: Serum ALT, AST, TG, and TC levels were detected using commercially available reagent kits;

[0066] 2.2 sTREM1 quantification: Serum sTREM1 was detected by ELISA (Reagent kit: Human / Mouse sTREM1 ELISA Kit, R&D Systems).

[0067] 2.3 Organizational Analysis:

[0068] a) RT-qPCR / Western Blot detection of TREM1 expression in liver tissue (method described in Example 3);

[0069] b) Pathological section staining to assess lipid deposition and degree of inflammation.

[0070] 3. Output Results

[0071] 3.1 Expression characteristics:

[0072] a) TREM1 mRNA and protein expression in the liver injury group were >2-fold higher than in the control group. Figure 1 In A and B), serum sTREM1 levels were synchronously elevated ( Figure 1 (C in the middle)

[0073] b) sTREM1 was significantly positively correlated with liver weight, ALT, AST, TG, and TC (p<0.05). Figure 1 (D in the middle)

[0074] 3.2 Advantages of early diagnosis: 1 week after administration, sTREM1 was significantly elevated (p<0.05), while ALT had not yet changed. Figure 2 (A, B, C in the table); The ROC curve shows that sTREM1 has an AUC of 0.85, which is significantly better than ALT (AUC = 0.57). Figure 2 (D in the middle).

[0075] Example 2: Validation of the mechanism by which TREM1 regulates hepatic lipid metabolism

[0076] 1. Transcriptomics and proteomics analysis

[0077] 1.1 Total RNA and protein were extracted from the livers of mice in the control group and the liver injury group (methods are described in Example 3). After passing quality control, the samples were sent to Novogene for transcriptomics and proteomics sequencing analysis.

[0078] 1.2 Differential Gene Screening: Hypothesis testing algorithms were used to identify differentially expressed genes for screening analysis, which revealed a significant increase in the expression of the key differentially expressed protein TREM1. Figure 3 (A in the middle)

[0079] 1.3 Analysis of biological functional pathways: Combined transcriptomic and proteomic KEGG enrichment results indicated significant activation of lipid metabolism-related pathways ( Figure 3 (B and D in the text).

[0080] 2. Functional localization of TREM1 in macrophages

[0081] a.1 Immunofluorescence / flow cytometry detection of major TREM1 expression localization in liver tissue:

[0082] a) The proportion of TREM1 expression in CD86+ cells was significantly increased in the liver injury group (p<0.05). Figures 4-7 );

[0083] b) The expression of TREM1 protein in liver tissue was strongly correlated with serum sTREM1 levels (r=0.83, p<0.01). Figure 7 ).

[0084] 3. Gene and pharmacological intervention

[0085] 3.1 Cell Model:

[0086] a) Pretreatment of hepatocytes with siRNA knockdown of TREM1 or LR12 (5 μM) significantly decreased TG / TC levels (p<0.01). Figure 9 A in Figure 10 (A in the middle)

[0087] b) After transfection with TREM1 overexpression plasmid, lipid accumulation was significantly increased (p<0.05). Figure 11 ).

[0088] 3.2 Animal Models:

[0089] a) DILI modeling was performed on TREM1 knockout mice (Trem1flox / flox / Lyz2-Cre+ / -) and wild-type control mice (Trem1flox / flox / Lyz2-Cre- / -): Hepatic lipid deposition was significantly reduced, and serum sTREM1 levels decreased (p<0.01). Figure 9 (B, C, D in the text)

[0090] b) LR12 inhibitor (5 mg / kg, intraperitoneal injection) for DILI modeling: significantly reduced hepatic lipid accumulation ( Figure 10 (B in the middle)

[0091] c) DILI model was established in TREM1-overexpressing mice (AAV9-F4 / 80-m-Trem1 tail vein injection): liver lipid deposition and serum sTREM1 were significantly increased (p<0.05). Figure 12 and Figure 13 ).

[0092] Example 3: Diagnostic validation of sTREM1 in patients with clinical liver injury

[0093] 1. Queue Design

[0094] 1.1 Patient source: This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine (approval number: 2021234), and all patients signed informed consent forms.

[0095] 1.2 Grouping criteria (based on EASL guidelines for drug-induced liver injury):

[0096] a) DILI group (n=49): Patients who met any of the following criteria during voriconazole treatment

[0097] i. ALT ≥ 5 times the upper limit of normal value (ULN);

[0098] ii. ALT ≥ 3 times ULN and T-Bil > 2 times ULN;

[0099] iii. ALP ≥ 2 times ULN and γ-GT > ULN, while excluding known skeletal diseases that cause elevated ALP.

[0100] b) Control group (n=30): Patients who did not meet the above criteria were included in the control group. The two groups of patients were matched in baseline characteristics, and their baseline liver function indicators before medication were consistent, that is, both were within the normal reference range or slightly abnormal but the difference between the groups was not statistically significant.

[0101] 2. Detection Method

[0102] 2.1 Serum sTREM1: ELISA quantification (procedure as in Example 1);

[0103] 2.2 Traditional indicators: ALT, AST, γ-GT, TG, CRP, etc. (biochemical analysis using commercial kits).

[0104] 3. Output results (diagnostic thresholds and effectiveness)

[0105] 3.1 Serum sTREM1 levels in the liver injury group were 3.2 times higher than those in the control group (p<0.001). Figure 14 (A in the middle)

[0106] 3.2 Diagnostic Criterion: ROC curve analysis showed that the diagnostic value (recognition ability) of sTREM1 for liver injury was AUC=0.877 (95% CI: 0.812-0.956). Figure 14 (B) The area under the curve (AUC) was statistically significantly different from the reference line (p<0.001). The optimal diagnostic threshold, calculated using the Youden index, was 159.6 pg / mL; a sTREM1 concentration > 159.6 pg / mL was considered positive. At the optimal cutoff value, the positive predictive value was 86.7%, and the negative predictive value was 71.4%.

[0107] 3.3 sTREM1 was significantly positively correlated with ALT, AST, TG, CRP, etc. (p<0.05). Figure 14 (C in the middle).

[0108] Example 4: Construction and Application of the sTREM1 Combined Diagnostic Model

[0109] 1. Multi-parameter model design

[0110] Key indicators: sTREM1 + ALT + TG + IL-1β (sTREM1 assay kit: R&D System; IL-1β ELISA kit: Raybiotech).

[0111] 2. Diagnostic efficacy

[0112] A multi-parameter model was constructed by combining sTREM1 with inflammation and lipid metabolism-related indicators to improve the differential diagnostic efficacy for liver injury. The combined model had an AUC of 0.998 in distinguishing DILI. Figure 15 ).

[0113] Example 5: Standardized Procedures for Key Experimental Methods

[0114] 1. RNA extraction (animal liver tissue)

[0115] Tissue was ground in liquid nitrogen → TRIzol lysis → chloroform separation → isopropanol precipitation → washing with 75% ethanol → RNA dissolved in RNase-free water (A260 / A280 = 1.8–2.0).

[0116] 2. Protein extraction (animal liver tissue)

[0117] The liver tissue was ground into powder in liquid nitrogen, and then lysed on ice with RIPA lysis buffer containing protease inhibitors. After centrifugation, the supernatant was collected to obtain the total protein.

[0118] 3. Western Blot

[0119] Protein lysis (RIPA buffer) → BCA quantification → 30 μg sample loading → SDS-PAGE electrophoresis → PVDF transfer → 5% skim milk blocking → anti-TREM1 primary antibody (1:1000, Abcam ab241332) incubation overnight at 4℃ → HRP secondary antibody (1:5000) incubation at room temperature for 1 h → ECL development.

[0120] 4. ELISA

[0121] Standard serial dilution → 100 μL / well added → Incubate at 37℃ for 2 h → Wash plate 4 times → Add detection antibody (1:1000) → TMB color development → Measure OD value at 450 nm → Calculate concentration using four-parameter fitting.

[0122] Key Reagents and Source Declaration:

[0123] The reagents and equipment mentioned in the above embodiments are all commercially available, including but not limited to:

[0124] TREM1 inhibitor LR12 (sequence: LQEEDAGEYGCM, purity >95%, purchased from MedChemExpress).

[0125] AAV9 lentiviral vector (purchased from Hanheng Biotechnology (Shanghai) Co., Ltd., HBAAV2 / 9-F4 / 80-m-Trem1);

[0126] Flow cytometry antibodies: anti-mouse CD86-PE (BioLegend), anti-TREM1-APC (R&D).

[0127] sTREM1 ELISA kit (R&D Systems, catalog number: KE10080 / mouse, DTRM10C / human);

[0128] IL-1β ELISA kit (Raybiotech, catalog number: ELM-IL1b).

[0129] Statistical methods:

[0130] Data are expressed as mean ± standard deviation. The Mann-Whitney U test (non-normal distribution) or Student's t test (normal distribution) was used to compare the DILI group with the control group. One-way ANOVA was used for comparison of multiple groups. p < 0.05 was considered to be significant (GraphPad Prism 9).

[0131] The above embodiments are merely preferred embodiments of the present invention, intended to illustrate the core innovation of the present invention (the application of sTREM1 as a diagnostic biomarker and combined model), and are not intended to limit the present invention. Any equivalent transformations or substitutions made based on the technical solutions of the present invention are within the scope of protection of the present invention.

[0132] In summary, the applicant collected blood samples from patients clinically using voriconazole. Using the European Association for the Study of the Liver (EASL) Clinical Practice Guidelines for drug-induced liver injury (DILI), the patients' blood samples were divided into a liver injury group and a control group. Non-targeted metabolomics analysis was performed, and differences in liver function indicators such as ALT, AST, and blood lipid levels were analyzed using clinical information. The study identified and validated the pathological characteristics of lipid metabolism disorders in patients with liver injury. Simultaneously, by establishing parallel animal models of voriconazole-induced liver injury with the same pathological features of hepatic steatosis, transcriptomic analysis of liver tissue revealed significant enrichment of lipid accumulation-related pathways. Oil Red O staining was used to measure the degree of lipid accumulation, sTREM1, and TREM1 levels in liver tissue in both the control and liver injury groups. By analyzing their correlation with clinical liver function indicators, a novel diagnostic biomarker, sTREM1, with specificity, sensitivity, non-invasiveness, and stability for DILI was proposed, providing a reference for the early detection and screening of DILI.

Claims

1. Application of soluble myeloid cell triggering receptor-1 as a biomarker in the preparation of products for diagnosing drug-induced liver injury.

2. Application of soluble myeloid cell triggering receptor-1 as a biomarker in the preparation of products for early diagnosis of drug-induced liver injury with hepatic steatosis.

3. Application of soluble myeloid cell triggering receptor-1 as a biomarker in the preparation of products for diagnosing drug-induced liver injury induced by triazole antifungals.

4. A kit for detecting soluble myeloid cell trigger receptor-1, characterized in that, The kit contains an antibody that can bind to the soluble myeloid cells to trigger receptor-1 immunity.

5. The reagent kit according to claim 4, characterized in that, The kit is selected from one of the following: enzyme-linked immunosorbent assay kit, chemiluminescent immunoassay kit, and lateral flow immunochromatographic kit.

6. The reagent kit according to claim 4, characterized in that, The kit also includes reagents for detecting additional biomarkers selected from at least one of alanine aminotransferase, triglycerides, and interleukin-1β.

7. A method for detecting soluble myeloid cell trigger receptor-1 in blood for non-diagnostic purposes, comprising the following steps: a) Obtain serum or plasma samples from the subject; b) The level of soluble myeloid cell triggering receptor-1 in the sample was detected using a kit containing an antibody that can bind to the soluble myeloid cell triggering receptor-1.

8. The method according to claim 7, characterized in that, The method for detecting the level of soluble myeloid cell trigger receptor-1 in the sample is selected from one of enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or lateral flow immunochromatography.

9. The method according to claim 7, characterized in that, It also includes the following steps: c) Detect the levels of additional biomarkers in the sample; The additional biomarker is selected from at least one of alanine aminotransferase, triglycerides, and interleukin-1β.

10. The method according to claim 9, characterized in that, The additional biomarker is selected from at least one of alanine aminotransferase, triglycerides, and interleukin-1β.

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