A set of sepsis-specific biomarkers and uses thereof

Abstract

The application discloses a set of sepsis-specific biomarkers and application thereof, and belongs to the medical detection field.The marker is composed of the combination of alpha 1-microglobulin / Bikunin precursor protein and one or more of antithrombin III protein, apolipoprotein A1 or apolipoprotein H.Through TMT proteomics screening of serum of clinical sepsis patients, seven core proteins, APOA1, APOH, AMBP, SERPINC1, F2, FGG and FGB, are screened out.Further screening of the seven proteins in a sepsis cell model and clinical sepsis patients obtains four more important proteins, APOA1, APOH, AMBP and SERPINC1.The four proteins are verified again in a sepsis mouse animal experiment and serum of clinical sepsis patients on a large scale, and the potential of the four core proteins as serum biomarkers for early diagnosis of sepsis is determined.

Description

Technical Field

[0001] This invention belongs to the field of medical testing, specifically relating to specific biomarkers for sepsis and their applications. Background Technology

[0002] Sepsis is a life-threatening syndrome of organ dysfunction caused by a dysregulated host response to infection. As the leading cause of death in intensive care unit patients, it has become a major public health problem worldwide. The disease is characterized by its rapid onset, rapid progression, and numerous complications, often leading to multiple organ dysfunction and severely threatening patients' lives. Given the high incidence and mortality rates of sepsis, its prevention, early diagnosis, and effective treatment have become major challenges in the global public health field. According to the clinical practice guidelines published in the 2021 Surviving Sepsis Campaign (SSC), the prognosis of patients with sepsis is closely related to the timing of diagnosis and treatment. The guidelines emphasize that early identification, timely diagnosis, and standardized treatment can significantly improve patient clinical outcomes and reduce the 28-day mortality rate, highlighting early diagnosis of sepsis as crucial for improving survival rates.

[0003] The high similarity of clinical symptoms in infectious diseases poses significant challenges to the diagnosis and prognosis of sepsis. Many patients remain undiagnosed due to a lack of clear clinical evidence, delaying treatment. Currently, the clinical diagnosis of sepsis primarily relies on the SOFA score to assess organ dysfunction in the context of infection, which is one of the core tools for clinical diagnosis. However, this method is essentially an organ function assessment, not an infection screening tool, and therefore unsuitable for early infections that do not cause obvious organ dysfunction. Furthermore, the so-called systemic inflammatory response syndrome criteria are often relied upon clinically to screen for possible sepsis, but the patient population is heterogeneous. These criteria (hypothermia or hyperthermia, tachycardia, tachypnea, and abnormal white blood cell count) are present in most critically ill patients; however, they are not specific to infection and therefore limit their application in differentiating patients with sepsis from those with other inflammatory diseases. The key to successful treatment of sepsis lies in early diagnosis and rapid selection of an effective treatment plan. However, commonly used serum biomarkers such as C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), and interleukin-10 (IL-10) lack sufficient sensitivity and specificity, failing to fully and accurately reflect the true state of sepsis. This makes them unsuitable for early diagnosis of sepsis or septic shock, hindering clinical decision-making for patients with sepsis and impeding timely diagnosis and treatment, thus limiting patient management. The lack of clarity regarding serum biomarkers poses a significant challenge to the early diagnosis of sepsis in clinical practice; therefore, the search for novel serum biomarkers for sepsis is crucial. Currently, there are no reliable serum biomarkers in clinical practice that can accurately diagnose and differentiate sepsis in healthy individuals. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a set of specific biomarkers for the early diagnosis of sepsis and their applications.

[0005] The technical solution of this invention is as follows:

[0006] The application of reagents for detecting marker levels in the serum of subjects in the preparation of diagnostic agents or kits for diagnosing sepsis, wherein the markers consist of one or more of the following proteins: α1-microglobulin / Bikunin precursor protein (AMBP) and antithrombin III protein (SERPINC1), apolipoprotein A1 (APOA1), or apolipoprotein H (APOH).

[0007] Furthermore, the biomarker is composed of four proteins: antithrombin III protein, apolipoprotein A1, α1-microglobulin / Bikunin precursor protein, and apolipoprotein H.

[0008] Furthermore, the reagent is an ELISA detection reagent or a Western blot detection reagent.

[0009] A diagnostic kit for sepsis includes reagents for detecting marker levels, said marker being a combination of one or more of the following proteins: α1-microglobulin / Bikunin precursor protein (AMBP) and antithrombin III protein (SERPINC1), apolipoprotein A1 (APOA1), or apolipoprotein H (APOH).

[0010] Furthermore, the biomarker is composed of four proteins: antithrombin III protein, apolipoprotein A1, α1-microglobulin / Bikunin precursor protein, and apolipoprotein H.

[0011] Furthermore, the reagent is an ELISA detection reagent or a Western blot detection reagent.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] This invention screened seven core proteins in the TMT proteomics of serum from patients with clinical sepsis: APOA1, APOH, AMBP, SERPINC1, F2, FGG, and FGB. Further screening of these seven proteins in a sepsis macrophage model and clinical sepsis patients revealed four more important proteins: APOA1, APOH, AMBP, and SERPINC1. These four proteins were then validated in mouse experiments with sepsis and in a large-scale clinical sepsis trial, confirming their potential as serum biomarkers for early diagnosis of sepsis.

[0014] Experimental data show that the levels of the four core proteins APOA1, APOH, AMBP, and SERPINC1 screened in this invention are significantly lower in the serum of patients with sepsis compared to healthy individuals. Therefore, they can all be used as serum biomarkers for the early diagnosis of sepsis.

[0015] In particular, α1-microglobulin / Bikunin precursor protein has not yet been reported to be associated with sepsis, so it is used as a core biomarker; to improve accuracy, combining it with one or more of the other three proteins can further improve diagnostic accuracy. Attached Figure Description

[0016] Figure 1 mRNA expression levels of 7 core targets in THP-1 and RAW264.7 sepsis cell models (n=3); results are expressed as mean ± standard deviation; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0017] Figure 2 Protein levels of central nervous system targets in the culture supernatants of THP-1 and M1 sepsis cell models were detected by ELISA (n=3); all data are expressed as mean ± standard deviation; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0018] Figure 3 Protein levels of central nervous system targets in the serum of healthy subjects and patients with sepsis were detected by ELISA; all data are expressed as mean ± standard deviation, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns=NoSignificant.

[0019] Figure 4 The diagnostic capabilities of the core targets screened through ROC curve analysis.

[0020] Figure 5 Western blot analysis of SERPINC1, APOH, AMBP (α1-MG) and APOA1 protein levels in THP-1 cells (n=3); all data are expressed as mean ± standard deviation, **P<0.01.

[0021] Figure 6 Western blot analysis of SERPINC1, APOH, AMBP (α1-MG) and APOA1 protein levels in RAW264.7 cells (n=3); all data are expressed as mean ± standard deviation, *P<0.05.

[0022] Figure 7 Western blot was used to detect the serum protein levels of SERPINC1, APOH, AMBP (α1-MG), and APOA1 in the clinical pilot group (n=3).

[0023] Figure 8 Western blot analysis of serum SERPINC1, APOH, AMBP (α1-MG), and APOA1 protein levels in the clinical pilot group (n=3); all data are expressed as mean ± standard deviation, ****P<0.0001.

[0024] Figure 9 Core protein levels in mouse serum were detected by ELISA (n=6); all data are expressed as mean ± standard deviation. P <0.01, *** P <0.001, ns=No Significant.

[0025] Figure 10 Western blot analysis of core protein levels in mouse serum (n=3); all data are expressed as mean ± standard deviation. P <0.01, *** P <0.001, ns=No Significant. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from commercial channels.

[0027] Example 1: TMT proteomics analysis of serum from patients with sepsis

[0028] 1. Clinical screening criteria for patients with sepsis:

[0029] Sepsis is defined as an acute increase in a SOFA score ≥2 representing organ dysfunction, with a suspected or documented infection (microbiological evidence or clinical standard diagnosis of infection). Blood samples were collected from patients diagnosed with sepsis upon admission to the ICU. Exclusion criteria included (1) patients not aged between 18 and 80 years; (2) patients with infectious diseases, infection-acquired immunodeficiency syndrome, those who had received antimicrobial therapy before admission, tumors, severe chronic liver disease, chronic renal failure, immunosuppression, those who had recently taken drugs that affect coagulation function, those with coagulation dysfunction caused by other diseases, and those who had recently undergone organ transplantation; (3) patients who had been in the ICU for less than 24 hours or died within 24 hours; (4) patients with insufficient or incomplete clinical or laboratory data; and (5) pregnant and lactating women.

[0030] 2. Clinical sample collection:

[0031] For early screening of serum biomarkers, serum was collected from patients diagnosed with sepsis and admitted to the ICU within 24 hours before the start of treatment. The serum was then separated and stored to avoid biases caused by treatment factors such as antibiotic use. Serum from healthy individuals undergoing routine checkups at the hospital during the same period was selected as a control, and factors such as lipemia and jaundice were excluded.

[0032] First, albumin / IgG was removed from the sample. After vacuum lyophilization, SDS was added for lysis and reconstitution, and the supernatant containing total protein was extracted. Protein concentration was quantified using the BCA method. Equal volumes of total protein were analyzed using 12% SDS-PAGE gel chromatography to ensure parallelism of protein extraction and accuracy of quantification results. After trypsin digestion, peptide labeling, reverse chromatography separation, and column elution, mass spectrometry was performed to obtain a total ion chromatogram. After LC-MS / MS analysis, reliable proteins were screened using a database. Based on these reliable proteins, two standards were selected to calculate the differences between samples. The difference screening criteria were: Foldchange ≥ 2 or Foldchange ≤ 1 / 2 and p-value < 0.05. 169 differentially expressed proteins (DEPs) were identified. To further identify interactions between common target proteins, a protein-protein interaction (PPI) network of the 169 differentially expressed proteins was constructed using the STRING database and Cytoscape version 3.9.0. Proteins with a critical value > 0.9 (high-confidence interaction score) were used as the source of important PPIs for network visualization. Using the CytoNCA plugin, topological indices such as median centrality (BC), proximity centrality (CC), degree centrality (DC), and network centrality (NC) in PPI networks were calculated to preliminarily screen central targets of differentially expressed proteins in the proteomics of apolipoprotein A1 (APOA1), prothrombin (F2), antithrombin III (SERPINC1), fibrinogen γ chain (FGG), fibrinogen β chain (FGB), α1-microglobulin / Bikunin precursor protein (AMBP), and apolipoprotein H (APOH) in sepsis.

[0033] Example 2: Real-time quantitative polymerase chain reaction (qRT-PCR) to verify the gene expression of core proteins in a sepsis cell model.

[0034] THP-1 cell line (human monocytic leukemia cell line) was cultured and plated, then induced with 100 ng / mL PMA for 24 h to obtain M0 cells (resting macrophages). The culture medium was then discarded, the adherent cells were washed, and fresh complete culture medium was added. The cells were then induced with 100 ng / mL phorbol ester (PMA), 100 ng / mL lipopolysaccharide (LPS), and 20 ng / mL interferon-γ (IFN-γ) for 48 h to obtain M1 cells (classical macrophages). The supernatant and cell extracts were then collected for ELISA, WB, and RT-qPCR detection. RAW264.7 cell line (mouse monocytic macrophage leukemia cell line) was cultured and plated, then induced with 100 ng / mL LPS for 6 h. The supernatant and cell extracts were then collected for ELISA, WB, and RT-qPCR detection.

[0035] RT-qPCR results confirmed that LPS significantly increased the expression of inflammatory factor mRNA in THP-1 cells and RAW264.7 cells, and the cell morphology showed changes similar to M1 cells, indicating that the LPS-induced differentiation model of macrophages was successfully established.

[0036] Human monocytic leukemia cell line THP-1 was cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. Mouse monocytic macrophage leukemia cell line RAW264.7 was cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a 5% CO2 incubator. The two cell lines with good growth were cultured at 5 × 10⁶ cells / plate. 6 Cells were seeded in 6cm cell culture dishes. When the cell density reached 80%, cells were induced with PMA, LPS, and IFN-γ, respectively. Total RNA was then extracted from the cells. cDNA was synthesized using reverse transcription reagents, and real-time fluorescent PCR was performed using qPCRMix (No ROX) reagent. The expression levels of target genes were normalized to GAPDH. -DDCt Computational methods were used to assess mRNA expression.

[0037] The results are as follows Figure 1 As shown: RT-qPCR was used to detect the mRNA expression of seven core targets in a sepsis cell model. In THP-1 cells, the mRNA expression trends of the five targets AMBP, SREPINC1, F2, FGG, and FGB were consistent with previous analyses, with AMBP, SREPINC1, and F2 showing a decreasing trend, and FGG and FGB showing an increasing trend. However, APOA1 and APOH showed a decreasing trend in previous proteomics analysis, while their mRNA expression showed an increasing trend. In RAW264.7 cells, the mRNA expression trends of the six targets APOH, AMBP, SREPINC1, F2, FGG, and FGB were consistent with previous analyses, with APOH, AMBP, SREPINC1, and F2 showing a decreasing trend, and FGG and FGB showing an increasing trend. However, APOA1 showed a decreasing trend in previous proteomics analysis, while its mRNA expression showed an increasing trend. The inconsistency between mRNA and protein changes may be related to post-transcriptional RNA regulation mechanisms, protein modification, or changes in the course of sepsis. Further quantitative detection of the core targets at the protein level is needed to verify these changes.

[0038] Example 3: Enzyme-linked immunosorbent assay (ELISA) and Western blot were used to verify the expression of the core protein.

[0039] (1) ELISA was used to verify the expression of core proteins from THP-1, RAW264.7 cell culture supernatants and clinical serum. Following the kit manufacturer's instructions, diluted samples were added to 96-well plates, followed by the addition of enzyme-labeled secondary antibody and incubation at 37°C for 60 min. TMB chromogenic buffer was added, and chromogenic incubation was terminated after half an hour in the dark. The absorbance at 450 nm was read using an ELISA reader within 5 minutes. The results showed that in THP-1 cells, the protein expression of the four targets APOA1, APOH, AMBP, and SREPINC1 was consistent with previous analysis results, showing a decreasing trend. However, the protein expression trends of F2, FGG, and FGB were inconsistent. Previous proteomics analysis showed a decreasing trend for F2, while ELISA showed an increasing trend; previous proteomics analysis showed an increasing trend for FGG and FGB, while ELISA showed a decreasing trend. Figure 2 ).

[0040] To further analyze the diagnostic ability of the selected central nervous system targets for sepsis, the expression of core target proteins in 8 patients with sepsis and 6 healthy volunteers was studied based on proteomics. Figure 3 The ROC curve of the target protein was plotted. Figure 4 Table 1 shows detailed information on the ROC curves of each target protein, including cut-off value, AUC value, sensitivity, specificity, and Youden index.

[0041] Table 1 ROC Curve Information Table

[0042] Predictor variables Cut-off value AUC (Area Under the Curve) Sensitivity Specificity Yoden Index APOA1 8.097 μg / mL 1.0000 100% 100% 1.000 APOH 339.2 ng / mL 0.8542 62.5% 100% 0.625 AMBP 32.13 mg / L 0.9792 83.33% 100% 0.833 SERPINC1 660.5 ng / mL 0.9792 83.33% 100% 0.833 F2 8.111 nmol / L 0.8750 66.67% 100% 0.667 FGG 7723 ng / mL 0.6250 75% 66.67% 0.417 FGB 51.65 ng / mL 0.6875 62.5% 83.33% 0.458

[0043] like Figure 4 The ROC curve analysis results show that the AUC values ​​of APOA1, APOH, AMBP, SREPINC1, and F2 are all greater than 0.8, indicating that they have certain diagnostic potential as biomarkers in distinguishing between patients with sepsis and healthy controls.

[0044] Based on the expression trends of the seven core targets in the THP-1 and RAW264.7 cell polarization models and their protein expression in the serum of clinical patients with sepsis, combined with the analysis results of ROC curves, APOA1, APOH, AMBP, and SERPINC1 have higher diagnostic value for sepsis. Therefore, these four proteins are selected as core biomarkers for subsequent experimental verification and mechanism research.

[0045] (2) After completing the cell culture procedure described above, harvest THP-1 and RAW264.7 cells and extract total protein. Measure the protein concentrations in cells and clinical serum, add loading buffer, and heat to denature. Use a 10% SDS-PAGE gel for Western blotting, separate proteins by electrophoresis at 80V for 100min, transfer to a PVDF membrane at 100V for 80min, block with 5% skim milk powder at room temperature for 1 hour, incubate with primary antibody overnight at 4℃, incubate with secondary antibody at room temperature for 2 hours, and then use enhanced chemiluminescence (ECL) reagent to detect protein bands to verify the expression of the core proteins screened above.

[0046] Experimental results showed that in the cell model simulating sepsis, the expression of APOA1, APOH, AMBP, and SERPINC1 proteins was decreased, and the differences were statistically significant. Figure 5 and Figure 6 ).

[0047] To validate the clinical reliability of the core biomarker, we recruited more clinical patients with sepsis, expanding the patient cohort, and measured the expression levels of the core biomarker.

[0048] The experimental results showed that, compared with the healthy group, the serum levels of APOA1, APOH, AMBP, and SERPINC1 in patients with sepsis were significantly decreased, with statistically significant differences. Figure 7 and Figure 8 This study confirmed that the four proteins APOA1, APOH, AMBP, and SERPINC1 are important participants in sepsis, ensuring their accuracy and reliability as biomarkers.

[0049] Example 4 verifies the expression of the core protein in sepsis-affected mice.

[0050] 1. Animals used to construct a sepsis disease model:

[0051] C57 mice, aged 6-8 weeks and weighing 20-23g, were purchased from Chengdu Dashuo Biotechnology Co., Ltd., Sichuan Province. Mice were divided into the following groups: Blank group (blank control), saline-only group (negative control), and LPS-only group (positive control), with 10 mice in each group. LPS was dissolved in saline, and a mouse model of LPS-induced sepsis was established by intraperitoneal injection of 10 mg / kg LPS. Mice in the LPS group were challenged with a single intraperitoneal injection of LPS, while the control group received an equal volume of saline. The blank group received no treatment. Twelve hours after intraperitoneal injection, all mice were anesthetized with sodium pentobarbital, and blood and organs were harvested. The effectiveness of the model was verified by serological indicators of liver and kidney function, qRT-PCR of inflammatory factors, and HE staining. The results showed that an early animal model of sepsis was successfully established.

[0052] After successfully establishing an animal model of early sepsis, the four core biomarkers previously identified in this study were validated in this model. The protein levels of APOA1, APOH, α1-MG, and SERPINC1 in the serum of mice in each group were detected using ELISA and Western blotting, respectively.

[0053] The results are as follows Figure 9 and Figure 10 As shown, compared with the Blank and Control groups, the levels of APOA1, APOH, α1-MG, and SERPINC1 proteins in the serum of mice in the LPS group were all decreased, and the differences were statistically significant. Simultaneously, Western blot (WB) experiments were performed on serum samples from three mice in each group. The results showed that the relative expression levels of these four proteins were also decreased in the LPS group mice, and the differences were statistically significant, consistent with the trend observed in the ELISA experiments.

Claims

1. The application of reagents for detecting the levels of biomarkers in the serum of subjects in the preparation of diagnostic agents or kits for diagnosing sepsis, wherein the biomarkers are composed of four proteins: antithrombin III protein, apolipoprotein A1, α1-microglobulin / Bikunin precursor protein, and apolipoprotein H. When the levels of all four proteins are significantly decreased, sepsis is determined.

2. The application according to claim 1, characterized in that, The reagents are ELISA or Western blot reagents.

Images