Sepsis biomarker FABP5 protein and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a sepsis biomarker FABP5 protein and application thereof. Serum samples of clinical non-sepsis patients and sepsis patients are collected, the content of FABP5 protein in the serum samples is detected, and it is found that the FABP5 protein in serum of the sepsis patients is remarkably increased and has a positive correlation relation with severity of sepsis. The FABP5 protein is used as a biomarker to predict the occurrence and clinical manifestation of sepsis, a more accurate diagnosis basis is provided, and powerful treatment decision support is provided for clinicians. In addition, detection of the FABP5 protein can help timely identify high-risk patients suffering from sepsis, so that support is provided for early intervention and treatment, and the death rate of sepsis is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a sepsis biomarker FABP5 protein and its application. Background Art

[0002] Sepsis is a life-threatening disease caused by the body's excessive response to infection and is a major global health challenge. Despite the progress of medical science, sepsis remains one of the main causes of death and disease burden worldwide and has a high incidence in intensive care units. Early diagnosis and timely intervention are crucial for improving the prognosis of patients and reducing sepsis-related mortality.

[0003] Sepsis-3.0 uses a Sequential Organ Failure Assessment (SOFA) score of ≥2 in infected patients as the diagnostic criterion for sepsis organ dysfunction. The indicators included in the SOFA score are PaO2 / FiO2 (respiratory system), platelet count (coagulation system), bilirubin (liver), mean arterial pressure and the dose of vasopressor used (cardiovascular system), Glasgow score (central nervous system), creatinine and urine output (kidney). Due to the excessive number of SOFA score indicators, the European Society of Intensive Care Medicine and the American College of Critical Care Medicine proposed the quick SOFA (qSOFA) score (altered mental status; systolic blood pressure ≤100 mmHg; respiratory rate ≥22 breaths / min). However, the latest Surviving Sepsis Campaign guidelines published in 2021 pointed out that the qSOFA has limited ability to predict adult septic shock and other screening indicators are needed in combination. Blood lactate level has been incorporated into the diagnostic criteria for septic shock. In addition, the diagnostic methods for sepsis rely not only on clinical signs and symptoms but also on the detection of inflammatory markers such as C-reactive protein and procalcitonin. However, these traditional biomarkers lack specificity and sensitivity and often lead to delayed or inaccurate diagnoses in the early stages of the disease. Therefore, there is an urgent need for more reliable and accurate biomarkers to help detect and prognose sepsis early.

[0004] Fatty acid binding proteins (FABPs) are cytoplasmic proteins highly expressed in mammalian tissues. With a molecular weight of approximately 15 kDa, their main function is to bind and transport fatty acids, regulating the influx, storage, and metabolism of fatty acids, thus participating in energy generation and the balance of fatty acid metabolism, and affecting the structure and function of cell membranes. In addition to fatty acid metabolism, FABPs also play important roles in signal transduction, gene expression regulation, and immune responses, especially in the regulation of inflammatory responses and cell proliferation. Currently, 9 subtypes of the FABPs family are known, including L-FABP (FABP1) - liver, I-FABP (FABP2) - intestine, H-FABP (FABP3) - heart, A-FABP (FABP4) - adipocyte, E-FABP (FABP5) - epidermis, IL-FABP (FABP6) - ileum, B-FABP (FABP7) - brain, M-FABP (FABP8) - myelin, and T-FABP (FABP9) - testis. FABPs are grouped and named according to the organ or tissue in which they were initially identified, isolated, and are most predominant, but their expression is not tissue-specific. Although the FABPs family is mainly involved in fatty acid transport and metabolism, recent studies have shown that the dysregulation of FABP proteins is associated with diseases such as obesity, cardiovascular diseases, cancer, and non-alcoholic fatty liver disease, and may be used as biomarkers for predicting diseases or targeted therapies. For example, a study by Northeastern University on September 25, 2023, showed that FABPs can be biomarkers for predicting dementia with Lewy bodies (DLB), where FABP3 is involved in the progression of DLB disease, FABP5 is involved in mitochondrial damage caused by brain inflammation, and FABP7 is involved in the degeneration of oligodendrocytes. H-FABP is clinically used as a biomarker for evaluating subclinical ischemia and predicting disease progression, and can also be combined with echocardiography to diagnose sepsis-induced cardiac insufficiency, and can also be used as a novel marker protein for acute myocardial injury. I-FABP can independently predict the 28-day mortality rate of sepsis patients. Urinary L-FABP can predict the progression of chronic glomerulonephritis and the occurrence of acute kidney injury. However, the potential use of FABP5 protein as a biomarker for predicting sepsis and assessing its severity has not been fully studied.

[0005] The present invention aims to fill this gap and proposes to use FABP5 protein to predict the occurrence and severity of sepsis. By analyzing the levels of FABP5 protein in the sera of sepsis patients and non-sepsis patients, and analyzing its predictive value through logistic regression analysis and receiver operating characteristic (ROC) curve analysis. The present invention aims to provide a new biomarker for the diagnosis and risk stratification of sepsis.

[0006] In summary, the development of accurate and sensitive sepsis biomarkers is crucial for improving patient prognosis and reducing the burden on the healthcare system. Utilizing the FABP5 protein as a sepsis prediction biomarker has great potential and represents a significant advancement in the field of critical care medicine. Summary of the Invention

[0007] The present invention first proposes using the FABP5 protein as a new biomarker for sepsis and explores its potential applications in the early diagnosis and severity assessment of sepsis. By detecting the level of FABP5 protein in the serum of sepsis patients and evaluating its clinical predictive value through univariate logistic regression analysis and ROC curves, the study shows that the FABP5 protein has significant diagnostic ability and can help identify high-risk patients at the early stage of sepsis. In addition, the validation results based on animal models further support the feasibility of the FABP5 protein as a sepsis biomarker, providing a new predictive tool and therapeutic target for the clinical management of sepsis. This discovery is expected to promote the improvement of early intervention measures for sepsis, enhance clinical treatment effects, and reduce the mortality rate of sepsis.

[0008] The technical solution adopted by the present invention is as follows:

[0009] In the first aspect, the present invention provides a biomarker for detecting sepsis, and the biomarker is the FABP5 protein, also known as the E-FABP protein, which belongs to one of the fatty acid binding proteins (FABPs).

[0010] Preferably, the degree of sepsis risk is determined by detecting the content of FABP5 protein in human serum. The human serum sample is selected from at least one of whole blood, serum, and plasma.

[0011] When the content of FABP5 protein in human serum ≥ 4.892 ng / mL, it indicates sepsis. The content of FABP5 protein in human serum is positively correlated with the severity of sepsis.

[0012] In the second aspect, the present invention provides the above biomarker FABP5 protein for detecting sepsis and its severity, including the following:

[0013] (1) Collect serum samples from clinical non-sepsis patients and sepsis patients and detect the concentration of FABP5 protein therein.

[0014] (2) Evaluate FABP5 through logistic regression analysis and ROC curves to verify its accuracy as a sepsis prediction biomarker.

[0015] (3) Further analyze the correlation between FABP5 protein and the severity of sepsis, SOFA score, Acute Physiology and Chronic Health Evaluation (APACHE II) score, and other clinical indicators, and provide more comprehensive clinical application value.

[0016] (4) Detect the content of FABP5 protein in the peripheral blood of different sepsis models at the animal level.

[0017] (5) Detect the effect of FABP5 protein on the progression of sepsis induced by cecal ligation and puncture (CLP) at the animal level.

[0018] In a third aspect, the present invention also provides the application of the biomarker FABP5 protein in the preparation of a kit for predicting the risk of sepsis. The kit is used to determine the content of FABP5 protein in a biological sample of a subject.

[0019] According to some embodiments of the present invention, the biomarker FABP5 protein exacerbates organ damage caused by sepsis by enhancing the inflammatory response or directly affecting the cell functions of the liver and kidneys.

[0020] According to some embodiments of the present invention, the biomarker FABP5 protein participates in multiple organ damage caused by sepsis through its specific molecular mechanism.

[0021] According to some embodiments of the present invention, the biomarker FABP5 protein significantly increases the levels of inflammatory factors TNF-α, IL-1β, and IL-6 by promoting the release of inflammatory factors.

[0022] In the present invention, the serum of sepsis patients is collected, and the FABP5 protein is used as a biomarker. It is found through detection that the increase in the level of FABP5 protein in the serum of sepsis patients is positively correlated with sepsis-related indicators such as SOFA score, APACHE II score, procalcitonin level, and lactate level. Through statistical analysis, it is found that the FABP5 protein as a biomarker can reasonably and accurately predict the severity of sepsis patients. The detection of FABP5 protein can help identify high-risk patients with sepsis in a timely manner, thereby providing support for early intervention and treatment and reducing the mortality rate of sepsis.

[0023] The beneficial effects of the present invention are:

[0024] Based on the above technical solutions, the present invention provides an innovative and reliable method for predicting the occurrence and severity of sepsis by using FABP5 protein as a biomarker, for sepsis risk prediction and sepsis prognosis assessment. This method can provide more accurate diagnostic basis for clinicians and effectively support early intervention and personalized treatment for patients. Through the correlation analysis with other clinical indicators related to sepsis, the relationship between FABP5 protein and these indicators can be deeply explored, which helps to further reveal the pathogenesis of sepsis and provide more information for clinical work. More importantly, FABP5 protein as a potential therapeutic target provides a new direction for the treatment of sepsis and opens up new technical means for treating sepsis. Based on the innovative application of FABP5 protein as a biomarker, the present invention not only helps to improve the early diagnosis of sepsis, but also provides new ideas for improving the clinical management of sepsis, optimizing the treatment plan for patients, and enhancing the clinical treatment effect. Therefore, the present invention has important clinical value in the clinical diagnosis and treatment of sepsis and is expected to be widely promoted in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below with reference to the accompanying drawings.

[0026] Figure 1 It is a graph of the content of FABP5 protein in the sera of non-sepsis patients and sepsis patients. Figure A is a graph of the content of FABP5 protein in the sera of non-sepsis patients and sepsis patients; Figure B is a graph of the content of FABP5 protein in the sera of non-septic shock sepsis patients and septic shock sepsis patients; Figure C is a graph of the content of FABP5 protein in the sera of surviving and deceased patients in the hospital with sepsis; the data was analyzed by Student's t-test, *p < 0.05, ***p < 0.001.

[0027] Figure 2 It is a graph of the content of FABP5 protein in the sera of different infection sites. The data was analyzed by One-way ANOVA test, *p < 0.05, ****p < 0.0001.

[0028] Figure 3 It is a univariate logistic regression analysis based on the content of FABP5 protein in the sera of non-sepsis patients and sepsis patients. Figure A is a logistic regression analysis graph, hazard ratio = 1.49; Figure B is an ROC curve, area under the curve = 0.8013, and the optimal cut-off value of the content of FABP5 protein is 4.892 ng / mL; Figure C is a proportional distribution graph of the content of FABP5 protein in the sera of non-sepsis patients, non-septic shock sepsis patients and septic shock sepsis patients, where FABP5 低 represents that the FABP5 content ≤ 4.892 ng / mL, represents FABP5 高Indicates that the FABP5 content > 4.892 ng / mL.

[0029] Figure 4 It is the correlation analysis of the serum FABP5 protein content with the SOFA score, APACHE II score, procalcitonin level, and lactate level in sepsis patients. The data was analyzed using Spearman correlation analysis.

[0030] Figure 5 It is the graph of the serum FABP5 protein content in different sepsis model mice. Panel A is the sepsis model induced by CLP; Panel B is the model of bronchial infection with Pseudomonas aeruginosa; Panel C is the model of bronchial infection with Staphylococcus aureus; Panel D is the model of bloodstream infection with Staphylococcus aureus; Panel E is the model of bloodstream infection with Candida albicans. The data was analyzed using Student's t-test, **p < 0.01, ****p < 0.0001.

[0031] Figure 6 It is the effect of FABP5 protein on the survival rate of CLP-induced sepsis mice. The data was statistically analyzed using the Kaplan-Meier method combined with the log-rank test, *p < 0.05.

[0032] Figure 7 It is the effect of FABP5 protein on the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine (Cr) in the serum of CLP-induced sepsis mice. The data was analyzed using One-way ANOVA test, *p < 0.05, **p < 0.01.

[0033] Figure 8 It is the effect of FABP5 protein on the hematoxylin and eosin (H&E) stained pathological sections of CLP-induced sepsis mice. Panel A is the typical pathological section diagram stained with H&E; Panel B is the statistical graph of pathological damage in liver, lung, and kidney tissues. The data was analyzed using One-way ANOVA test, **p < 0.01. Scale bar: 100 μm.

[0034] Figure 9 It is the effect of FABP5 protein on the contents of TNF-α, IL-1β, and IL-6 in the serum of CLP-induced sepsis mice. The data was analyzed using One-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001. Specific implementation methods

[0035] The biomarker involved in the present invention is FABP5 protein, which is used to predict the occurrence and development of sepsis. The technical solution of the present invention includes the following steps:

[0036] (1) Clinical sample collection: Serum samples are collected from clinical non-sepsis patients and sepsis patients.

[0037] (2) Detection of FABP5 protein content: The enzyme-linked immunosorbent assay (ELISA) technique is used to detect the content of FABP5 protein in serum samples.

[0038] (3) Clinical data analysis:

[0039] Comparison of content differences: The content of FABP5 protein in serum samples of non-sepsis patients and sepsis patients is compared and analyzed to determine the difference between the two. And the differences in the serum FABP5 protein content of patients with different infection sites or different pathogen infection types are compared.

[0040] Evaluation of predictive value: Logistic regression analysis and ROC curve analysis are used to evaluate the value of FABP5 protein as a sepsis prediction index, and its accuracy and reliability in the diagnosis of sepsis are determined.

[0041] Correlation analysis: The correlation between FABP5 protein and sepsis-related indicators (such as SOFA score, APACHE II score, PCT, lactic acid, etc.) is analyzed.

[0042] (4) Construction of different mouse sepsis models to detect the content of FABP5 protein in peripheral blood: Sepsis models induced by CLP, bronchial infection with Pseudomonas aeruginosa, bronchial infection with Staphylococcus aureus, blood-borne infection with Staphylococcus aureus, and blood-borne infection with Candida albicans are constructed respectively. Serum is collected, and the ELISA technique is used to detect the content of FABP5 protein in the serum.

[0043] (5) Detection of the effect of FABP5 protein on the progression of CLP-induced sepsis at the animal level: The FABP5 protein is purified through the prokaryotic system and injected into mice through the tail vein, and then the CLP operation is performed. The survival rate of the mice is observed, and the levels of ALT, AST, and Cr in the serum are detected using a kit; the pathological damage of liver, kidney, and lung tissues is evaluated by HE staining; the contents of TNF-α, IL-1β, and IL-6 in the serum are detected using an ELISA kit.

[0044] The present invention will be described in detail below in conjunction with specific embodiments, but the implementation of the present invention is not limited thereto.

[0045] Example 1: Clinical sample collection

[0046] Fifty-five serum samples from non-sepsis patients and one hundred serum samples from sepsis patients were collected from Nanfang Hospital of Southern Medical University. The inclusion and exclusion criteria for the patients were as follows:

[0047] Inclusion criteria for sepsis patients: (1) Age ≥ 18 years old; (2) Patients meeting the sepsis 3.0 diagnostic criteria; (3) Patients / family members signed the informed consent form and voluntarily entered the cohort. Inclusion criteria for non-sepsis patients: (1) Age ≥ 18 years old; (2) Trauma patients without a history of obvious pathogen infection; (3) Patients / family members signed the informed consent form and voluntarily entered the cohort. Exclusion criteria: (1) Age < 18 years old; (2) Pregnant, lactating or menstruating women; (3) Those who received antibiotic, microbial agent or immunosuppressant treatment within 3 months before enrollment; (4) Presence of other diseases that affect the research results (such as active cancer or end-stage liver disease).

[0048] Example 2: Detection of serum FABP5 protein content

[0049] The human FABP5 enzyme-linked immunosorbent assay kit (product number EH0863) produced by Wuhan Fine Biotech Co., Ltd. was used to detect the FABP5 protein content in serum samples from non-sepsis patients and sepsis patients. The experimental steps were carried out according to the instructions as follows: The serum was diluted 20 times with the sample diluent. Different concentrations of FABP5 standards were prepared according to the instructions: 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL and 0.313 ng / mL. 100 μL of the diluted sample and standard were added to the wells respectively, and after covering with a sealing film, it was placed at 37 °C and incubated for 90 min. The plate was washed 2 times. 100 μL of the biotin antibody working solution was added to each well, and after covering with a sealing film, it was placed at 37 °C and incubated for 60 min. The plate was washed 3 times. 100 μL of the HRP-streptavidin (SABC) working solution was added to each well, and after covering with a sealing film, it was placed at 37 °C and incubated for 30 min. The plate was washed 5 times. 90 μL of the TMB chromogenic substrate was added to each well, and after covering with a sealing film, it was placed at 37 °C and incubated for 15 min. 50 μL of the reaction termination solution was added to each well, and the value was immediately read at 450 nm on an enzyme-linked immunosorbent assay instrument and calculated to obtain the FABP5 protein content of the sample.

[0050] Example 3: Clinical data analysis

[0051] As Figure 1 shown, first, the difference in the FABP5 protein content in the sera of non-sepsis patients and sepsis patients was analyzed. The results showed that the FABP5 protein content in the sera of sepsis patients was significantly higher than that of non-sepsis patients( Figure 1in A). Then, sepsis patients were divided into non-septic shock patients and septic shock patients, as well as in-hospital survivors and non-survivors. The results showed that the serum FABP5 protein level in septic shock patients was significantly higher than that in non-septic shock patients ( Figure 1 in B), and the serum FABP5 protein level in non-surviving sepsis patients was significantly higher than that in surviving sepsis patients ( Figure 1 in C). This indicates that the serum FABP5 protein level is correlated with the severity of sepsis.

[0052] As Figure 2 shown, sepsis patients were classified according to the site of infection, and their serum FABP5 levels were compared with those of non-sepsis patients. The results showed that the serum FABP5 protein levels in sepsis patients with different infection sites were all higher than those in non-sepsis patients.

[0053] As Figure 3 shown, univariate logistic regression analysis was performed on the serum FABP5 protein levels of non-sepsis patients and sepsis patients. The results showed that the hazard ratio was 1.49, indicating that the level of serum FABP5 protein is a risk factor for sepsis. The ROC curve was used to evaluate the clinical accuracy of FABP5 protein in predicting sepsis. The area under the curve (AUC) was 0.8013, indicating that the serum FABP5 protein concentration has good diagnostic efficacy and high accuracy in the clinical diagnosis of sepsis. According to the maximum value of the Youden index, the optimal cut-off point was determined to be 4.892 ng / mL, with a sensitivity of 0.62 and a specificity of 0.89. To verify whether the level of FABP5 protein can effectively reflect the different severities of sepsis, especially whether sepsis progresses to shock, we based on 4.892 ng / mL as the cut-off value (FABP5 高 and FABP5 低 respectively represent the high and low severities of sepsis. An FABP5 level ≤ 4.892 ng / mL is denoted as FABP5 低 , and an FABP5 level > 4.892 ng / mL is denoted as FABP5 高 ), and a proportional distribution map of non-sepsis patients, non-septic shock patients, and septic shock patients was drawn ( Figure 3 in C). The results showed that the FABP5 protein level is related to the severity of sepsis. As the severity of sepsis increases, the proportion of high FABP5 gradually increases, being 10.91%, 52.83%, and 72.34% in turn.

[0054] As Figure 4As shown, due to the small amount of data, Spearman rank correlation analysis was used to detect the correlation between the serum FABP5 content in sepsis patients and sepsis-related indicators. The Spearman correlation coefficient (r s ) is between -1 and 1. If r s is greater than 0, it is a positive correlation; if r s is less than 0, it is a negative correlation. The larger the absolute value of r s , the stronger the correlation. The results showed that the Spearman correlation coefficient between serum FABP5 and SOFA score was 0.4439, the Spearman correlation coefficient with APACHE II score was 0.3530, the Spearman correlation coefficient with procalcitonin was 0.5097, and the Spearman correlation coefficient with lactate was 0.4344, all showing positive correlation.

[0055] Example 4: Construct different sepsis models to detect the serum FABP5 protein content

[0056] The SPF-grade C57BL / 6 male mice (6 - 8 weeks old) used in the experiment were purchased from Beijing Speyford Biotechnology Co., Ltd., and the experimental animal license number is: SYXK (Jing) 2024 - 0010.

[0057] The mice were modeled by cecal ligation and puncture (CLP) to construct a sepsis mouse model. The specific steps for constructing a sepsis mouse model by CLP are as follows: First, the mice were anesthetized by intraperitoneal injection of 60 mg / kg pentobarbital sodium, fixed on the operating board and the skin was prepared. The abdominal skin was disinfected with 75% alcohol, and an incision of 1 - 2 cm was made in the midline of the abdomen to expose the abdominal cavity. Then, the cecum was ligated, and a puncture was made 3 / 4 of the way from the end of the cecum using an 18G injection needle to squeeze out a small amount of cecal contents to prevent the puncture hole from closing. Subsequently, the cecum was returned to the abdominal cavity, and the abdominal muscles and skin were sutured layer by layer. After the operation, the mice were given subcutaneous injection of normal saline for fluid resuscitation and placed on a heating pad for warming. Ten C57BL / 6 mice were divided into a sham operation group and a CLP model group, with 5 mice in each group. In the sham operation group, only the abdominal cavity was opened without cecal treatment. Twelve hours after modeling, peripheral blood was collected to obtain serum.

[0058] Bronchial infection with Pseudomonas aeruginosa model and Staphylococcus aureus model: First, the mice were anesthetized by intraperitoneal injection of 300 mg / kg tribromoethanol, fixed on the operating board, and the tracheal orifice was exposed with a tongue depressor with a light for oral tracheal intubation. Then, 2×10 7 CFU / mouse of Pseudomonas aeruginosa (P. aeruginosa) or 2×10 8Staphylococcus aureus (S. aureus) per mouse. Fifteen C57BL / 6 mice were divided into a PBS control group and a bacterial infection group. There were 4 mice in the P. aeruginosa infection group and 4 corresponding mice in the PBS control group; 4 mice in the S. aureus infection group and 3 corresponding mice in the PBS control group. Twelve hours after modeling, peripheral blood was collected to obtain serum.

[0059] Blood-borne infection models of Staphylococcus aureus and Candida albicans: 2×10 9 CFU / mouse of S. aureus or 1.5×10 7 CFU / mouse of Candida Albicans (C. Albicans). Twenty-two C57BL / 6 mice were divided into a PBS control group and a bacterial infection group. There were 5 mice in the S. aureus infection group and 5 corresponding mice in the PBS control group; 6 mice in the C. Albicans infection group and 6 corresponding mice in the PBS control group. Twelve hours after modeling, peripheral blood was collected to obtain serum.

[0060] The mouse FABP5 enzyme-linked immunosorbent assay kit (product number EM0374) from Wuhan Fine Biotech Co., Ltd. was used to detect the FABP5 content in the sera collected from the above different models. The serum dilution factor and experimental procedures were the same as before.

[0061] As Figure 5 shown, compared with the sham operation group, 12 hours after constructing the sepsis model, the content of FABP5 protein in the serum of mice increased significantly, and the difference was statistically significant ( Figure 5 A in it). Moreover, the content of FABP5 protein in the sera of mice in different types of bacterial infection groups was significantly higher than that in their corresponding PBS control groups ( Figure 5 B to E in it). This result indicates that the level of FABP5 protein is significantly increased in septic mice and may be closely related to the occurrence and development of sepsis. Considering its consistent increase in different infection types, FABP5 has the potential to be used as a diagnostic biomarker for sepsis. By detecting the content of FABP5 protein in the serum, an effective biomarker can be provided for the early diagnosis of sepsis, which helps to provide more accurate guidance for the diagnosis and treatment of sepsis in clinical practice.

[0062] Example 5: Detection of the effect of FABP5 protein on the progression of CLP-induced sepsis at the animal level

[0063] (1) Prokaryotic expression and purification of murine FABP5 recombinant protein: Mix 1 μL of pReceiver-B11-FABP5 plasmid with 100 μL of BL21(DE3) competent cells, place on ice for 30 min, incubate at 42 °C for 90 s, place on ice for 5 min, add 800 μL of LB medium, incubate at 37 °C for 1 h to resuscitate the competent cells, centrifuge at 3000 rpm for 3 min, discard 800 μL of the supernatant, resuspend the bacteria, spread on an LB plate containing ampicillin, and culture overnight at 37 °C. Pick a single colony into 5 mL of LB medium, add ampicillin at a final concentration of 1 μg / mL, and culture with shaking at 37 °C for 10 h. Expand the culture 1:100 into 300 mL of LB medium containing ampicillin, and culture with shaking at 37 °C until OD600 = 0.5 - 0.8. Add isopropyl-β-D-thiogalactoside to a final concentration of 1 mM, and induce with shaking overnight at 16 °C. Centrifuge at 6,000 rcf for 5 min to collect the bacteria, lyse the bacteria by sonication, centrifuge at 12,000 rcf for 30 min using a 4 °C centrifuge, collect the supernatant and transfer it to a chromatography column containing Ni-NTA resin, incubate with rotation at 4 °C for 2 h, discard the supernatant, wash multiple times with 6 times the resin volume of 1×LEW buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), then elute the protein with 2 mL of 1×elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH = 8.0). Transfer the protein into a dialysis bag and place it in PBS solution, dialyze for 24 h to remove imidazole, centrifuge at 12,000 rcf at 4 °C for 30 min to collect the supernatant, remove endotoxin using an endotoxin removal kit, and detect the endotoxin content. Determine the protein concentration using the BCA protein quantification method, and aliquot and store the protein at -80 °C in a refrigerator.

[0064] (2) Observation of mouse survival rate

[0065] Randomly divide 48 male C57BL / 6 mice mentioned above into 4 groups, with 12 mice in each group, namely the CLP model group, the murine FABP5 recombinant protein 10 μg + CLP group, the murine FABP5 recombinant protein 50 μg + CLP group, and the murine FABP5 recombinant denatured protein (FABP5△) + CLP group. The murine FABP5 recombinant denatured protein was obtained by heating 50 μg of murine FABP5 recombinant protein at 98 °C for 5 min and collecting the supernatant by centrifugation at 12,000 rcf for 30 min. Perform the CLP surgery 1 h after injecting the corresponding FABP5 recombinant protein via the tail vein, and observe and record the survival of the mice for 36 h.

[0066] As Figure 6As shown, after supplementing 50 μg of FABP5 protein, the course of sepsis in mice was significantly accelerated and the mortality rate was significantly increased, indicating that FABP5 protein may promote the progression of sepsis by promoting inflammatory responses or altering immune responses. The experimental results suggest that FABP5 protein plays an important role in the pathological process of sepsis and exacerbates the deterioration of the condition. In addition, the FABP5 recombinant denatured protein treatment group did not significantly affect the survival rate of septic mice, and this comparative experiment further confirmed the function of FABP5 protein. This finding provides a new perspective on FABP5 protein as a potential therapeutic target for sepsis. It is demonstrated that serum FABP5 protein has a good prompting effect on the diagnosis of sepsis and is an ideal risk warning marker for sepsis.

[0067] (3) Detection of mouse serum biochemical indicators

[0068] Sixteen male C57BL / 6 mice were randomly divided into three groups: the CLP group (n = 5), the mouse-derived FABP5 recombinant protein 50 μg + CLP group (n = 6), and the FABP5△ + CLP group (n = 5). One hour after intravenous injection of the corresponding FABP5 recombinant protein via the tail vein, the CLP surgery was performed, and after 12 h, peripheral blood was collected to obtain serum. The levels of ALT, AST, and Cr in the serum were detected using the ALT detection kit (product number C009-3-1), AST detection kit (product number C010-3-1), and Cr detection kit (product number C011-2-1) from Nanjing Jiancheng Bioengineering Institute, and the operation steps were strictly carried out according to the instructions attached to the kit.

[0069] As Figure 7 shown, exogenous supplementation of FABP5 protein significantly increased the levels of ALT, AST, and Cr in the serum of septic mice, and these indicators are usually used to evaluate liver and kidney function damage. ALT and AST are markers of liver damage, while Cr reflects the impairment of kidney function. The supplementation of FABP5 led to an increase in these key indicators, indicating that FABP5 protein may exacerbate organ damage in septic mice by enhancing inflammatory responses or directly affecting the cell functions of the liver and kidneys. However, when the FABP5 protein was denatured, the abnormal increase in these biochemical indicators no longer occurred.

[0070] (4) Mouse tissue pathology detection

[0071] Twenty male C57BL / 6 mice were randomly divided into three groups: the CLP group (n = 7), the murine FABP5 recombinant protein 50 μg + CLP group (n = 6), and the FABP5△ + CLP group (n = 7). One hour after intravenous injection of the corresponding FABP5 recombinant protein, the CLP surgery was performed. After 12 h, liver, lung, and kidney tissues were collected and immediately placed in 10% neutral formalin for fixation for 24 h. After fixation, the tissues were dehydrated successively with 70%, 80%, 90%, 95%, and 100% alcohol solutions, with each alcohol solution treatment for 30 min. Then, the tissues were cleared with xylene, treated twice, each time for 10 - 15 min. The cleared tissues were placed in molten paraffin at a temperature of 60 °C and soaked for 1 - 2 h to ensure complete penetration of the paraffin. After the paraffin cooled and solidified, the tissues were cut into 5-μm-thick sections using a microtome. The sections were then dewaxed, dewaxed twice with xylene, each time for 10 - 15 min, and then hydrated successively with 100%, 95%, 90%, 80%, and 70% alcohol, each time for 5 min. The hydrated sections entered the HE staining step. First, they were stained with hematoxylin staining solution for 5 - 10 min, and after staining, they were rinsed with running water for 2 - 3 minutes to remove excess hematoxylin. Then, they were stained with eosin staining solution for 2 - 5 min, and after staining was completed, they were rinsed with running water for 2 - 3 min. The stained sections needed to be dehydrated again, dehydrated with alcohol solutions of different concentrations, and then cleared with xylene. Finally, a neutral gum mounting solution was dropped, and the coverslip was gently covered for mounting. After the mounting solution dried, the sections were observed under a microscope for histological analysis.

[0072] Figure 8 Figure A in [reference] shows the hematoxylin and eosin (H&E) stained pathological sections of CLP-induced septic mice with FABP5 protein. Figure 8Figure B in [reference] is the corresponding statistical chart of pathological damage. Significant pathological changes were observed in the liver, lungs, and kidneys of septic mice. Specifically, compared with the simple CLP group, the FABP5 recombinant protein + CLP group showed more obvious hepatic interstitial edema in the liver, accompanied by infiltration of a large number of inflammatory cells, especially the aggregation of neutrophils and monocytes. The lung tissue showed more severe alveolar wall thickening, with inflammatory exudates filling the alveolar cavity, and partial alveolar epithelial cell shedding. The pathological changes in the kidneys were manifested as more damage and shedding of renal tubular epithelial cells, and cell debris and inflammatory cell infiltration were visible in the renal tubular lumen. Overall, exogenous supplementation of FABP5 protein significantly aggravated the pathological damage of the liver, lungs, and kidneys in septic mice, indicating that FABP5 protein may lead to dysfunction and damage of multiple organs in septic mice by promoting inflammatory responses and cell damage. However, when the FABP5 protein was denatured, its effect on organ damage in septic mice was no longer significant, suggesting that the functional activity of FABP5 protein is closely related to its original structure. This finding indicates that FABP5 protein may be involved in multiple organ damage caused by sepsis through its specific structure or molecular mechanism, providing new ideas for the future development of intervention strategies against FABP5 protein in diseases.

[0073] (5) Detection of serum inflammatory factors in mice

[0074] Nineteen male C57BL / 6 mice were randomly divided into three groups: the CLP group (n = 8), the murine FABP5 recombinant protein 50 μg + CLP group (n = 5), and the FABP5△ + CLP group (n = 6). One hour after intravenous injection of the corresponding FABP5 recombinant protein via the tail vein, the CLP surgery was performed, and serum was collected from peripheral blood 12 hours later. The contents of TNF-α, IL-1β, and IL-6 in the serum were detected using murine TNF-α ELISA kits (product number EMC102a.96), murine IL-1β ELISA kits (product number EMC001b.96), and murine IL-6 ELISA kits (product number EMC004.96) from Shenzhen Xinbosheng Biotechnology Co., Ltd. The experimental procedures were carried out according to the instructions as follows: Dilute the serum with sample diluent, dilute 400-fold for detecting IL-6, and dilute 10-fold for detecting TNF-α and IL-1β. Prepare standard products with different concentrations according to the instructions. Add 100 μL of the diluted sample and standard product to the wells, cover with a sealing film, and incubate at 37°C for 90 min. Wash the plates 3 times. Add 100 μL of biotin antibody working solution to each well, cover with a sealing film, and incubate at 37°C for 60 min. Wash the plates 3 times. Add 100 μL of HRP-streptavidin working solution to each well, cover with a sealing film, and incubate at 37°C for 30 min. Wash the plates 5 times. Add 100 μL of TMB chromogenic substrate to each well, cover with a sealing film, and incubate at 37°C for 15 min. Add 100 μL of reaction termination solution to each well, immediately read the values at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader and calculate to obtain the contents of TNF-α, IL-1β, and IL-6 in the samples.

[0075] As Figure 9 shown, exogenous supplementation of FABP5 protein significantly increased the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum of septic mice, suggesting that FABP5 protein may enhance the inflammatory response and induce "cytokine storm" by promoting the release of inflammatory factors in septic mice. This phenomenon indicates that FABP5 protein not only plays an important role in the occurrence of sepsis, but may also further exacerbate the pathological process of sepsis by regulating the intensity of the inflammatory response.

Claims

1. A biomarker for detecting sepsis, characterized in that, The biomarker is FABP5 protein.

2. Use of the biomarker FABP5 protein described in claim 1 in the preparation of a kit for predicting the risk of sepsis.

3. The application according to claim 2, characterized in that Determine the degree of sepsis risk by detecting the content of FABP5 protein in human serum.

4. The application according to claim 3, characterized in that, When the content of FABP5 protein in human serum is detected to be ≥ 4.892 ng / mL, it indicates sepsis.

5. The application according to claim 3, wherein There is a positive correlation between the content of FABP5 protein in human serum and the severity of sepsis.

6. The application according to claim 3, wherein The human serum sample is selected from at least one of whole blood, serum, and plasma.

7. The application according to claim 2, characterized in that, The biomarker FABP5 protein can enhance the inflammatory response or directly affect the cell functions of the liver and kidneys.

8. The application according to claim 2, wherein The biomarker FABP5 protein can participate in multi-organ damage caused by sepsis through its specific molecular mechanism.

9. The application according to claim 2, wherein The biomarker FABP5 protein can promote the release of inflammatory factors.

10. The application according to claim 9, wherein The inflammatory factors include TNF-α, IL-1β, and IL-6.

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