Application of 4-GBA as biomarker in evaluation of infection tolerance of sepsis patient

Abstract

The invention relates to application of 4-GBA (guanidinobutyric acid) as a biomarker in evaluation of infection tolerance of sepsis patients, the biomarker is 4-GBA (guanidinobutyric acid) which is obtained by detecting differential metabolites of peripheral blood serum of sepsis patients and healthy people through high-throughput target metabonomics, and the differential metabolites are detected and verified through the target metabonomics; experiments of the applicant prove that for relatively healthy people, the content of 4-GBA in serum of a sepsis patient is low in expression, and the 4-GBA is in positive correlation with the infection endurance capacity of the sepsis patient; the 4-GBA is in negative correlation with the disease severity of a sepsis patient. The application of the 4-GBA as the molecular marker in evaluating the infection endurance capacity of the sepsis patient has great clinical value and practical significance in the aspects of early diagnosis, subtype classification, related treatment target assessment and prediction of patient prognosis of sepsis, and provides a new direction for early diagnosis, treatment and prediction prognosis of the sepsis patient.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of 4-GBA as a biomarker in evaluating the infection tolerance of sepsis patients. Background Art

[0002] Sepsis refers to life-threatening organ dysfunction caused by a dysregulated host response to infection. Every year, 19 million people suffer from sepsis worldwide, of which 11 million are related to sepsis. Deaths from sepsis account for 19.7% of the total global death population, ranking first, and it has become an important public health issue facing all mankind.

[0003] At present, the biomarkers used in clinical practice for infection in patients with sepsis include procalcitonin (PCT), C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-8 (IL-8), heparin-binding protein (HBP), etc. The above markers can be used to detect the prognosis of patients with sepsis, but they are limited due to their low specificity or low sensitivity.

[0004] The course of sepsis is accompanied by immune dysfunction. After encountering bacterial invasion, the human immune system is activated, triggering an inflammatory response to eliminate the invading bacteria. In this case, if the bacteria are completely eliminated, the immune system can return to normal. However, if the bacteria are not completely eliminated, the overactivation of the immune system will form a "cytokine storm", leading to multiple organ dysfunction and even early death in patients. Patients who survive the early stage will enter the late stage of sepsis, at which time the patient's immune system function will be impaired, and its ability to present antigens and eliminate bacteria will be greatly reduced. Multiple organ dysfunction caused by bacterial infection again is the main cause of late death in sepsis patients. In addition to the resistance mechanism driven by the immune response to kill and eliminate pathogens, the human body's anti-infection response to bacteria can also maintain the body's homeostasis and protect the patient's survival through a tolerance mechanism aimed at slowing down the pathological damage caused by pathogens and immune responses to the host. To date, the tolerance molecules with "positive" regulatory effects reported include: ferritin heavy chain in hepatocytes, autophagy-related proteins in the lungs, insulin-like growth factor in white adipose tissue, aryl hydrocarbon receptor in splenic dendritic cells, amphiregulin produced by natural lymphocytes 2 or regulatory T cells in the lungs, macrophage nuclear factor erythroid 2-related factor 2, and NADPH oxidase subunit Cybb. The above molecules can alleviate target organ damage, improve the survival rate of animals in different infection models, and improve prognosis through different regulatory mechanisms without affecting the pathogen load. However, there is currently a lack of biomarkers for evaluating the infection tolerance of patients with sepsis. Therefore, it is of great practical significance to seek new biomarkers for evaluating the infection tolerance of patients with sepsis.

[0005] 4-guanidinobutyric acid (4-GBA) is widely distributed in various tissues and organs of the human body. The main sources of 4-GBA are the catabolism of food by intestinal microorganisms and the catabolism of agmatine in the body catalyzed by diamine oxidase (DAO) and aldehyde dehydrogenase (AIDdh). 4-GBA has the effects of inhibiting the growth of Helicobacter pylori and preventing and treating acute and chronic gastritis. However, there are no reports on the study of 4-GBA in sepsis. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the present invention provides the use of 4-GBA as a biomarker in evaluating the infection tolerance of sepsis patients.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] Application of 4-GBA as a biomarker in evaluating infection tolerance in patients with sepsis.

[0009] Furthermore, the 4-GBA content is reduced in patients with sepsis.

[0010] A kit for detecting the infection tolerance of a sepsis patient, wherein the kit contains a reagent for detecting the content of 4-GBA in the patient's peripheral blood serum.

[0011] In certain specific embodiments, the kit distinguishes the infection tolerance of the test subject by detecting the 4-GBA content in the peripheral blood serum samples of sepsis patients.

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

[0013] 1. The present invention relates to the use of 4-GBA (guanidinobutyric acid) as a biomarker in evaluating the infection tolerance of sepsis patients. The marker is obtained by detecting the differential metabolites of peripheral blood serum of sepsis patients and healthy people through high-throughput targeted metabolomics, and is verified by targeted metabolomics detection. The applicant's experiment confirmed that the expression of 4-GBA in the serum of sepsis patients is low compared with that of healthy people, and the content of 4-GBA is negatively correlated with the disease severity of sepsis patients; the content of 4-GBA is negatively correlated with the APACHE-II score and GCS score of sepsis patients, the content of 4-GBA is negatively correlated with AST and Cre, the liver and kidney function markers of sepsis patients, the content of 4-GBA is negatively correlated with the percentage of neutrophils in sepsis patients, and is positively correlated with the percentage of lymphocytes, indicating that the content of 4-GBA is positively correlated with the infection tolerance of sepsis patients.

[0014] 2. The application of 4-GBA as a molecular marker in the evaluation of infection tolerance of sepsis patients has great clinical value and practical significance for the early diagnosis of sepsis, subtype classification, evaluation of related treatment targets, and prediction of patient prognosis, and provides a new direction for the early diagnosis, treatment and prediction of prognosis of sepsis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0016] Figure 1 This is a volcano plot of metabolite differences in serum samples of 5 healthy controls and 5 sepsis patients detected by high-throughput targeted metabolomics in Example 1 of the present invention.

[0017] Figure 2 This is a box plot of serum 4-GBA levels in 5 healthy controls and 5 sepsis patients in Example 1 of the present invention.

[0018] Figure 3 This is a box plot of serum 4-GBA levels in 10 healthy controls and 10 sepsis patients in Example 2 of the present invention.

[0019] Figure 4 In Example 2 of the present invention, the serum 4-GBA content of sepsis patients was negatively correlated with the APACHE-II score and GCS score representing the severity of the disease of sepsis patients. P<0.05, with statistical significance.

[0020] Figure 5 In Example 2 of the present invention, the serum 4-GBA level of sepsis patients was negatively correlated with the aspartate aminotransferase (AST) level and creatinine (Cre) level of liver and kidney function. P<0.05, with statistical significance.

[0021] Figure 6 In Example 2 of the present invention, the serum 4-GBA content of sepsis patients was negatively correlated with the neutrophil percentage of sepsis patients, P < 0.05, with statistical significance. The 4-GBA content was positively correlated with the lymphocyte percentage, P < 0.01, with statistical significance. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any equivalent changes in form made based on the concept of the present invention should be regarded as the scope of the present invention.

[0023] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0024] Sources

[0025] Methanol and acetonitrile were purchased from Thermo.

[0026] Formic acid was purchased from TCI.

[0027] Sodium carbonate (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ) and dansyl chloride were purchased from Sigma.

[0028] The standard guanidine butyric acid (4-GBA) was purchased from Sigma.

[0029] Example 1 Analysis of differentially expressed metabolites in sepsis patients (Case) and healthy subjects (Control) by high-throughput targeted metabolomics

[0030] Peripheral venous sera from 5 patients clinically diagnosed with sepsis and 5 healthy subjects were collected, and high-throughput targeted metabolomics was used to analyze the differentially expressed metabolites between sepsis patients and healthy subjects. The technical process and analysis of serum metabolomics are as follows:

[0031] 1. Serum Sample Collection

[0032] After the diagnosis of the included patients was confirmed, 2 ml of peripheral venous blood was drawn using a coagulant tube; all the collected venous blood was separated into serum and blood cells using a low-temperature centrifuge within 2 hours; the centrifugation conditions were: 4°C, 3000rpm, 15min; the upper layer of serum was aspirated with a pipette and packaged and frozen in a -80°C refrigerator for later use; repeated freezing and thawing of the samples were avoided.

[0033] 2. Serum Metabolomics Sample Preparation

[0034] 2.1 Take the sample out of the -80℃ freezer, place it on ice to thaw, and vortex for 30 seconds to mix;

[0035] 2.2 Add 100ul of sample to a 2ml EP tube, then add 200ul of water and vortex to mix;

[0036] 2.3 Add 1200ul of liquid extract (formaldehyde:acetonitrile = 1:1) and vortex to mix;

[0037] 2.4 Ultrasonicate in an ice-water bath for 15 min, vortex mix, and stand at -40°C for 2 h;

[0038] 2.5 Centrifuge at 4°C, 12000 rpm for 15 min; extract 1200 ul of supernatant into a new EP tube, and concentrate by centrifugation to dryness;

[0039] 2.6 Redissolve in 120 μL acetonitrile: water, v:v = 6:4, vortex until the dry powder is dissolved, sonicate in an ice-water bath for 30 s, and centrifuge at 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) at 4°C for 15 min.

[0040] 3. Mass Spectrometry Analysis

[0041] 3.1 Carefully take out 60-70 μL of supernatant into LC injection vial and test on the machine;

[0042] 3.2 Take the standard mixed solution as the QC quality control sample and test it on the machine;

[0043] The present invention uses a SCIEX 6500QTRAP+ triple quadrupole mass spectrometer to perform mass spectrometry analysis in multiple reaction monitoring (MRM) mode. All mass spectrometry data acquisition and target compound quantitative analysis in the present invention are completed by SCIEX Analyst Work Station Software (1.7.2) and Data Driven Flow (v-1.0.1). Calculation formula: C [nmol / L] = C [μmol / L] * V [μL] * CF / V [mL]. Finally, the concentration of the target metabolite in the sample is obtained.

[0044] 4. Data Analysis

[0045] For metabolomics data, SIMCA (V16.0.2) software was used to visualize metabolizable products.

[0046] 5. Expression of differential metabolites 4-GBA in peripheral blood serum between sepsis patients and healthy subjects

[0047] In this example, high-throughput targeted metabolomics was used to detect the difference in metabolites in serum samples of 5 healthy controls and 5 sepsis patients. Figure 1 As shown in the figure, the variable projection importance (VIP) of the first principal component of the OPLS-DA model is greater than 1, and P < 0.05 is used as the standard to screen and distinguish up-regulated metabolites from down-regulated metabolites. As can be seen from the figure, red is up-regulated metabolites and blue is down-regulated metabolites, indicating that there are significant differences in metabolites between serum samples of sepsis patients and healthy controls.

[0048] The box plot of serum 4-GBA levels of 5 healthy controls and 5 sepsis patients in this example is shown in Figure 2 As shown in the figure, it can be seen that the expression level of serum 4-GBA in sepsis patients (Case) is lower than that in healthy people (Control), P < 0.01, indicating that there is a statistical difference between the two, and the difference is significant.

[0049] Example 2: Expanding the sample size for validation and analysis through targeted metabolomics analysis

[0050] According to the inclusion and exclusion criteria, 10 patients diagnosed with sepsis and 10 healthy controls were included. Peripheral blood plasma was collected for targeted metabolomics analysis.

[0051] 1. After the diagnosis of the included patients is confirmed, 2 ml of peripheral venous blood is drawn using a coagulant tube; all the collected venous blood is separated into serum using a low-temperature centrifuge (4°C, 3000 rpm, 15 min) within 2 hours; the upper layer of serum is aspirated with a pipette and packaged and frozen in a -80°C refrigerator for later use; avoid repeated freezing and thawing of the samples.

[0052] 2. Serum Sample Preparation

[0053] 2.1 Take 100 μL of serum sample and place it in a 2 ml centrifuge tube, add 400 μL of 1% formic acid acetonitrile, vortex and sonicate at room temperature for 3 min;

[0054] 2.2 Centrifuge at 12000rpm and 4℃ for 10min. After centrifugation, take 300μL of supernatant and concentrate it to dryness;

[0055] 2.3 Add 100 μL of 1 mg / mL dansyl chloride derivatization agent to the spin-dried sample;

[0056] 2.4 Add 100 μL 0.1 M Na 2 CO 3 +NaHCO 3 Buffer, vortex thoroughly;

[0057] 2.5 Mix well and place in a 60℃ water bath for 30min. After the reaction is complete, cool to room temperature, vortex and filter into a sample vial;

[0058] 3. Preparation of Standard Stock Solution

[0059] 3.1 Weigh an appropriate amount of 4-GBA and prepare the 4-GBA standard stock solution;

[0060] 3.2 Dilute with 1% formic acid and acetonitrile to an appropriate concentration to prepare a working standard solution;

[0061] 3.3 Take 300 μL of each standard curve concentration point and concentrate to dryness, then add 100 μL of 1 mg / mL dansyl chloride derivatization agent;

[0062] 3.4 Add 100 μL 0.1 M Na 2 CO 3 +NaHCO 3 Buffer, vortex thoroughly;

[0063] 3.5 Mix well and place in a 60℃ water bath for 30min. After the reaction is complete, cool to room temperature, vortex and filter into a sample vial;

[0064] 4. On-machine testing

[0065] 5. Calculation formula: content = C*0.5*2 / 3 / sampling volume*1000. Content unit: nmol / L; C unit: nmol / L; sampling volume unit: μL.

[0066] 6. Results Analysis

[0067] For the high-throughput target metabolomics detection results, SIMCA (V16.0.2) software was used for visualization analysis. For the targeted metabolomics detection results, GraphPad Prism 8.02 software was used for analysis.

[0068] This example analyzes the correlation between the serum 4-GBA content of patients with sepsis and the disease severity scores of patients with sepsis (APACHE-II score, GCS score), the correlation with the biomarkers of liver and kidney function damage (aspartate aminotransferase (AST), creatinine (Cre) content, and the correlation with immune cells (neutrophil percentage, lymphocyte percentage).

[0069] In this example, targeted metabolomics was used to detect the 4-GBA content in the peripheral blood serum of 10 patients with sepsis and 10 healthy people. Figure 3 As shown in the figure, it can be seen that there is a difference in the expression level of 4-GBA as a biomarker in the peripheral blood serum of sepsis patients and healthy people. The serum 4-GBA level of sepsis patients is lower than that of healthy people, P < 0.001, and there is a statistical difference between the two.

[0070] In this example, the 4-GBA levels in peripheral blood serum of 10 sepsis patients and 10 healthy subjects are shown in Table 1.

[0071] Table 1 4-GBA concentration in serum

[0072]

[0073] The correlation between serum 4-GBA content in patients with sepsis and APACHE-II score and GCS score in patients with sepsis is as follows Figure 4 As shown in the results, the serum 4-GBA content in patients with sepsis was negatively correlated with the APACHE-II score of the patients (Y=-121.9X+8286, R=0.4169, P<0.05), which was statistically significant; the serum 4-GBA content in patients with sepsis was negatively correlated with the GCS score (Y=-179.9X+8400, R=0.4542, P<0.05), which was statistically significant. This indicates that the lower the serum 4-GBA content in patients with sepsis, the higher the severity of the disease and the worse the prognosis.

[0074] The correlation between serum 4-GBA level and aspartate aminotransferase (AST) level and creatinine (Cre) level of liver and kidney function in patients with sepsis is as follows Figure 5 As shown in the results, serum 4-GBA content was negatively correlated with aspartate aminotransferase (AST) content (Y=-9.962X+7411, R=0.4342, P<0.05), and there was a statistical difference; serum 4-GBA content was negatively correlated with creatinine (Cre) content (Y=-6.196X+7524, R=0.4658, P<0.05), and there was a statistical difference. This shows that the lower the serum 4-GBA content of patients with sepsis, the more severe the tissue and organ damage, and the lower the ability of patients' tissues and organs to tolerate infection.

[0075] The correlation between serum 4-GBA content in patients with sepsis and the percentage of neutrophils and lymphocytes in patients with sepsis is as follows: Figure 6 As shown, the serum 4-GBA content was negatively correlated with the neutrophil percentage (Y=-52.38X+10605, R=0.5752, P<0.01), and there was a statistical difference, indicating that the lower the serum 4-GBA content of patients with sepsis, the more severe the infection of the patients; in the lymphocyte percentage, the serum 4-GBA content was positively correlated with the lymphocyte percentage (Y=65.83X+5508, R=0.6704, P<0.05), and there was a statistical difference, indicating that the lower the serum 4-GBA content of patients with sepsis, the lower the tolerance of lymphocyte infection.

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. 4-GBA is used as a biomarker to evaluate the infection tolerance of patients with sepsis.

2. The use according to claim 1, characterized in that: The 4-GBA is lowly expressed in sepsis patients.

3. A kit for detecting the infection tolerance of sepsis patients, characterized in that: The kit contains a reagent for detecting the content of 4-GBA in the peripheral blood serum of a patient.

4. The kit according to claim 3, characterized in that The kit distinguishes the infection tolerance of the test subject by detecting the 4-GBA content in the peripheral blood serum samples of sepsis patients.

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