Application of metabolite of arachidonic acid in mycoplasma pneumonia

Abstract

The invention discloses application of a metabolite of arachidonic acid in mycoplasma pneumonia, and relates to the technical field of biological detection. According to the application of the arachidonic acid metabolite in mycoplasma pneumonia, the arachidonic acid metabolite is detected, so that the content of the arachidonic acid metabolite in a biological sample is accurately detected through chromatography-mass spectrometry according to the change of the level of the metabolite in inflammatory reaction, and the arachidonic acid metabolite can be used as an auxiliary index for diagnosis of inflammatory diseases such as pneumonia; if in a pneumonia patient, the concentration may be higher than that of a healthy person; in different types of inflammatory diseases, metabolite change modes of arachidonic acid are different, and detection of the metabolite in combination with other clinical indexes is helpful for distinguishing pneumonia and other inflammatory diseases with similar symptoms, such as differential diagnosis of bronchitis and the like; meanwhile, in the pneumonia development process, the inflammation progress can be known by detecting the content of metabolites.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to the application of arachidonic acid metabolites in mycoplasma pneumonia. Background Art

[0002] Arachidonic acid is mainly concentrated in the muscles, organs and blood of mammals, especially in the brain and nerve tissues. Most of it is bound to the second carbon atom of the glycerol skeleton of the cell membrane phospholipids. Under the stimulation of various chemical and physical damages and biological factors (cytokines, growth factors, endotoxins), phospholipase A2 can hydrolyze cell membrane phospholipids and release free arachidonic acid after activation. Arachidonic acid has three metabolic pathways: (1) cyclooxygenase COX; (2) lipoxygenase LOX; (3) cytochrome P450 (CYP) metabolic pathway. It is the direct precursor of twenty carbon derivatives such as prostaglandins, thromboxanes and leukotrienes. These bioactive substances participate in the regulation of inflammatory response and immune system, regulate vascular tension and platelet aggregation, regulate tumor cell growth, affect hormone synthesis and release of endocrine system, and play a very important role in the development of central nervous system and retinal nerve. Therefore, targeted detection of arachidonic acid expression in children infected with Mycoplasma pneumoniae (MP) is of great significance for further exploring the pathogenesis of MP and finding markers and effective therapeutic targets.

[0003] 12(S)-HETE, full name 12(S)-hydroxyeicosatetraenoic acid, is a metabolite of arachidonic acid (AA) and is catalyzed by 12-lipoxygenase (LOX). It plays an important role in the occurrence and development of many diseases, including inflammation, oxidative stress, cancer, diabetes and hypertension. In pneumonia, 12(S)-HETE may be associated with the development of the disease through multiple mechanisms. For example, it may be involved in deterioration and exudation during the inflammatory response. In patients with severe trauma, changes in plasma 12(S)-HETE levels are associated with the severity of systemic injury and can be used as a biomarker to identify the severity of trauma and predict the short-term and long-term prognosis of patients with severe trauma. In addition, research progress on 12(S)-HETE in inflammation and oxidative stress shows that it plays a role in the inflammatory response process and may be related to the occurrence and development of pneumonia.

[0004] 12(S)-HETE may play a role in inflammatory diseases such as pneumonia by participating in inflammatory response, affecting immune cell function, etc., but its specific mechanism of action and its role in Mycoplasma pneumonia need further research to clarify; to this end, the present invention provides the use of arachidonic acid metabolites in Mycoplasma pneumonia to solve the above-mentioned problems. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of arachidonic acid metabolites in Mycoplasma pneumonia, which solves the problems mentioned in the above background technology.

[0006] To achieve the above objectives, the present invention is implemented through the following technical scheme: application of targeted detection of arachidonic acid metabolites in Mycoplasma pneumonia.

[0007] Preferably, the targeted detection comprises a reagent for detecting the content of the metabolite marker in a sample from a subject.

[0008] Preferably, the targeted detection comprises detection by liquid chromatography-tandem mass spectrometry.

[0009] Preferably, the targeted detection diagnoses whether pneumonia occurs by detecting the content level of metabolic markers.

[0010] Preferably, the samples are separated using an Agilent 1290 Infinity LC ultra high performance liquid chromatography system.

[0011] Preferably, the detection conditions of the chromatography are: the sample is placed in an automatic sampler at 4°C, the column temperature is 35°C, the mobile phase A: 0.1% formic acid aqueous solution, the mobile phase B: 0.1% formic acid acetonitrile solution, the flow rate is 400 μL / min, and the injection volume is 2 μL; the liquid phase gradient is as follows: 0-1min, phase B is maintained at 30%; 1-9min, phase B changes linearly from 30% to 90%; 9-11min, phase B is maintained at 90%; 11-11.1min, phase B changes linearly from 90% to 20%; 11.1-14min, phase B is maintained at 20%.

[0012] Preferably, mass spectrometry analysis is performed using a 5500QTRAP mass spectrometer in negative ion mode.

[0013] Preferably, the negative ion conditions of 5500QTRAP ESI source are as follows: source temperature: 500°C; ion Source Gas1 (Gas1): 50; Ion Source Gas2 (Gas2): 50; Curtain gas (CUR): 30; ion Sapary Voltage Floating (ISVF): -4500V.

[0014] Preferably, the ion pairs to be detected are detected in MRM mode.

[0015] Beneficial Effects

[0016] The present invention provides the use of arachidonic acid metabolites in Mycoplasma pneumonia. Compared with the prior art, it has the following beneficial effects: the use of the arachidonic acid metabolites in Mycoplasma pneumonia, by detecting the metabolites of arachidonic acid, the level of the metabolites changes during the inflammatory response, and the content of the metabolites in biological samples is accurately detected by chromatography-mass spectrometry, which can be used as an auxiliary indicator for the diagnosis of inflammatory diseases such as pneumonia. For example, in patients with pneumonia, its concentration may be higher than that of healthy people; in different types of inflammatory diseases, the change pattern of arachidonic acid metabolites is different. Combined with other clinical indicators, the detection of the metabolites is helpful to distinguish pneumonia from other inflammatory diseases with similar symptoms, such as differential diagnosis of bronchitis; at the same time, during the development of pneumonia, the detection of the content of metabolites can understand the progression of inflammation, such as the content Continuous increase may indicate worsening inflammation, whereas decrease may indicate improvement of the condition. During treatment, regular testing of metabolite levels can evaluate the effectiveness of the treatment plan. If levels decrease after treatment, it indicates that the treatment is effective in controlling inflammation. If levels increase instead of decrease, the treatment plan may need to be adjusted. Chromatography-mass spectrometry can not only measure the E content of metabolites, but also analyze other related metabolites in the entire arachidonic acid metabolic pathway. By studying their changes, we can gain an in-depth understanding of the abnormalities in the arachidonic acid metabolic pathway in inflammatory diseases, providing a basis for revealing the pathogenesis of the disease. Clarifying the mechanism of action of 12(S)-HETE and related metabolites in diseases will help discover new drug targets and treatment directions, laying the foundation for the development of more effective therapeutic drugs and methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A box plot of differential metabolites between HC group and MPP group of children provided for the application of the metabolites of arachidonic acid of the present invention in Mycoplasma pneumonia;

[0018] Figure 2 A heat map of differential metabolites between HC group and MPP group of children provided for the application of the metabolites of arachidonic acid of the present invention in Mycoplasma pneumonia;

[0019] Figure 3 ROC curves of HC group and MPP group provided for the application of the metabolites of arachidonic acid of the present invention in Mycoplasma pneumonia;

[0020] Figure 4 A comparative diagram of differential metabolite analysis of three groups of children provided for the application of the metabolites of arachidonic acid of the present invention in Mycoplasma pneumonia;

[0021] Figure 5 The ROC curve of 12(S)-HETE in children with mild and severe MPP is provided for the application of the metabolites of arachidonic acid of the present invention in Mycoplasma pneumonia. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] In this technical scheme, 9 healthy control children (HC) and 12 children with Mycoplasma pneumoniae infection (MPP) were selected for arachidonic acid targeted mass spectrometry detection.

[0024] Example 1

[0025] Metabolite extraction

[0026] The sample extraction method is as follows: take out the sample at -80℃, take 100ul of the sample, add 500ul of BHT protein precipitant, add 10ul of internal standard with a concentration of 1ug / ml, vortex for 20s, centrifuge at 14000rcf, 4℃ for 10min, take 400ul of the supernatant, add 1000ul of pure water, use Oasis HLB 96-well Plate to purify the sample, add 2mL of methanol to activate the well plate, add 1mL each time, add 2mL of pure water to balance the well plate, add 1mL each time, load the sample multiple times, then add 2mL of Washingsolution A twice, add 1mL each time, add 2mL of Washing solution B twice, add 1mL each time, discard the filtrate, add 1mL of pure methanol to elute the sample, elute twice, collect the eluate, blow dry with nitrogen, and freeze at -80℃.

[0027] Example 2

[0028] Chromatography-mass spectrometry detection conditions

[0029] 1) Chromatographic conditions: The samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system. The samples were placed in an autosampler at 4°C, the column temperature was 35°C, the mobile phase A was 0.1% formic acid in water, the mobile phase B was 0.1% formic acid in acetonitrile, the flow rate was 400 μL / min, and the injection volume was 2 μL; the relevant liquid phase gradients were as follows: 0-1min, phase B was maintained at 30%; 1-9min, phase B changed linearly from 30% to 90%; 9-11min, phase B was maintained at 90%; 11-11.1min, phase B changed linearly from 90% to 20%; 11.1-14min, phase B was maintained at 20%; a QC sample was set for every certain number of experimental samples in the sample queue to detect and evaluate the stability and repeatability of the system; the sample queue was set with a standard mixture of the target substance for the correction of the chromatographic retention time.

[0030] 2) Mass spectrometry conditions: A 5500QTRAP mass spectrometer (SCIEX) was used for mass spectrometry analysis in negative ion mode. The negative ion conditions of the 5500QTRAP ESI source were as follows: source temperature: 500°C; ion Source Gas1 (Gas1): 50; Ion Source Gas2 (Gas2): 50; Curtain gas (CUR): 30; ion Sapary Voltage Floating (ISVF): -4500 V. The MRM mode was used to detect the ion pairs to be measured. The ion pair information of all target substances is shown in Table 1.

[0031] Table 1 MRM results of HC group and MPP group

[0032]

[0033]

[0034] Note: (1) HC group is the healthy control group, and MPP group is the Mycoplasma pneumonia group;

[0035] (2) RSD is the relative standard deviation of the metabolite in the QC sample. It is generally believed that the quantitative results obtained when the RSD value is less than 0.3 are accurate;

[0036] (3) Foldchange is the fold change. For example, in the comparison group A_vs_B, it represents the mean of group A divided by the mean of group B. When p < 0.05 is met, >1 indicates upregulation and <1 indicates downregulation;

[0037] (4) * indicates P < 0.05, indicating significant difference.

[0038] Table 2 ROC curve analysis of metabolites in HC group and MPP group

[0039]

[0040]

[0041] Note: * indicates P < 0.05, with significant difference.

[0042] Table 3 Comparison of metabolites among three groups of children

[0043]

[0044]

[0045] Note: One-way ANOVA analysis method, * indicates P < 0.05, there is a significant difference.

[0046] like Figure 1 As shown in the figure; Box-plot is a statistical graph commonly used to show the dispersion of a set of data. It can intuitively show the changes in metabolites between groups and the distribution within the group. Arrange a set of data from large to small, and calculate its upper edge (maximum value), upper quartile Q3, median, lower quartile Q1, and lower edge (minimum value). The minimum and maximum values ​​form intervals that can reflect the degree of variation in the data. In the box-plot, the horizontal axis is the group name, and the vertical axis is the content information. The significant difference between the groups is expressed by the P value calculated by the t-test or variance analysis. In the figure, ****, **, *, and * represent p<0.0001, 0.001, 0.01, and 0.05, respectively.

[0047] like Figure 2 As shown, in order to more comprehensively and intuitively display the differences in the expression patterns of metabolites in different samples, the expression levels of all metabolites are calculated by distance matrix, and cluster analysis is performed using hierarchical clustering. The cluster analysis diagram of the example group is as above. In the figure, the horizontal axis is the sample number, the vertical axis is the metabolite, and the depth of the color reflects the level of expression. The redder the color, the higher the expression level of the metabolite, and the bluer the color, the lower the expression level of the metabolite. Generally speaking, when the candidate metabolites screened are reasonable and accurate, samples in the same group can appear in the same cluster through clustering. At the same time, metabolites clustered in the same cluster have similar expression patterns and may be in relatively close reaction steps in the metabolic process.

[0048] like Figure 3As shown, dodeca-S hydroxyeicosatetraenoic acid 705.0014, sensitivity 1, specificity 1; 15-S hydroxyeicosatetraenoic acid 66.1399, sensitivity 0.75, specificity 1; 13-S hydroxyoctadecadienoic acid 31.1499, sensitivity 0.833, specificity 0.889; leukotriene B 41.9714, sensitivity 1, specificity 0.667; thromboxane B 244.1288, sensitivity 0.833, specificity 1.

[0049] like Figure 4 As shown in the figure, ****, **, *, *, ns represent p < 0.0001, 0.001, 0.01, 0.05, respectively, indicating no significant difference.

[0050] like Figure 5 As shown, the area under the curve was 0.875, P = 0.042, 944.4987, sensitivity 0.875, and specificity 0.75.

[0051] Conclusion: The 12(S)-HETE in the MPP pneumonia group was significantly higher than that in the HC group, and that in the severe MPP group was higher than that in the mild group, with strong sensitivity and specificity. It has clinical significance for the prediction of severe diseases, in-depth study of pathogenesis, and development of new drug targets.

[0052] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0053] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0054] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of targeted detection of arachidonic acid metabolites in Mycoplasma pneumonia.

2. The use according to claim 1, characterized in that: The targeted detection includes a reagent for detecting the content of the metabolite marker in a sample from a subject.

3. The use according to claim 2, characterized in that: The targeted detection includes detection by liquid chromatography-tandem mass spectrometry.

4. The use according to claim 3, characterized in that: The targeted detection diagnoses whether pneumonia occurs by detecting the content level of metabolic markers.

5. The use according to claim 4, characterized in that: The samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system.

6. The use according to claim 5, characterized in that: The detection conditions of the chromatography are as follows: the sample is placed in an automatic sampler at 4°C, the column temperature is 35°C, the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is 0.1% formic acid acetonitrile solution, the flow rate is 400 μL / min, and the injection volume is 2 μL; the liquid phase gradient is as follows: 0-1min, the B phase is maintained at 30%; 1-9min, the B phase changes linearly from 30% to 90%; 9-11min, the B phase is maintained at 90%; 11-11.1min, the B phase changes linearly from 90% to 20%; 11.1-14min, phase B is maintained at 20%.

7. The use according to claim 6, characterized in that: Mass spectrometry analysis was performed using a 5500QTRAP mass spectrometer in negative ion mode.

8. The use according to claim 7, characterized in that: in, The negative ion conditions of 5500QTRAP ESI source are as follows: source temperature: 500℃; ion Source Gas1 (Gas1): 50; Ion Source Gas2 (Gas2): 50; Curtain gas (CUR): 30; ion Sapary Voltage Floating (ISVF): -4500V.

9. The use according to claim 8, characterized in that: in, The MRM mode was used to detect the ion pairs.

Images