A biomarker and its application in diagnosis of fulminant myocarditis

Abstract

This invention discloses a biomarker and its application in the diagnosis of fulminant myocarditis. This invention is the first to discover that the expression levels of chemokines CXCL2 and CXCL3 in the blood of patients with fulminant myocarditis are significantly higher than those in normal individuals, and the difference is statistically significant. By detecting the expression levels of chemokines CXCL2 and CXCL3 in human blood, fulminant myocarditis can be effectively detected. Verification has shown that CXCL2 and CXCL3, as biomarkers for diagnosing fulminant myocarditis, have high accuracy, specificity, and sensitivity. Therefore, they can be used as detection targets in the diagnosis of patients with fulminant myocarditis, demonstrating good application value.

Description

Technical Field

[0001] This invention relates to the field of protein detection technology, and more specifically, to a biomarker and its application in the diagnosis of fulminant myocarditis. Background Technology

[0002] Fulminant myocarditis is the most severe and unique type of myocarditis. Its main characteristics are a sudden onset and extremely rapid disease progression. Patients quickly develop hemodynamic abnormalities and may be accompanied by respiratory failure and liver and kidney failure. The early mortality rate is over 50%.

[0003] The clinical characteristics and current status of diagnosis and treatment of fulminant myocarditis have the following features:

[0004] 1. The clinical understanding and diagnostic ability of fulminant myocarditis are seriously insufficient, resulting in a very high rate of missed diagnosis and misdiagnosis of these patients, and treatment is often delayed.

[0005] 2. There are currently no effective treatment guidelines worldwide. Fulminant myocarditis has mostly been treated with empirical regimens, including anti-infection therapy, blood pressure control, and cardiac assist devices, but patient survival rates have not been substantially improved.

[0006] 3. The effective treatment of fulminant myocarditis has immense medical and social value. Fulminant myocarditis commonly affects healthy young adults without underlying diseases. Due to its high early mortality rate, it causes significant harm to patients' families and society. However, it is noteworthy that once patients with fulminant myocarditis survive the critical period, their long-term survival rate and quality of life are almost identical to those of the general population. An 11-year follow-up study showed that the survival rate of fulminant myocarditis was significantly higher than that of ordinary acute myocarditis.

[0007] Fulminant myocarditis is clinically extremely dangerous, characterized by rapid disease progression and high mortality. Previous treatments focused on enhancing myocardial contractility and raising blood pressure, but these efforts have not effectively reduced mortality. The fundamental reason for this is a lack of accurate, or even completely erroneous, understanding of the pathophysiology and pathogenesis of fulminant myocarditis, hindering the development of effective treatments and leading to the severe consequences of continuous disease progression and high mortality rates. Therefore, it is urgent to thoroughly research and understand the pathogenesis of fulminant myocarditis, identify new therapeutic targets, and adopt more effective measures to improve or delay its progression, thereby reducing mortality and hospitalization rates. Furthermore, the diagnosis of fulminant myocarditis still lacks specific indicators, necessitating the search for new biomarkers.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to provide a biomarker and its application in the diagnosis of fulminant myocarditis, so as to accurately, quickly and non-invasively determine whether a subject is at risk of fulminant myocarditis.

[0010] This invention is implemented as follows:

[0011] In a first aspect, the present invention provides a biomarker for diagnosing fulminant myocarditis, comprising CXCL2 and / or CXCL3. Preferably, the biomarker is a combination of CXCL2 and CXCL3.

[0012] CXCL2, also known as GRO2 or MIP-2α, and CXCL3, also known as GRO gamma (MIP-2beta), are both neutrophil chemokines belonging to the CXC chemokine subfamily. Mature CXCL2 / 3 proteins bind with high affinity to the type B IL-8 receptor and are effective neutrophil attractants and activators. These chemokines primarily act on leukocytes, regulating their transport.

[0013] Experiments have shown that CXCL2 and / or CXCL3 can effectively distinguish between healthy individuals and those with fulminant myocarditis. In particular, the combination of CXCL2 and CXCL3 demonstrates higher sensitivity and specificity as a biomarker.

[0014] In some embodiments, the biomarkers described above are derived from test samples of the subject, including plasma, serum, cell culture supernatant, urine, and tissue specimens.

[0015] Secondly, the present invention also provides the use of reagents for detecting the above-mentioned biomarkers in the preparation of products for the diagnosis of fulminant myocarditis.

[0016] Thirdly, the present invention also provides a product comprising reagents for detecting the aforementioned biomarkers, the product being used to diagnose whether a subject has fulminant myocarditis.

[0017] In some embodiments, the products described above include kits, devices, and / or equipment for detecting the content of biomarkers in a sample to be tested.

[0018] In some embodiments, the kit described above includes CXCL2 antibody and / or CXCL3 antibody.

[0019] The kits described above also include other diagnostically acceptable auxiliary reagents; wherein the diagnostically acceptable auxiliary reagents are selected from: routine ELISA assay reagents or routine flow cytometry assay reagents.

[0020] Fourthly, the present invention also provides the application of the above-mentioned biomarkers as targets for screening drugs to prevent or treat fulminant myocarditis.

[0021] In some embodiments, the drug includes an active ingredient that can reduce, downregulate, or inhibit the biomarker.

[0022] In this invention, the above-mentioned drug targets CXCL2 and / or CXCL3, and achieves the effect of preventing and treating fulminant myocarditis by inhibiting the expression level of these biomarkers.

[0023] In some embodiments, the active ingredient is selected from inhibitors, neutralizing antibodies, or peptides of the biomarker.

[0024] In this invention, the above-mentioned drugs reduce the expression level of biomarkers in the plasma of patients with fulminant myocarditis by targeting biomarker inhibitors, neutralizing antibodies or peptides, thereby achieving the effect of preventing and treating fulminant myocarditis.

[0025] In some embodiments, the aforementioned drug may also include pharmaceutically acceptable excipients.

[0026] Pharmaceutically acceptable excipients include: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, flavoring agents, anti-adhesion agents, binding agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0027] In some embodiments, the aforementioned fulminant myocarditis is selected from fulminant myocarditis caused or triggered by viruses, bacteria, allergies, drugs, upper respiratory tract infections, and / or intestinal infections.

[0028] Fifthly, the present invention also provides the application of the above-mentioned biomarkers in constructing computational models for predicting or monitoring fulminant myocarditis.

[0029] The input variable for the computational model is the content of biomarkers for fulminant myocarditis.

[0030] The present invention has the following beneficial effects:

[0031] This invention is the first to discover that the expression levels of chemokines CXCL2 and CXCL3 in the blood of patients with fulminant myocarditis are significantly higher than those in normal individuals, and the difference is statistically significant. By detecting the expression levels of chemokines CXCL2 and CXCL3 in human blood, fulminant myocarditis can be effectively detected. Verification has shown that CXCL2 and CXCL3, as biomarkers, have high accuracy, specificity, and sensitivity in diagnosing fulminant myocarditis. Therefore, they can be used as detection targets for the diagnosis of patients with fulminant myocarditis, demonstrating good application value. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The graph shows the changes in the expression of CXCL2 and CXCL3 in the plasma of patients with fulminant myocarditis in Example 1. In the graph, HC represents the control group and FM represents fulminant myocarditis.

[0034] Figure 2 This is a graph showing the survival rate analysis of the four groups of mice in Example 2;

[0035] Figure 3 This refers to the detection of cardiac function in the four groups of mice in Example 2;

[0036] Figure 4 To predict and differentiate fulminant myocarditis from normal controls using CXCL2 and CXCL3, Figure A represents the ROC curve of CXCL2, Figure B represents the ROC curve of CXCL3, and Figure C represents the ROC curve of high-sensitivity C-reactive protein (hs_CRP). Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038] In this invention, the term "biomarker" is also referred to as "biomarker" in general, referring to a biochemical indicator that can mark changes or potential changes in the structure or function of a system, organ, tissue, cell, or subcellular structure. It can be used for disease diagnosis, disease staging, or to evaluate the safety and efficacy of new drugs or therapies in target populations.

[0039] In this invention, the term "diagnosis" and similar terms refer to the identification of a specific disease. Specifically, diagnosis in this invention includes determining whether or not a person has fulminant myocarditis, or assessing the prognosis of fulminant myocarditis. More specifically, diagnosis in this invention refers to determining whether or not a person has fulminant myocarditis, or assessing the prognosis of fulminant myocarditis, based on the expression levels of biomarkers in a combination of biomarkers in the subject's body.

[0040] In this invention, the term "prediction" and related terms refer to a description of the possible outcomes of a particular condition (e.g., fulminant myocarditis).

[0041] Embodiments of the present invention include "monitoring" subjects who may be at risk of developing fulminant myocarditis. These subjects may be patients not diagnosed with fulminant myocarditis, but who may be at risk due to various clinical or medical assessments.

[0042] In this invention, the terms "sample," "biological sample," "test sample," "sample," "sample from subject," and "patient sample" are used interchangeably, and can be samples of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. Samples can be obtained directly from the patient using one of the methods discussed in this invention or other methods known in the art, or samples can be pretreated to alter their properties (e.g., by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, and addition of reagents).

[0043] This invention can use a variety of nucleic acid and protein technologies known to those skilled in the art to detect the level of biomarkers.

[0044] In this invention, the reagent for detecting biomarkers can be antibodies. By using immunological methods based on antigen-antibody reactions, the expression level of the corresponding biomarkers can be detected. Analytical methods used for this purpose include Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and protein chips. These methods are merely illustrative of antibody-antigen immune reactions, and this invention is not limited to these methods.

[0045] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0046] Example 1

[0047] This embodiment describes the detection and analysis of elevated CXCL2 and CXCL3 expression levels in the peripheral blood of patients with fulminant myocarditis.

[0048] The subjects of this study were 10 patients with fulminant myocarditis and 6 control group subjects whose baseline data were matched according to gender, age and other factors.

[0049] Study population and design: From April 2017 to March 2019, hospitalized patients with fulminant myocarditis were continuously enrolled at Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei Province. Inclusion criteria included: ① age greater than 18 years; ② diagnosis of fulminant myocarditis: myocardial biopsy indicating myocarditis or meeting clinical diagnostic criteria (rapidly developing severe hemodynamic instability symptoms, elevated cTnI, echocardiography showing a rapid decrease in left ventricular ejection fraction or below 40%). Exclusion criteria included: ① acute myocardial infarction; ② ordinary acute myocarditis; ③ severe neurological diseases (Alzheimer's disease, progressive Parkinson's syndrome), lower limb amputation, or deafness.

[0050] Inflammatory factor expression profile detection: Peripheral blood was drawn into EDTA anticoagulant tubes after fasting, and centrifuged immediately at 3000 rpm for 8 minutes. Serum was then frozen and stored at -80°C for analysis. Plasma CXCL2 and CXCL3 expression were detected using ELISA kits from QuantiCyto and MULTI SCIENCES, respectively.

[0051] Figure 1 The test results showed that, compared with the control group, the levels of CXCL2 and CXCL3 in the peripheral blood of patients with fulminant myocarditis were significantly increased. In the figure, control represents the control group, and FM represents fulminant myocarditis.

[0052] Example 2

[0053] This example demonstrates how anti-CXCL2 and CXCL3 neutralizing antibodies can inhibit myocardial injury in CVB3-induced A / J fulminant myocarditis mice.

[0054] Mouse model preparation: Male A / J mice aged 4-6 weeks were purchased from the Model Animal Center of Nanjing University and housed in the SPF-grade animal facility of Tongji Medical College, Huazhong University of Science and Technology. One week after acclimatization, mice were administered CVB3 virus via intraperitoneal injection (10 mg / L). 4 Dissolved in 0.1 ml PBS, this group served as the CVB3 fulminant myocarditis model group. Another group of mice with fulminant myocarditis received intraperitoneal injections of anti-CXCL2 and CXCL3 neutralizing antibodies, serving as the treatment group; the third group was a blank control group receiving no treatment; and the fourth group received goat IgG antibody (10... 4 The negative control group was injected intraperitoneally with a solution dissolved in 0.1 ml PBS.

[0055] Mouse cardiac hemodynamics were assessed using a Millar pressure-volume system from Millar Instrument PowerLab. After anesthetizing the animals to an appropriate depth, a midline incision was made in the neck, the right common carotid artery was dissected, its distal end was ligated, and its proximal end was clamped. A V-shaped incision was made in the artery using microscissors, and a microcatheter was inserted until it reached the left ventricle. Signals were recorded via the conduction system to obtain heart rate (HR), left ventricular end-diastolic pressure (PED), left ventricular end-systolic pressure (PES), and the maximum rate of dp / dt of left ventricular pressure descent. min ) and the maximum rate of rise of left ventricular pressure (+dP / dt) max Hemodynamic data, such as hemodynamic data.

[0056] Experimental Results: Seven days after intraperitoneal injection of anti-CXCL2 and CXCL3 neutralizing antibodies, cardiac catheterization was performed. Animals were then sacrificed, and tissue samples were collected for the aforementioned tests. The results showed that, compared with the control group, the survival rate of mice with fulminant myocarditis after CVB3 infection was significantly decreased, while the survival rate of anti-CXCL2 and CXCL3 neutralizing antibodies could increase it (e.g., Figure 2 (As shown). CVB3 infection significantly reduced cardiac function in mice with fulminant myocarditis, while anti-CXCL2 and CXCL3 neutralizing antibodies could enhance its function (e.g., Figure 3 (As shown in the figure). * indicates p < 0.05.

[0057] Example 3

[0058] This example assesses the diagnostic efficacy of peripheral blood CXCL2 and CXCL3 levels for fulminant myocarditis.

[0059] In this embodiment, another group of subjects (31 patients with fulminant myocarditis and 17 control subjects matched for baseline data with no differences according to gender, age, etc.) were selected as the validation group. The study population and study design were the same as in Embodiment 1.

[0060] Inflammatory factor level detection: Peripheral blood was drawn into EDTA anticoagulant tubes after fasting, and immediately centrifuged at 3000 rpm for 8 minutes. Serum was then frozen and stored at -80°C for analysis. Plasma CXCL2 and CXCL3 expression were detected using ELISA kits from QuantiCyto and MULTISCIENCES, respectively.

[0061] like Figure 4 As shown, CXCL2 and CXCL3 have better diagnostic efficacy compared to high-sensitivity C-reactive protein, indicating that CXCL2 and CXCL3 have a significant advantage in distinguishing normal controls from fulminant myocarditis.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting a biomarker in the manufacture of a product for the diagnosis of fulminant myocarditis, characterized in that, The biomarker is CXCL3 protein; The biomarker is derived from the subject's test sample, which is serum.

2. The application of biomarkers as targets for screening drugs to prevent or treat fulminant myocarditis, characterized in that, The biomarker is CXCL3 protein in serum; The drug includes an active ingredient that can reduce, downregulate, or inhibit the biomarker; The active ingredient is selected from the neutralizing antibody of the biomarker.

3. The application according to claim 2, characterized in that, The drug also includes pharmaceutically acceptable excipients.

4. The application of biomarkers in constructing computational models for predicting or monitoring fulminant myocarditis, characterized in that, The biomarker is the CXCL3 protein in serum.

5. The application according to claim 4, characterized in that, The input variable of the computational model is the content of the biomarker.

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