Peripheral blood marker and system for identifying severity of viral myocarditis or coronary stenosis

Abstract

The invention belongs to the field of biological medicine industry, and discloses a peripheral blood marker and system for identifying the severity of viral myocarditis or coronary artery stenosis. The inventor finds for the first time that the average value of LRG1 in blood is not obviously changed compared with that of a normal person when mild and moderate myocarditis and microcirculation angina pectoris with normal coronary artery or coronary artery stenosis smaller than 10% exist, and the blood LRG1 is obviously increased when the coronary artery stenosis of the coronary heart disease exceeds 70%; when severe myocarditis occurs, the blood LRG1 is also obviously increased, so that the increase of the blood LRG1 is a blood marker for patients with severe myocarditis or severe coronary artery lesion, and the blank that blood indexes are lacked in the prior art to prompt the patients to have severe coronary artery stenosis is filled. By detecting the LRG1 content of the peripheral blood and combining with other conventional examinations, the severity of myocarditis or coronary stenosis can be simply, accurately and quickly distinguished, and a corresponding basis is provided for subsequent accurate treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine industry, and particularly relates to a peripheral blood marker for identifying the severity of viral myocarditis or coronary artery stenosis and a system. BACKGROUND

[0002] Overview of viral myocarditis: a variety of viruses can cause myocarditis, among which viral infection of the intestinal tract and upper respiratory tract is most common. Coxsackie virus group A, coxsackie virus group B, ECHO virus, poliovirus are common myocarditis-causing viruses, among which coxsackie virus group B virus is the most common virus. Other viruses include adenovirus, measles virus, mumps virus, hepatitis virus, herpes zoster virus, cytomegalovirus and HIV, etc.

[0003] 1. Clinical manifestations The clinical manifestations of viral myocarditis patients depend on the extent and location of the lesions. Mild cases can have no symptoms, while severe cases can have heart failure, cardiogenic shock and sudden death.

[0004] Patients often have a history of upper respiratory tract or intestinal tract infection 1-3 weeks before the onset, have prodromal symptoms of viral infection, such as fever, general muscle soreness, sore throat, fatigue, nausea, vomiting, diarrhea, and other digestive symptoms, followed by palpitations, chest tightness, chest pain or precordialgia, dizziness, dyspnea, edema, and even Adams-Stokes syndrome; a small number of patients have heart failure or cardiogenic shock. Many symptoms are not directly related to heart discomfort, so it is very easy to ignore. Some patients have very subtle prodromal symptoms and directly enter the acute attack symptoms. Acute attack symptoms: moderate to severe fatigue, palpitations, chest tightness, shortness of breath, chest pain, dizziness, which can worsen significantly within 1-3 days; severe cases can also have symptoms of acute left heart failure, including dyspnea, lower extremity edema, etc.; more critically ill or fulminant patients can directly appear syncope or even sudden death without many prodromal symptoms or discomfort. The most common discomfort for most viral myocarditis patients is palpitations, chest tightness, and occasional "heart stop" (premature beat, arrhythmia). Some myocarditis patients change from acute to recurrent or persistent, with a course of more than 6 months. Patients show easy fatigue, palpitations, chest tightness, chest pain, occasional "heart stop" (premature beat, arrhythmia), and increased discomfort after exercise. Persistent illness can further worsen, showing shortness of breath, edema, etc.

[0005] When the patient appears after the upper respiratory tract infection, diarrhea and other viral infection symptoms, 3 weeks after a series of cardiac abnormalities, such as severe fatigue, palpitations, chest tightness, shortness of breath, chest pain, dizziness, even syncope and other symptoms, should be vigilant myocarditis possible, need to go to the hospital as soon as possible. Mainly in the diagnosis and treatment of cardiovascular medicine: palpitations, chest tightness, chest pain, dyspnea, lower extremity edema and other patients can be treated in the heart. Some patients with critical illness, syncope, severe dyspnea, chest pain and other emergency situations, need to go to the emergency department immediately, or call the emergency number in time. In addition, due to the patient's early upper respiratory tract infection or diarrhea and other conditions, sometimes they will be treated in the fever clinic, respiratory medicine, infectious diseases and other departments.

[0006] 2. Related examination 2.1 General physical examination: doctors will ask about symptoms, medical history, heart auscultation and other symptoms. Physical examination can find: ①cardiac enlargement: mild cases usually have no cardiac enlargement, and severe cases may have mild to moderate cardiac enlargement; ②Changes in heart rate and rhythm: tachycardia, abnormal slow heart rate and various arrhythmias not parallel to fever, among which ventricular premature beats are most common; ③Changes in heart sound: first heart sound is weakened or split, and heart sound can be fetal heart rhythm; ④If there is pericardial involvement, pericardial friction rub can be heard; ⑤Other signs of heart failure: pulmonary rales, jugular venous distension, liver enlargement and bilateral lower extremity edema; ⑥Severe cases may have signs of cardiogenic shock.

[0007] 2.2 Electrocardiogram: various arrhythmias, ST-T changes and other symptoms can be found, common T wave inversion or reduction, and ST segment shift can also be seen, ventricular arrhythmias and atrioventricular block are more common.

[0008] 2.3 X-ray examination: can detect the morphological changes of the heart. Most patients have no large heart shadow; some patients may have enlarged heart shadow; left heart failure may have signs of pulmonary congestion and pulmonary edema; pericardial effusion may have a flask-like change in heart shadow.

[0009] 2.4 Echocardiography: can determine whether there is cardiac enlargement, ventricular wall thickness and movement, cardiac contractility, pericardial effusion, etc. Mild cases may be normal, severe cases may have left ventricular enlargement, ventricular wall motion reduction, cardiac contractility abnormalities, and ventricular filling abnormalities.

[0010] 2.5 Radionuclide myocardial imaging can show the location and extent of myocardial cell necrosis, with high sensitivity and low specificity.

[0011] 2.6 Endomyocardial biopsy Endomyocardial biopsy is an invasive test and is mainly used in patients with severe illness, poor response to treatment, and unknown etiology. Positive results are reliable evidence for the diagnosis of myocarditis. Due to the focal nature of viral myocarditis lesions, false negative results can occur due to sampling errors. Endomyocardial biopsy: Myocardial biopsy is still the gold standard for the diagnosis of myocarditis. As an invasive test, it is not routinely performed, but it is helpful in the diagnosis of myocarditis in patients with severe illness, poor response to treatment, and unknown etiology.

[0012] 2.7 Laboratory tests: Patients with myocarditis may have elevated troponin (T or I), creatine kinase isoenzyme (CK-MB), and increased erythrocyte sedimentation rate. At the same time, the levels of B-type natriuretic peptide (BNP) or N-terminal pro-B-type natriuretic peptide (NT-proBNP) can also indicate the degree of impaired cardiac function in patients. During the acute phase, white blood cell count, erythrocyte sedimentation rate, C-reactive protein, serum creatine phosphokinase isoenzyme (CK-MB), serum troponin T, and serum troponin I may increase.

[0013] 2.8 Virological examination Viruses can be isolated from throat swabs, feces, and myocardial tissue, or viral RNA can be detected using PCR techniques. Serum can be tested for specific anti-viral antibody titers.

[0014] 2.9 Coronary angiography: Patients with suspected myocarditis but with ischemic or infarction changes on electrocardiogram, or older patients who need to rule out acute myocardial infarction, should undergo coronary angiography to confirm the diagnosis.

[0015] 2.10 Cardiac magnetic resonance imaging: This test has great value in the diagnosis of myocarditis. Typical findings include gadolinium delayed enhancement and patchy myocardial enhancement on scanning.

[0016] 3. Diagnosis The clinical diagnosis of viral myocarditis is mainly based on: a history of intestinal infection or respiratory infection before the onset of the disease, clinical manifestations of cardiac damage, positive markers of myocardial injury, other auxiliary tests showing myocardial damage, and positive etiological tests. Viral myocarditis should be considered in the clinical diagnosis. Definitive diagnosis relies on endomyocardial biopsy.

[0017] 3.1 Diagnostic criteria for viral myocarditis First part: History and physical examination Abnormal cardiac manifestations may occur within 3 weeks after viral infection of the upper respiratory tract, diarrhea, etc. Symptoms such as severe fatigue, chest tightness, dizziness, etc. may be found during the doctor's examination: significant reduction in first heart sound at the apex, diastolic gallop rhythm, pericardial friction rub, cardiac enlargement, congestive heart failure, or Arthus syndrome, etc.

[0018] Second part: Cardiac-related abnormalities appear 3 weeks after infection Newly developed arrhythmias or ECG changes within 3 weeks after the infection: sinus tachycardia, atrioventricular block, sinus arrest or bundle branch block. Multifocal, paired ventricular premature beats, spontaneous atrial or junctional tachycardia, paroxysmal or non-paroxysmal ventricular tachycardia, atrial or ventricular flutter or fibrillation. ST segment depression > 0.01 mV or ST segment elevation or abnormal Q wave in more than two leads.

[0019] Part III: Reference index of myocardial injury Significant increase of serum cardiac troponin I or T (emphasized quantitative determination), creatine kinase isoenzyme (CK-MB) during the course of the disease; Echocardiography shows cardiac chamber enlargement or abnormal wall motion, and / or radionuclide cardiac function test confirms left ventricular systolic or diastolic dysfunction.

[0020] Part IV: Etiological basis Detection of virus, viral gene fragments or viral protein antigens in endocardium, myocardium, pericardium or pericardial puncture fluid during the acute phase; Virus antibody positive, which should be judged according to different detection methods; Virus-specific IgM positive (according to the diagnostic criteria of each laboratory, under strict quality control conditions). If enterovirus nucleic acid is detected in the patient's blood at the same time, it is more supportive of recent viral infection in the patient.

[0021] For patients with any one of the above Part I, Part II, and any two of Part III, and after excluding other causes of myocardial disease, acute viral myocarditis can be clinically diagnosed. If the first item in Part IV is met, acute viral myocarditis can be directly diagnosed etiologically; if only the second and third items in Part IV are met, acute viral myocarditis can only be tentatively diagnosed etiologically. If only a few premature beats or mild T wave changes occur within 3 weeks after viral infection, the doctor will not easily diagnose acute viral myocarditis. For patients who are difficult to diagnose, the doctor will arrange long-term follow-up and, if conditions permit, will recommend endomyocardial biopsy for viral gene detection and pathological examination.

[0022] 3.2 Diagnosis criteria for fulminant myocarditis Fulminant myocarditis is characterized by rapid clinical progression, severe illness, and direct threat to life. Medically, fulminant myocarditis is generally defined as an inflammatory disease of the myocardium with a sudden onset and severe hemodynamic disturbances. Diagnosis is made by combining the patient's clinical manifestations with laboratory and imaging examinations. A clinical diagnosis of fulminant myocarditis can be made when there is a sudden onset, rapid development of severe hemodynamic disturbances (syncope, sudden death), laboratory tests showing severe myocardial damage, and echocardiography showing diffuse wall motion deterioration. If the patient also presents with one or more of the following: Adams-Stokes syndrome, congestive heart failure with or without myocardial infarction-like electrocardiographic changes, cardiogenic shock, acute renal failure, persistent ventricular tachycardia with hypotension, or myocarditis / pericarditis, a diagnosis of severe fulminant viral myocarditis can be made.

[0023] 3.3 Differential Diagnosis While considering a diagnosis of viral myocarditis, it is important to rule out hyperthyroidism, mitral valve prolapse syndrome, coronary artery disease (such as acute myocardial infarction), stress cardiomyopathy (Takotsubo syndrome), and other myocardial disorders such as connective tissue diseases, metabolic diseases, and Keshan disease. It is also necessary to exclude β-receptor hyperfunction, other myocardial disorders, and cardiac damage caused by drugs and toxins.

[0024] 4. Treatment There is no specific treatment for myocarditis. Treatment mainly focuses on symptomatic and supportive care, supplemented by treatment for viral infection and myocardial inflammation.

[0025] 4.1 General Treatment Early bed rest is essential to reduce the burden on the heart. A diet of easily digestible and protein-rich foods is recommended. Patients should be instructed to rest in bed and avoid emotional stress and fluctuations. Blood pressure, heart rate, electrocardiogram, and myocardial enzyme levels should be monitored. During the acute phase of myocarditis, physical activity should be restricted for at least 6 months. A light, easily digestible, and nutritious diet, with frequent small meals, is recommended.

[0026] 4.2. Antiviral treatment It is mainly used in the early stages of disease.

[0027] 4.3. Nourishing the myocardium In cases of acute myocarditis, free radical scavengers are used, including intravenous or oral vitamin C, coenzyme Q10, vitamin B10, ATP, inosine, cyclic adenosine monophosphate, cytochrome C, and tanshinone. Other treatments include improving myocardial energy metabolism (e.g., creatine phosphate, coenzyme Q10); supplementing with water-soluble vitamins (e.g., vitamin C) and fat-soluble vitamins; and providing symptomatic supportive care.

[0028] 4.4. Glucocorticoids Not for routine use. Consider using it 10–30 days after onset if other treatments are ineffective.

[0029] 4.5. Symptomatic treatment When cardiogenic shock, heart failure, bradycardia, or tachyarrhythmia occurs, appropriate symptomatic treatment should be given.

[0030] 4.6 Treatment of fulminant myocarditis This type of myocarditis is a critical condition, and the current recommended treatment is a comprehensive approach based on life support. Doctors will initiate aggressive, comprehensive treatment methods as early as possible. General treatment includes strict bed rest and nutritional support; common drug treatments include nourishing the myocardium, reducing cardiac load, and protecting the gastric mucosa; and anti-infective and antiviral treatments. Glucocorticoid therapy; intravenous immunoglobulin support; plasma and blood purification therapy; A range of life support measures, such as intra-aortic balloon pump (IABP), extracorporeal membrane oxygenation (ECMO), mechanical ventilation, and temporary pacemaker implantation; Heart transplantation may be necessary if required.

[0031] 5. Prognosis Early diagnosis and treatment of viral myocarditis are crucial. Most cases of acute myocarditis have a good prognosis, with approximately 50% recovering within 2-4 weeks. However, a small percentage of patients may experience prolonged illness, developing chronic or persistent myocarditis or cardiomyopathy. Due to the lack of a cure for viral infection and individual differences in reactivity, a small number of patients may develop dilated cardiomyopathy. Patients who have developed dilated cardiomyopathy should receive standardized treatment as for this condition. The mortality rate in the acute phase of fulminant myocarditis can be as high as 80%, but once the acute danger period is over, the long-term prognosis is good. A very small number of patients die from severe arrhythmias, heart failure, or cardiogenic shock.

[0032] 6. Differential diagnosis between coronary heart disease and viral myocarditis Coronary artery disease (CAD) is a heart condition caused by lesions in the coronary arteries. If the coronary arteries are narrowed by more than 70%, treatment such as stent placement is necessary. At this stage, patients may experience symptoms such as palpitations, chest tightness, chest pain, upper abdominal pain, nausea, fatigue, dizziness, and insomnia, which are similar to the symptoms of myocarditis. In some cases, severe CAD can lead to myocardial infarction, characterized by changes in cardiac enzymes and troponin levels. Coronary angiography will show coronary artery stenosis exceeding 70% or complete blockage. In some cases, CAD may not yet lead to myocardial infarction or myocardial necrosis, but coronary angiography and coronary CT angiography will show coronary artery stenosis exceeding 70%. At this stage, there are no elevated cardiac enzymes or troponin levels in the patient's blood, and currently, no other blood indicators suggest that the patient has developed coronary artery stenosis requiring interventional treatment.

[0033] 7. The relationship between peripheral blood proteins in viral myocarditis and severe coronary artery stenosis Patients with viral myocarditis and severe coronary artery stenosis exhibit related symptoms. Every symptom has a material basis, manifesting as changes in gene expression and proteins. Proteins are the functional executors of genes; research into protein structure, location, and protein-protein interactions will provide a direct foundation for elucidating the nature of life phenomena. Almost all physiological and pathological processes, as well as the effects of drugs and environmental factors, depend on proteins and cause changes in the proteome. Before any disease manifests any perceptible symptoms, certain proteins have certainly already changed. Therefore, identifying key and marker proteins for various diseases is of great significance for disease diagnosis, pathological research, and drug screening.

[0034] Leucine-rich repeat (LRR) proteins are a family of proteins consisting of eight leucine-rich repeat sequences. Studies have shown that LRR proteins are transmembrane proteins that mainly participate in normal physiological activities as cell adhesion molecules or ligand-binding proteins.

[0035] Leucine-rich α2-glycoprotein 1 (LRG1) is a member of the leucine-rich repeat family and belongs to the LRR protein family. Previous studies have shown that LRG1 participates in important physiological and pathological processes, such as protein-protein interactions, signal transduction, and cell adhesion.

[0036] Currently, various proteins with leucine-rich repeat structures have been found in the nervous system. In some neurodegenerative diseases, decreased concentrations of LRG1 in cerebrospinal fluid (CSF) have been detected, and human LRG1 expression has been observed in the anatomically examined cerebral cortex. Previous studies have performed proteomic analysis on CSF samples from healthy individuals and patients with idiopathic normal pressure hydrocephalus (INPH), finding a specific increase in LRG1 expression in the CSF of INPH patients. Therefore, LRG1 has potential value in the clinical diagnosis of INPH. Abnormal LRG1 expression is associated with the development of various tumors, such as glioma, lung cancer, colon cancer, epithelial ovarian cancer, and hepatocellular carcinoma.

[0037] In studies of retinopathy caused by vascular remodeling, LRG1 was found to promote endothelial cell mitosis and angiogenesis. This hypothesis was further confirmed in in vitro human vascular endothelial cell (HUVEC) lumen formation assays. Providing recombinant human LRG1 as a mediator resulted in a significant increase in lumen formation and branching, while anti-LRG1 antibodies significantly blocked lumen formation. Other studies have shown that the angiogenic activity of LRG1 is not limited to the eye and may play important roles in other major biological processes, including tumors and immune responses. The regulatory role of LRG1 on the TGF-β1 signaling pathway provides strong evidence for the function of LRG1. LRG1 is a very promising therapeutic target for controlling ocular angiogenesis and may play an important role in other diseases such as cancer.

[0038] The function of LRG1 may be related to its LRR structure. LRG is thought to regulate the function of other proteins, such as cytochrome C, by binding to them, thereby limiting the function of some proteins. Gliomas originate from glial cells and are the most common tumors of the central nervous system, accounting for 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors. Related experiments have shown that LRG1 plays an important role in promoting the proliferation, migration, and invasion of glioma cells. These functions are achieved by mediating the transforming growth factor β1 (TGF-β1) signaling pathway. Furthermore, LRG1 can promote endothelial cell proliferation and tumor angiogenesis through the TGF-β signaling pathway, suggesting that LRG1 may be involved in the occurrence and development of astrocytic malignancies. Previous studies have shown that inhibiting LRG1 expression inhibits the in vitro growth of glioma cells and delays the occurrence of gliomas in xenograft mouse models, providing evidence for the tropism of LRG1 on glioma pathological changes.

[0039] The inventor's prior Chinese patent application CN117110626A discloses that the average value of LRG1 in the blood is higher in individuals with carotid artery disease than in normal individuals, and that the detachment of plaques from carotid vessels increases more significantly as the disease progresses. As a peripheral blood biomarker for dangerous carotid artery disease, it exhibits high sensitivity and specificity and can be used in conjunction with ultrasound results for the diagnosis of carotid artery disease. CN115856325A discloses that the level of peripheral blood LRG1 is closely related to lower limb arterial injury. By detecting the level of LRG1 in peripheral blood, lower limb arterial injury, especially lower limb arteriosclerosis or plaque, lower limb arterial stenosis, thrombosis, or occlusion, can be well predicted. In particular, it can effectively predict lower limb arteriosclerosis or plaque, lower limb arterial stenosis, thrombosis, or occlusion caused by hypertension, hyperlipidemia, or hyperglycemia. CN110261617A discloses that LRG1 can be used as a peripheral blood biomarker for cerebral hemorrhage. Summary of the Invention

[0040] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide a peripheral blood marker and system for identifying the severity of viral myocarditis or coronary artery stenosis.

[0041] The technical solution adopted in this invention is: The first aspect of the present invention provides: Application of LRG1 quantitative reagent in the preparation of diagnostic or auxiliary diagnostic reagents for differentiating the severity of viral myocarditis or coronary artery stenosis.

[0042] In some instances, the LRG1 quantification reagent is an ELISA reagent, an immunofluorescence reagent, or a mass spectrometry analysis reagent.

[0043] In some instances, the samples tested by the LRG1 quantitative reagent are peripheral blood.

[0044] In some instances, a peripheral blood LRG1 level below 5 ng / mL is considered mild to moderate viral myocarditis or coronary artery stenosis of less than 10%; a peripheral blood LRG1 level above 5 ng / mL is considered coronary artery stenosis of more than 70% or severe myocarditis.

[0045] In some cases, coronary artery stenosis of more than 70% includes patients with elevated blood levels of cardiac enzymes and troponin who have already experienced myocardial infarction, as well as patients with normal levels of cardiac enzymes and troponin who have not yet experienced myocardial infarction.

[0046] A second aspect of the present invention provides: A diagnostic or auxiliary diagnostic system for differentiating the severity of viral myocarditis or coronary artery stenosis, comprising: The LRG1 quantification device is used to quantify the amount of LRG1 in samples from patients with viral myocarditis. The analytical device, combined with the LRG1 content, determines the severity of viral myocarditis or the severity of coronary artery stenosis. The result output device is used to output the analysis results of the analysis device.

[0047] When conducting the analysis, other known methods can be combined for preliminary identification to achieve better differentiation.

[0048] In some instances, the LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

[0049] In some cases, high LRG1 levels are used to differentiate between severe viral myocarditis and coronary artery stenosis of 70% or more.

[0050] In some cases, high LRG1 levels refer to levels exceeding 5 ng / mL in peripheral blood samples.

[0051] In some instances, a peripheral blood LRG1 level below 5 ng / mL is considered mild to moderate viral myocarditis or coronary artery stenosis of less than 10%; a peripheral blood LRG1 level above 5 ng / mL is considered coronary artery stenosis of more than 70% or severe myocarditis.

[0052] In some cases, coronary artery stenosis of more than 70% includes patients with elevated blood levels of cardiac enzymes and troponin who have already experienced myocardial infarction, as well as patients with normal levels of cardiac enzymes and troponin who have not yet experienced myocardial infarction.

[0053] The beneficial effects of this invention are: The inventors' research is the first to discover that in mild to moderate myocarditis and microcirculatory angina with normal coronary arteries or coronary artery stenosis less than 10%, the average serum LRG1 level is not significantly different from that of normal individuals. However, serum LRG1 levels rise significantly when coronary artery stenosis exceeds 70%, regardless of whether cardiac enzymes and troponin are elevated (indicating myocardial infarction) or cardiac enzymes are not elevated (indicating no myocardial infarction yet). Serious myocarditis also shows significantly elevated serum LRG1 levels, indicating that elevated serum LRG1 is a blood marker for severe myocarditis or severe coronary artery stenosis, filling a gap in existing technologies that lack blood indicators to suggest severe coronary artery stenosis. Severe myocarditis, especially fulminant myocarditis, is a disease with a high mortality rate, and the elevated serum LRG1 levels in patients with severe myocarditis provide a new approach for rapid screening of this condition. Therefore, by detecting peripheral blood LRG1 levels, combined with other routine examinations, it is possible to easily, accurately, and quickly differentiate the severity of coronary artery stenosis caused by severe myocarditis, coronary artery plaques, thrombosis, etc., providing a basis for accurate subsequent treatment. Detailed Implementation

[0054] The technical solution of the present invention will be further explained below with reference to experiments.

[0055] Screening and collection of clinical samples: We collected data from nearly 8 years of study, including 30 cases of mild to moderate myocarditis and 22 cases of severe myocarditis. Thirty cases had coronary artery stenosis exceeding 70% due to plaque, thrombosis, or spasm, accompanied by elevated cardiac enzymes and troponin. Twenty-six cases had coronary artery stenosis exceeding 70% due to plaque, thrombosis, or spasm, but without elevated cardiac enzymes or troponin. Thirty cases presented with chest pain and normal coronary angiography and CT scans, or with coronary artery stenosis less than 10%. Gender and age were recorded. All patients underwent blood pressure, blood lipids, blood glucose, liver function, kidney function, coagulation parameters, and ECG. All cases were confirmed by ultrasound, chest CT, coronary CTA, echocardiography, and coronary angiography. Severe myocarditis was also confirmed by coronary CTA and coronary angiography to be without coronary artery stenosis. Sixty age- and gender-matched healthy individuals were selected as controls. Specimen collection: 5 mL of venous blood was collected from patients who met the inclusion criteria, allowed to stand for 15 min, centrifuged at 3000 r / min for 15 min, the serum was extracted, placed in a sterile cryovial, and stored at -80℃ for research.

[0056] Study of blood samples from case patients The method for separating serum is as follows: serum and cells are separated by high-speed centrifugation.

[0057] Main equipment, materials, and reagents: homogenizer, high-speed centrifuge, 3kDa ultrafiltration centrifuge tubes, ICP-MS. The water used in the experiment was ultrapure water with a concentration of 18.2 MΩ / cm. Sample pretreatment methods: 1) Fully automated biochemical analyzer (one each of Bekmann DXC800 and AU400), 0.01% electronic analytical balance (Shimadzu), ELISA reader, Roche electrochemiluminescence automated immunoassay analyzer (Elecsys 2010), specific protein analyzer (imported), purified water system (MEDICA60), electric thermostatic water bath (DK-600), low-temperature high-speed centrifuge (Sigma), ultra-low temperature freezer (Thermo Fisher Scientific), low-temperature freezer (HFC350, Germany), etc. The above equipment fully guarantees the implementation of this project. Reagent kits: Shenzhen-made ELISA LRG1 detection kit.

[0058] Statistical methods: The t-test was used to compare the means of each group. SPSS statistical software was used, and P<0.05 was considered statistically significant.

[0059] result: Record gender, age, and blood pressure, blood lipids, blood glucose, liver function, kidney function, coagulation parameters, ECG, etc. for all patients. All cases were confirmed by ultrasound, chest CT, coronary CTA, echocardiography, and coronary angiography. Severe myocarditis was also confirmed by coronary CTA and coronary angiography to be without coronary artery stenosis. The normal range was determined by the mean plus 2 standard deviations. The upper limit of normal cutoff value of the Shenzhen-made LRG1 test kit (LRG1 Shenzhen) was 5 ng / ml. The Shenzhen-made reagent was used to test 30 cases of mild to moderate myocarditis, 30 cases of coronary artery stenosis of more than 70% with elevated cardiac enzymes and troponin, 26 cases of coronary artery stenosis of more than 70% without elevated cardiac enzymes and troponin, 22 cases of severe and fulminant myocarditis, and 30 patients with chest pain and normal coronary angiography and coronary CT or coronary artery stenosis of less than 10%. The changes in peripheral blood LRG1 in patients with myocarditis, angina pectoris, and myocardial infarction are shown in Table 1.

[0060] Table 1. Changes in serum LRG in myocarditis, angina pectoris, and myocardial infarction (X±SD, unit ng / ml) Group Example LRG1 / ng / ml Normal 60 3.2±1.1 Mild to moderate myocarditis 30 3.8±2.2 Microcirculation angina pectoris with normal coronary angiography or less than 10% coronary stenosis 30 3.9±2.5 More than 70% coronary stenosis with high myocardial enzyme 30 19.8±2.6 More than 70% coronary stenosis without high myocardial enzyme 26 18.6±2.5 Severe myocarditis 22 20.3±2.8 In patients with mild to moderate myocarditis and those with chest pain and normal coronary angiography or stenosis of less than 10%, LRG1 levels were not significantly different compared to normal individuals (P>0.05); however, LRG1 levels were significantly elevated in patients with severe myocarditis (P<0.001). In patients with coronary artery plaque, thrombosis, or spasm leading to coronary artery stenosis exceeding 70%, regardless of the presence or absence of elevated cardiac enzymes and troponin, serum LRG1 levels were significantly elevated (P<0.001). The sensitivity and specificity of LRG1 in diagnosing myocarditis are shown in Table 2.

[0061] Sensitivity (positive concordance rate) = (Number of true positives / (Number of true positives + Number of false negatives)) * 100%, the percentage of patients correctly diagnosed. Specificity (negative concordance rate) = (Number of true negatives / (Number of true negatives + Number of false positives)) * 100%, the percentage of correctly identifying non-patients. False positive rate = Number of false positives / Number of negative results (gold standard) False negative rate = Number of false negatives / Number of gold standard positives The normal upper limit cutoff value of the Shenzhen-made LRG1 test kit is 5 ng / ml. The kit was used to test 30 cases of mild to moderate myocarditis, 30 cases of coronary artery stenosis exceeding 70% with elevated myocardial enzymes, 26 cases of coronary artery stenosis exceeding 70% but with normal myocardial enzymes, 22 cases of severe myocarditis, and 30 patients with chest pain and normal coronary angiography and coronary CT or coronary artery stenosis less than 10%. Results showed that in mild to moderate myocarditis, 2 cases had LRG1 levels exceeding the cutoff value, and 28 cases were below normal. In the 30 cases of coronary artery stenosis with elevated myocardial enzymes, 26 cases had elevated LRG1 levels, and 4 cases had normal levels. In the 26 cases of coronary artery stenosis exceeding 70% but with normal myocardial enzymes, 21 cases had elevated LRG1 levels, and 5 cases had normal levels. In the 22 cases of severe myocarditis, 21 cases had elevated LRG1 levels, and 1 case had no elevated levels. Three patients with chest pain and normal coronary angiography and coronary CT, or with coronary artery stenosis of less than 10%, had elevated serum LRG1 levels, while 27 had normal levels. Among 60 normal controls (asymptomatic and without physical signs), 12 had serum LRG1 levels exceeding the cutoff value (false positive rate of 20%), and 48 had normal serum LRG1 levels, with a specificity of 80%. The sensitivity and specificity of LRG1 in diagnosing myocarditis are shown in Table 2.

[0062] Table 2: Sensitivity and specificity of LRG1 (with a cutoff value of 5 ng / ml) in diagnosing myocarditis of different degrees and coronary artery stenosis exceeding 70%.

[0063] Table 3 shows the changes in LRG1 before and after treatment for severe myocarditis.

[0064] Table 3. Changes in serum LRG1 levels after recovery from severe myocarditis (X±SD, unit ng / ml) Group Example LRG1 / ng / ml Normal 60 3.2±1.1 Before treatment of severe myocarditis 22 20.3±2.8 After treatment of severe myocarditis 12 3.3± 2.1 Note: Severe myocarditis has a high mortality rate. Of the 22 cases of severe myocarditis, 10 died or were transferred to other hospitals during treatment, and their post-treatment LRG1 data were missing. The results in Table 3 show that LRG1 levels were significantly reduced after treatment in severe myocarditis compared to before treatment (p<0.001), and were comparable to normal levels (p>0.05).

[0065] The results of this study indicate that the average serum LRG1 level is not significantly different from that of normal individuals in mild to moderate myocarditis and microcirculatory angina with normal coronary arteries or coronary artery stenosis of less than 10%. Serum LRG1 levels are significantly elevated in patients with coronary artery stenosis exceeding 70% in coronary heart disease, regardless of whether myocardial enzymes and troponin are elevated (indicating myocardial infarction) or not elevated (indicating no myocardial infarction). Serum LRG1 levels are also significantly elevated in severe myocarditis. This helps in the clinical differential diagnosis of acute mild to moderate myocarditis, microcirculatory angina, myocardial infarction in patients with coronary heart disease, severe coronary artery stenosis without myocardial infarction, and severe myocarditis. This suggests that elevated serum LRG1 is a blood marker for severe myocarditis or severe coronary artery disease. In particular, our study fills a gap in the understanding of patients with severe coronary artery stenosis exceeding 70% but not elevated myocardial enzymes and troponin, as previously there were no corresponding blood indicators suggesting severe coronary artery stenosis. Severe myocarditis, especially fulminant myocarditis, is a disease with a high mortality rate. The elevated LRG1 level in the blood of patients with severe myocarditis has provided a new approach for the rapid screening of severe myocarditis.

[0066] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

[0067] References: [1] Tessa Gordon 1, Nasim Amirjani, David CEdwards, K Ming Chan, Briefpost-surgical electrical stimulation accelerates axon regeneration and musclereinnervation without affecting the functional measures in carpal tunnelsyndrome patients. Trial Exp Neurol. 2010 May;223(1):192-202. [2] Proteomic analysis of hippocampus in mice following long-term exposure to low levels of copper. Qian Sun, Ming Ying, Quan Ma, Zhijun Huang, Liangyu Zou, Jianjun Liu, Zhixiong Zhuang, Xifei Yang. Toxicol. Res., 2016, 4, 28 [3] Serumproteomic analysis reveals potential serum biomarkers for occupational medicamentosa-like dermatitis caused by trichloroethylene. Peiwu Huang a,d,1,Xiaohu Ren a,1,Zhijun Huang c,1,Xifei Yang a,Wenxu Hong a,Yanfang Zhang b,Hang Zhang a,d,Wei Liu a,Haiyan Huang a,Xinfeng Huang a,Desheng Wua,Linqing Yang a,Haiyan Tang b,Li Zhou a,Xuan Li a,Jianjun Liu. Toxicology Letters 2014,6,21(sci 3.75) [4] 1. Ditochondrial proteomic alterations caused by long-term low –dose copper exposure in mouse cortex. Xuemei Lin, Gang Wei, Zhijun Huang,etc. Toxicology Letter, 2016 [5]. Detection of coagulation factor VIIR353Q genotype in Han Chinese patients with cerebral hemorrhage in South China. Chinese Journal of Arteriosclerosis, 2007, 6. [6]. Research progress on the application of gene chips in the diagnosis and treatment of cardiovascular diseases, 2005, 10, *Chinese Journal of Medical Engineering*. [7] Application of factor DNA molecular genetic markers RFLP, STR, and SNP in coagulation genotype analysis. Chinese Journal of Misdiagnosis Medicine, 2006, 4.

Claims

1. Application of LRG1 quantitative reagent in the preparation of diagnostic or auxiliary diagnostic reagents for differentiating the severity of viral myocarditis or coronary artery stenosis.

2. The application according to claim 1, characterized in that, The LRG1 quantitative reagents are ELISA reagents, immunofluorescence reagents, and mass spectrometry analysis reagents.

3. The application according to claim 1, characterized in that, The sample tested by the LRG1 quantitative reagent is peripheral blood.

4. The application according to claim 3, characterized in that, A peripheral blood LRG1 level below 5 ng / mL is considered mild to moderate viral myocarditis or coronary artery stenosis of less than 10%; a peripheral blood LRG1 level above 5 ng / mL is considered coronary artery stenosis of more than 70% or severe myocarditis.

5. A diagnostic or auxiliary diagnostic system for differentiating the severity of viral myocarditis or coronary artery stenosis, comprising: The LRG1 quantification device is used to quantify the amount of LRG1 in samples from patients with viral myocarditis. The analytical device, combined with the LRG1 content, determines the severity of viral myocarditis or the severity of coronary artery stenosis. The result output device is used to output the analysis results of the analysis device.

6. The differential diagnosis or auxiliary diagnosis system according to claim 5, characterized in that, The LRG1 quantification device is an ELISA detection device, an immunoblotting device, an immunofluorescence device, or a mass spectrometry device.

7. The diagnostic or auxiliary diagnostic system according to claim 5, characterized in that, High levels of LRG1 are used to differentiate between severe viral myocarditis and coronary artery stenosis of 70% or more.

8. The diagnostic or auxiliary diagnostic system according to claim 7, characterized in that, High LRG1 content refers to its content in peripheral blood samples exceeding 5 ng / mL.

9. The diagnostic or auxiliary diagnostic system according to claim 5, characterized in that, The sample was peripheral blood.

10. The diagnostic or auxiliary diagnostic system according to claim 5, characterized in that, A peripheral blood LRG1 level below 5 ng / mL indicates mild to moderate viral myocarditis or chest pain with coronary artery stenosis of less than 10%; a peripheral blood LRG1 level above 5 ng / mL indicates coronary artery stenosis of more than 70% or severe myocarditis.

Images