Application of N-glycan as acute ischemic stroke biomarker

Abstract

The invention provides application of N-glycan as an acute ischemic stroke biomarker, and the application is as follows: serum characteristic N-glycan is taken as the human acute ischemic stroke biomarker, and the serum characteristic N-glycan is one or more of H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3; or the brain tissue characteristic N-glycan is used as a biomarker of the acute ischemic stroke of the mouse, and the brain tissue characteristic N-glycan is one or more of H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1. The marker provided by the invention has the advantages of strong specificity, small trauma and low cost when being used for predicting the early ischemic stroke.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of N-glycan as a biomarker for acute ischemic stroke. Background Art

[0002] Acute ischemic stroke (AIS) is characterized by high morbidity, mortality, disability, and recurrence rates, placing a significant burden on public health systems. Revascularization therapy is the only clinical treatment for AIS, but the therapeutic window (within 6 hours of onset) is short. Therefore, early and accurate diagnosis and timely revascularization therapy are crucial for patients with ischemic stroke (IS).

[0003] In clinical applications, although advances in imaging technologies such as dynamic CT and MRI enhanced scanning have promoted the detection and qualitative analysis of IS, the high cost and technical requirements make it unsuitable for large-scale detection and screening in clinical applications. The use of biomarkers to supplement IS detection and recanalization treatment of patients is a hot research direction. However, early blood markers of AIS are still limited in clinical practice, and the detection accuracy is low. The development of new and more specific blood biomarkers for accurate diagnosis and prediction of ischemic stroke, especially early progression, is of great significance.

[0004] Protein N-glycosylation is one of the most common post-translational modifications and plays a key role in various biological activities, including cell adhesion, cell migration, and signal transduction. Due to their unique bioselectivity, N-glycans attached to glycoproteins are directly involved in disease processes, making them an important source of blood-based biomarkers for many diseases. However, there are currently no N-glycan biomarkers for IS. Summary of the Invention

[0005] To solve the problems existing in the background technology, the present invention provides an application of N-glycan as a biomarker for acute ischemic stroke.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, the present invention provides a use of N-glycans as a biomarker for acute ischemic stroke, the use comprising:

[0008] Using human serum characteristic N-glycans as a biomarker for human acute ischemic stroke, wherein the human serum characteristic N-glycans are one or more of H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3; or

[0009] Using brain tissue characteristic N-glycans as a biomarker for acute ischemic stroke in mice, wherein the brain tissue characteristic N-glycans are one or more of H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1; or

[0010] The mouse serum characteristic N-glycans were used as biomarkers for acute ischemic stroke in mice, and the mouse serum characteristic N-glycans were H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1 and H6N5G4.

[0011] According to the above scheme, the human serum characteristic N-glycans in patients with acute ischemic stroke have significant changes compared with the human serum characteristic N-glycans in the normal population, the brain tissue characteristic N-glycans in mice with acute ischemic stroke have significant changes compared with the normal brain tissue characteristic N-glycans in mice, and the mouse serum characteristic N-glycans in mice with acute ischemic stroke have significant changes compared with the normal mouse serum characteristic N-glycans, p < 0.05.

[0012] According to the above scheme, the human serum characteristic N-glycan used as a biomarker for human acute ischemic stroke is a fucosylated N-glycan, which is at least one of H4N4F1, H5N4F1, H5N4F1S1 and H5N4F1S2.

[0013] According to the above scheme, the human serum characteristic N-glycan used as a biomarker for human acute ischemic stroke is fucosylated N-glycan H5N4F1S1 or H4N4F1, and the area under the curve of the receiver operating characteristic curve thereof is ≥0.71.

[0014] According to the above scheme, the human serum characteristic N-glycan used as a biomarker for human acute ischemic stroke is a composition consisting of fucosylated N-glycans H4N4F1, H5N4F1, H5N4F1S1 and H5N4F1S2, and the area under the curve of the receiver operating characteristic curve of the composition is ≥0.70.

[0015] According to the above scheme, the characteristic N-glycan of brain tissue serving as a biomarker for acute ischemic stroke in mice is a fucosylated N-glycan, including at least one of H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1.

[0016] According to the above scheme, the characteristic N-glycan of brain tissue serving as a biomarker for acute ischemic stroke in mice is a composition consisting of fucosylated N-glycans H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1, and the area under the curve of the receiver operating characteristic curve of the composition is ≥0.98.

[0017] According to the above scheme, the characteristic N-glycan of brain tissue used as a biomarker for acute ischemic stroke in mice is the fucosylated N-glycan H5N5F2, and the area under the curve of the receiver operating curve thereof is ≥0.96.

[0018] According to the above scheme, the mouse serum characteristic N-glycan H5N4G1 is used as a marker for the progression of acute ischemic stroke in mice.

[0019] In a second aspect, the present invention provides a kit for detecting acute ischemic stroke, comprising:

[0020] A reagent for detecting human serum characteristic N-glycans, wherein the serum characteristic N-glycans are at least one of the following: H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2, and H6N5S3; or

[0021] A reagent for detecting characteristic N-glycans of mouse brain tissue, wherein the characteristic N-glycans of the brain tissue are at least one of the following: H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1; or

[0022] A reagent for detecting mouse serum characteristic N-glycan, wherein the mouse serum characteristic N-glycan is at least one of the following: H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1 and H6N5G4.

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

[0024] The present invention detects and analyzes human serum characteristic N-glycans in patients with acute ischemic stroke and healthy people, and finds that the expression levels of six characteristic N-glycans, H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3, are significantly different. Any one or more combinations of these six characteristic N-glycans can be used as markers for human acute ischemic stroke and used to determine acute ischemic stroke. Receiver operating curve (ROC) analysis shows that the area under the curve is ≥0.63, and the better. The area under the curve was ≥0.70; by detecting and analyzing the characteristic N-glycans in the brain tissue of mice with acute ischemic stroke and healthy mice, it was found that the expression levels of 9 characteristic N-glycans, including H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1, were significantly different. Any one or more combinations of them can be used as markers for acute ischemic stroke in mice, and can be used to judge acute ischemic stroke. Receiver operating curve (ROC) analysis showed that the area under the curve was ≥0.78, the better, the area under the curve was ≥0.88, and the better, the area under the curve was ≥0.98; at the same time, the characteristic N-glycans of mouse serum were detected and analyzed in acute ischemic stroke mice and healthy mice, and it was found that H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1 and H6N5 There were significant differences in the expression levels of these 12 characteristic N-glycans of G4. Any one or more combinations of these 12 characteristic N-glycans can be used as markers for acute ischemic stroke in mice, and can be used to determine acute ischemic stroke. Receiver operating characteristic (ROC) analysis showed that the area under the curve was ≥0.75, and the best was ≥0.99, and even better was 1. Furthermore, it was found that the expression levels of H5N4G1 were significantly different at different stages of ischemic stroke, and can be used to indicate the progression of acute ischemic stroke in mice.

[0025] The marker of the present invention has high specificity, little trauma and low cost in predicting early ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the early ischemic stroke mouse modeling and its N-glycan map are shown in the following table: Figure 1 A is the result of establishing the mouse model of early ischemic stroke. Figure 1 B is the MALDI-MS mass spectrum of mouse brain tissue. Figure 1 C, D, and E show the distribution of N-glycans in mouse brain tissue;

[0027] Figure 2The results of the analysis of the N-glycans with significant changes in the brain tissue of the mice of the present invention are shown in FIG. Figure 2 A is a comparative diagram of the changes in 9 brain tissue characteristic N-glycans in the brain tissues of healthy mice and early ischemic stroke mice. Figure 2 B is the OPLS-DA analysis of N-glycans with significant changes in mouse brain tissue for early ischemic stroke and control groups. Figure 2 C is the ROC analysis of 9 N-glycans with significant changes in brain tissues of control and early ischemic stroke model mice, *, p < 0.05;

[0028] Figure 3 The results of the analysis of different glycosylation modification characteristics of N-glycans with significant changes in the brain tissue of mice of the present invention are as follows: Figure 3 A is the relative abundance of all detected mannosylated glycans and glycans with significant changes in mannosylated glycans in mouse brain tissues; 3B is the relative abundance of all detected fucosylated glycans and glycans with significant changes in fucosylated glycans in mouse brain tissues; 3C is the relative abundance of all detected sialylated glycans and glycans with significant changes in sialylated glycans in mouse brain tissues; 3D is the ROC curve of mannosylated, fucosylated, and sialylated glycans with significant changes in brain tissues of control and early ischemic stroke mice; *, p < 0.05;

[0029] Figure 4 The results of the analysis of the N-glycans with significant changes in the serum of the mice of the present invention are shown in FIG. Figure 4 A is the OPLS-DA analysis of N-glycans with significant changes in mouse serum for early ischemic stroke and control groups. Figure 4 B is the ROC analysis of 12 N-glycans with significant changes in the serum of control and early ischemic stroke model mice; Figure 4 C is the ROC curve of the expression of N-glycan H5N4G1 in mouse serum at 1, 2, and 3 hours after acute ischemic stroke, *, p < 0.05.

[0030] Figure 5 The present invention is the present invention's human serum N-glycan profile and the N-glycan analysis of significant changes in healthy people and early ischemic stroke patients, wherein Figure 5 A is the MALDI-MS mass spectrum of human serum. Figure 5 B is the N-glycans with significant changes in serum between controls and early ischemic stroke patients, *, p < 0.05;

[0031] Figure 6 ROC analysis of 6 N-glycans with significant changes in serum of healthy subjects and patients with early ischemic stroke;

[0032] Figure 7The results of the analysis of different glycosylation modification characteristics of N-glycans with significant changes in human serum of the present invention are as follows: Figure 7 A is the relative abundance of all detected fucosylated glycans and significantly changed fucosylated glycans in human serum, Figure 7 B is the relative abundance of all detected sialylated glycans and significantly changed sialylated glycans in human serum, Figure 7 C is the ROC curve of all fucosylated glycans and significantly changed fucosylated glycans in healthy subjects and patients with early ischemic stroke, *, p < 0.05. DETAILED DESCRIPTION

[0033] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0034] The present invention detects and analyzes N-glycans in the brain tissue and serum of early acute ischemic stroke (AIS) model mice and finds that the N-glycan fingerprint has undergone significant changes. Further analysis of the serum N-glycan profiles of acute ischemic stroke (AIS) patients and healthy people reveals significantly changed N-glycans, which can be used as blood markers for acute ischemic stroke, laying the foundation for the early diagnosis, monitoring, prognosis and identification of therapeutic targets for AIS.

[0035] In a specific embodiment of the present invention, early acute ischemic stroke in mice is defined as within 3 hours of onset of ischemic stroke, and samples are taken at 1 hour, 2 hours and 3 hours of onset respectively. Early acute ischemic stroke in humans is defined as within 24 hours of onset, and samples are taken at 6 hours and 24 hours of onset respectively.

[0036] 1. Sample Acquisition

[0037] 1. Construction of mouse middle cerebral artery occlusion (MCAO) model and acquisition of brain tissue samples

[0038] The male mice (C57BL6) used in the experiment, weighing 18-20 g and aged 3-4 weeks, were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. During surgery, the mice were anesthetized with isoflurane (3% for induction and 1-1.5% for maintenance), and the body temperature was maintained at 37.5°C ± 0.5°C using a heating pad. A suture plug was inserted into the internal carotid artery through the left common carotid artery to block the entrance of the middle cerebral artery to induce MCAO. After the operation, the marked mice were returned to their home cages and euthanized after 1 hour, 2 hours, or 3 hours of occlusion. To assess the success of the occlusion, cerebral blood flow was monitored using a laser Doppler flowmeter. In this study, the death rate of mice immediately after surgery was approximately 10% (intracranial hemorrhage occurred), and these mice were excluded from the study.

[0039] Euthanized mice were dissected, their brains removed, and cut into 1mm-thick Lenox coronal sections. Six to seven 1mm-thick brain slices were then cut 3mm from the olfactory bulb using a razor blade and stained with TTC. A 2% TTC solution preheated to 37°C was poured into a 10cm diameter Petri dish, and the freshly cut 1mm brain slices were immersed and arranged in an orderly manner. According to the experimental principle of TTC staining, the ischemic side appears white, and the non-ischemic side appears red. TTC staining of 1mm-thick coronal brain sections confirmed the successful establishment of the mouse cerebral ischemia-hypoxia model.

[0040] According to the experimental needs, the mouse brain tissue was divided into two groups: the non-ischemic side and the ischemic side. After completing the neurological assessment, the mice were deeply anesthetized to ensure unconsciousness, and then the cranial cavity was quickly opened from the cervical vertebrae depression using scissors, and the entire mouse brain was removed1. If the model is successfully established, a gray-white infarct boundary line can be observed with the naked eye. The brain tissue of the mice with successful experiments was placed on ice and divided into the non-ischemic side and ischemic side groups according to the sampling needs for collection. They were placed in clean and sterile EP tubes, labeled, and stored in a refrigerator at -80°C. Subsequently, the brain tissue samples were ultrasonically lysed to effectively break the cell walls to release intracellular components and prepare the samples for N-glycan pretreatment.

[0041] 2. Obtaining mouse serum samples

[0042] The healthy mice and mouse middle cerebral artery occlusion (MCAO) models were constructed as above. Serum was collected from healthy mice and mice 1 h, 2 h, and 3 h after the onset of acute ischemic stroke and stored in a -20°C refrigerator.

[0043] 2. Obtaining human serum samples

[0044] The study subjects were divided into a healthy control group and a non-thrombolytic stroke experimental group. The human serum samples used in this study were obtained from healthy individuals in the physical examination department of Shenzhen Second People's Hospital and patients with imaging-confirmed acute ischemic stroke who had not received revascularization therapy and were admitted to the emergency department's stroke green channel (approved by the hospital's ethics committee). In the stroke experimental group, 1-3 ml of venous blood was collected from patients immediately upon admission and before they received intravenous thrombolysis. After stagnating, the blood was centrifuged at 3000 rpm for 10 minutes, and the serum was retained and stored in a -20°C refrigerator. Basic clinical information of the patients, including age, gender, and NIHSS score at admission, was collected. Inclusion criteria included: 1. Age 18 years and older; 2. Confirmed acute ischemic stroke by emergency department physicians and imaging examinations; 3. Onset within 6 hours and between 6 and 24 hours without receiving revascularization therapy; 4. No bleeding was detected on a head CT scan. Exclusion criteria: 1. Mild or non-disabling neurological symptoms, possibly without transient ischemic attack or epileptic seizure; 2. Patients diagnosed with hemorrhagic stroke rather than ischemic stroke; 3. Patients who have received vascular recanalization treatment, such as thrombolytic therapy or interventional therapy; 4. Patients with severe comorbidities, severe heart or lung dysfunction, malignant tumors, or pregnancy.

[0045] 2. Obtaining the N-glycan profile of the sample to be tested

[0046] 1. Sample preprocessing

[0047] 1) Enzymatic digestion: Mix 5 μL of serum or tissue lysate (approximately 100 μg of protein) with 10 μL of 10x glycoprotein buffer and water to a total of 100 μL. After mixing, add 2.4 μL of 1x glycoprotein denaturation buffer and incubate at 100°C for 10 minutes. Then, add 12 μL of 10% NP-40 and 0.5 μL of PNGase F enzyme and incubate at 37°C for 16 hours.

[0048] 2) PGC Purification: Wash the column with 3 mL of ACN and 80% ACN containing 0.1% TFA, followed by equilibration with 3 mL of 5% ACN containing 0.1% TFA, leaving 2-3 mm of equilibration buffer on the PGC column. Add 500 μL of equilibration buffer to the sample, mix thoroughly, and load. Wash the column with 3 mL of 5% ACN containing 0.1% TFA, and elute with 1 mL of 40% ACN containing 0.1% TFA. Collect the eluate. Use a SpeedVac vacuum dryer at 45°C to dry.

[0049] 3) Methylation: Add 25 μL DMSO containing 1 M methylamine hydrochloride, 0.5 M 4-methylmorpholine, and 25 μL DMSO containing 50 mM PyAOP to the sample, mix well, and react at room temperature in the dark for 40 min.

[0050] 4) MCC Purification: Wash the column with 3 mL of water; equilibrate the column with 3 mL of a 4:1:1 solution of n-butanol, ethanol, and water, leaving 2-3 mm of equilibration solution on the column; add 500 μL of the equilibration solution to the sample, mix thoroughly, and load; wash the column with 3 mL of a 4:1:1 solution of n-butanol, ethanol, and water; elute with 1 mL of a 1:1 solution of ethanol and water, and collect the eluate. Use a SpeedVac vacuum dryer at 45°C to dry.

[0051] 2. N-glycan detection

[0052] N-glycan analysis was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS, AB SCIEX). The spectrum was accumulated by 1200 laser shots in positive light ion mode. The dried samples were dissolved in 50% acetonitrile and an equal amount of each sample was mixed with a freshly prepared Super-DHB solution (10 mg / mL in 50% acetonitrile containing 1 mM NaOH). The mixture was then loaded onto a MALDI plate for analysis. The MALDI-MS data of the N-glycans were further processed using Data Explorer 4.0 (AB SCIEX) to obtain an N-glycan profile.

[0053] 3. Data Analysis

[0054] A series of statistical analyses (independent sample T-test, one-way analysis of variance, regression analysis, etc.) were performed on the N-glycan data of mouse brain tissue, mouse serum, and human serum samples to determine the relative abundance of their N-glycans, identify the characteristic N-glycan profile of early ischemic stroke, construct receiver operating characteristic (ROC) curves, and determine the specificity and predictive efficacy of N-glycan biomarkers in mouse brain tissue, mouse serum, and human serum.

[0055] 1) Characteristic N-glycan profiles in mouse brain tissue and N-glycan biomarkers for distinguishing healthy mice from mice with early ischemic stroke

[0056] Figure 1 The results of early ischemic stroke mouse model and its N-glycan map, among which Figure 1 A is the result of establishing an early ischemic stroke mouse model, showing that the mouse cerebral ischemia-hypoxia model was successfully established; Figure 1 B is the MALDI-MS mass spectrum of mouse brain tissue. Figure 1C, D, and E are the distribution of N-glycans in mouse brain tissue. 1C is classified according to major categories (High mannose represents high-mannose N-glycan structures; C / H represents hybrid and complex N-glycan structures; C / H Fuc represents hybrid and complex N-glycan structures with fucosylation modification; C / H Sia&Fuc represents hybrid and complex N-glycan structures with both fucosylation and sialylation modification), 1D is classified for fucosylated N-glycans (+1 represents an N-glycan structure with a single fucose; +2 represents an N-glycan structure with two fucoses; +3 represents an N-glycan structure with three fucoses; +4 represents an N-glycan structure with four fucoses), and 1E is classified for sialylated N-glycans (+1 represents an N-glycan structure with a single sialic acid; +2 represents an N-glycan structure with two sialic acids).

[0057] By comparing the N-glycans in the brain tissues of control and early ischemic stroke model mice, nine mouse brain tissue characteristic N-glycans, including H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1, and H6N5F3S1, were obtained. These nine N-glycans showed significant changes in the brain tissues of control and early ischemic stroke model mice. A receiver operating characteristic (ROC) curve was constructed using these nine N-glycans with significant changes to evaluate their predictive ability for early ischemic stroke in mice. Figure 2 As shown, Figure 2 A is a comparative diagram of the changes in 9 brain tissue characteristic N-glycans in the brain tissues of healthy control mice and early ischemic stroke mice. Figure 2 B is the OPLS-DA analysis of N-glycans with significant changes in mouse brain tissue for early ischemic stroke and control groups, where I0-1, I0-2, and I0-3 represent three control samples, I1-1, I1-2, I1-3, I1-4, I1-5, and I1-6 represent six samples at 1 hour after ischemic stroke, I2-1, I2-2, I2-3, I2-4, I2-5, and I2-6 represent six samples at 2 hours after ischemic stroke, and I3-1, I3-2, I3-3, I3-4, I3-5, and I3-6 represent six samples at 3 hours after ischemic stroke. Figure 2C shows the receiver operating characteristic (ROC) analysis of nine N-glycans that were significantly altered in brain tissues of control and early ischemic stroke model mice. Among them, the AUCs for H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1, and H6N5F3S1 for distinguishing healthy mice from mice with early ischemic stroke were 0.78, 0.92, 0.84, 0.88, 0.76, 0.98, 0.88, 0.88, and 0.84, respectively, indicating that the above nine N-glycans can be used as biomarkers for early ischemic stroke in mice.

[0058] The glycosylation modification of N-glycans is mainly divided into mannosylation, fucosylation and sialylation. The different glycosylation modifications of all detected glycans and glycans with significant changes in mouse brain tissue were statistically analyzed. The results are as follows: Figure 3 As shown, Figure 3 A is the relative abundance of all detected mannosylated glycans and significantly changed mannosylated glycans in mouse brain tissue (wherein in MT-DM, MT represents mouse brain tissue, DM represents all detected mannosylated glycans, and in MT-SM, MT represents mouse brain tissue, SM represents detected mannosylated glycans with significant changes). 3B is the relative abundance of all detected fucosylated glycans and significantly changed fucosylated glycans in mouse brain tissue (wherein in MT-DF, MT represents mouse brain tissue, DF represents all detected fucosylated glycans, and in MT-SF, MT represents mouse brain tissue, SF represents detected fucosylated glycans with significant changes). 3C is the relative abundance of all detected sialylated glycans and significantly changed sialylated glycans in mouse brain tissue. For abundance (in MT-DS, MT represents mouse brain tissue, DS represents all sialylated glycans detected; in MT-SS, MT represents mouse brain tissue, and SS represents the sialylated glycans detected with significant changes), 3D is the ROC curve of the mannosylated, fucosylated, and sialylated glycans that changed significantly in the brain tissues of the control group and early ischemic stroke mice. The AUCs of the mannosylated, fucosylated, and sialylated glycans that changed significantly for distinguishing healthy mice from early ischemic stroke mice were 0.78, 0.98, and 0.94, respectively. The fucosylated glycans that changed significantly included H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1, and H6N5F3S1.

[0059] 2) Characteristic N-glycan profiles in mouse serum and N-glycan biomarkers for distinguishing healthy mice from mice with early ischemic stroke

[0060] By comparing the N-glycans in the serum of control and early ischemic stroke model mice, 12 mouse serum characteristic N-glycans, including H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1, and H6N5G4, were obtained. These 12 N-glycans with significant changes in serum were used to construct a receiver operating characteristic (ROC) curve to evaluate their predictive ability for early ischemic stroke in mice. The results are shown in Figure 2. Figure 4 As shown, Figure 4 A is the OPLS-DA analysis of the early ischemic stroke and control groups based on the significant changes in N-glycans in mouse serum, where I0-1, I0-2 and I0-3 represent three control samples, I1-1, I1-2, I1-3, I1-4, I1-5 represent five samples at 1 hour after ischemic stroke, I2-1, I2-2, I2-3, I2-4, I2-5 represent five samples at 2 hours after ischemic stroke, and I3-1, I3-2, I3-3, I3-4, I3-5 represent five samples at 3 hours after ischemic stroke. Figure 4 B shows receiver operating characteristic (ROC) analysis of 12 N-glycans significantly altered in the serum of control and early ischemic stroke mice; *, p < 0.05. Among them, the AUCs for H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1, and H6N5G4 for distinguishing healthy mice from mice with early ischemic stroke were 0.88, 0.99, 0.99, 0.99, 0.97, 0.91, 0.91, and 1.00, and 0.95, 0.84, 0.75, and 0.99, respectively, indicating that all 12 N-glycans can be used as biomarkers for early ischemic stroke in mice. Figure 4 C is the ROC curve of the expression of N-glycan H5N4G1 in mouse serum at 1, 2, and 3 hours after acute ischemic stroke, where C represents the control, I1 represents mice with ischemic stroke 1 hour later, I2 represents mice with ischemic stroke 2 hours later, and I3 represents mice with ischemic stroke 3 hours later. As can be seen in the figure, there are significant differences in the expression of N-glycan H5N4G1 at 1, 2, and 3 hours later. The areas under the curve of the receiver operating characteristic curves used to distinguish between the control and I1, I1 and I2, and I1 and I2 are 1.00, 0.92, and 0.96, respectively, indicating that N-glycan H5N4G1 can be used as a marker to indicate the progression of acute ischemic stroke in mice and to predict the onset time and severity of acute ischemic stroke in mice.

[0061] 3) Characteristic N-glycan profiles in human serum and N-glycan biomarkers for distinguishing healthy subjects from patients with early ischemic stroke

[0062] Figure 5 N-glycan analysis of the N-glycan profile and significant changes in human serum, including Figure 5 A is the MALDI-MS mass spectrum of human serum. Figure 5 B is the N-glycan that changes significantly in the serum of controls and early ischemic stroke patients. By comparing the N-glycans in the serum of healthy people and early ischemic stroke patients, six human serum characteristic N-glycans including H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3 were obtained. There were significant changes in the serum of healthy people and early ischemic stroke patients. The ROC curve was constructed using the above six N-glycans with significant changes to evaluate their predictive ability for early ischemic stroke in humans. The results are shown in Figure 2. Figure 6 As shown in the data, the AUCs of H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3 for distinguishing healthy subjects from patients with early ischemic stroke were 0.71, 0.67, 0.71, 0.69, 0.65 and 0.63, respectively, indicating that the above six N-glycans can be used as biomarkers for early ischemic stroke in humans.

[0063] The different glycosylation modifications of glycans detected in human serum and glycans with significant changes were statistically analyzed. Figure 7 As shown, Figure 7 A represents the relative abundance of all detected fucosylated glycans and significantly changed fucosylated glycans in human serum (wherein in HS-DF, HS represents human serum, DF represents all detected fucosylated glycans, and in HS-SF, HS represents human serum, and SF represents detected fucosylated glycans with significant changes). 7B represents the relative abundance of all detected sialylated glycans and significantly changed sialylated glycans in human serum (wherein in HS-DS, HS represents human serum, DS represents all detected sialylated glycans, and in HS-SS, HS represents human serum, and SS represents detected sialylated glycans with significant changes). Figure 7 C is the ROC curve of all fucosylated glycans and significantly changed fucosylated glycans in healthy subjects and patients with early ischemic stroke. The AUCs of all fucosylated glycans and significantly changed fucosylated glycans were 0.69 and 0.70, respectively, which can be used as biomarkers for early ischemic stroke. The significantly changed fucosylated glycans include H4N4F1, H5N4F1, H5N4F1S1 and H5N4F1S2.

[0064] The present invention detects and analyzes human serum characteristic N-glycans in patients with acute ischemic stroke and healthy people, and finds that the expression levels of six characteristic N-glycans, H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3, are significantly different. Any one or more combinations of these six characteristic N-glycans can be used as markers for human acute ischemic stroke and used to judge acute ischemic stroke. Receiver operating curve (ROC) analysis shows that the area under the curve is ≥0.63, and the better , the area under the curve ≥ 0.70; by detecting and analyzing the characteristic N-glycans in the brain tissue of mice with acute ischemic stroke and healthy mice, it was found that the expression levels of 9 characteristic N-glycans, including H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1, were significantly different. Any one or more combinations of these can be used as markers for acute ischemic stroke in mice and used to judge acute ischemic stroke. The receiver operating curve (ROC) analysis showed that the area under the curve was ≥0.78, the better, the area under the curve was ≥0.88, and the better, the area under the curve was ≥0.98; at the same time, the serum characteristic N-glycans in acute ischemic stroke mice and healthy mice were detected and analyzed, and it was found that H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1 and H6N5 There were significant differences in the expression levels of these 12 characteristic N-glycans of G4. Any one or more combinations of these 12 characteristic N-glycans can be used as markers for acute ischemic stroke in mice, and can be used to determine acute ischemic stroke. Receiver operating characteristic (ROC) analysis showed that the area under the curve was ≥0.75, and the best was ≥0.99, and even better was 1. Furthermore, it was found that the expression levels of H5N4G1 were significantly different at different stages of ischemic stroke, and can be used to indicate the progression of acute ischemic stroke in mice.

[0065] The marker of the present invention has high specificity, little trauma and low cost in predicting early ischemic stroke.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of N-glycans as biomarkers for acute ischemic stroke, characterized in that: The applications include: Using human serum characteristic N-glycan as a biomarker for human acute ischemic stroke, wherein the human serum characteristic N-glycan is one or more of H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2 and H6N5S3; or Using brain tissue characteristic N-glycan as a biomarker for acute ischemic stroke in mice, wherein the brain tissue characteristic N-glycan is one or more of H5N2, H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1; or The mouse serum characteristic N-glycans are used as biomarkers for acute ischemic stroke in mice, and the mouse serum characteristic N-glycans are H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1 and H6N5G4.

2. The use according to claim 1, characterized in that: The human serum characteristic N-glycans in patients with acute ischemic stroke have significant changes compared with the human serum characteristic N-glycans in the normal population, the brain tissue characteristic N-glycans in mice with acute ischemic stroke have significant changes compared with the brain tissue characteristic N-glycans of normal mice, and the mouse serum characteristic N-glycans in mice with acute ischemic stroke have significant changes compared with the serum characteristic N-glycans of normal mice, p < 0.

05.

3. The use according to claim 1, characterized in that: The human serum characteristic N-glycan as a biomarker for human acute ischemic stroke is a fucosylated N-glycan, which is at least one of H4N4F1, H5N4F1, H5N4F1S1 and H5N4F1S2.

4. The use according to claim 3, characterized in that: The human serum characteristic N-glycans used as biomarkers for human acute ischemic stroke are fucosylated N-glycans H5N4F1S1 or H4N4F1, and the area under the curve of the receiver operating characteristic curve is ≥0.

71.

5. The use according to claim 3, characterized in that: The human serum characteristic N-glycans used as biomarkers for human acute ischemic stroke are a composition consisting of fucosylated N-glycans H4N4F1, H5N4F1, H5N4F1S1 and H5N4F1S2, and the area under the curve of the receiver operating characteristic curve of the composition is ≥0.

70.

6. The use according to claim 1, characterized in that: The brain tissue characteristic N-glycans as biomarkers for acute ischemic stroke in mice are fucosylated N-glycans, including at least one of H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1.

7. The use according to claim 6, characterized in that: The brain tissue characteristic N-glycan as a biomarker for acute ischemic stroke in mice is a composition consisting of fucosylated N-glycans H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1, and the area under the curve of the receiver operating characteristic curve of the composition is ≥0.

98.

8. The use according to claim 6, characterized in that: The characteristic N-glycan in brain tissue that serves as a biomarker for acute ischemic stroke in mice is the fucosylated N-glycan H5N5F2, and the area under the curve of the receiver operating curve is ≥0.

96.

9. The use according to claim 1, characterized in that: The mouse serum characteristic N-glycan H5N4G1 is used as a marker for the progression of acute ischemic stroke in mice.

10. A kit for detecting acute ischemic stroke, characterized in that: include; A reagent for detecting human serum characteristic N-glycans, wherein the human serum characteristic N-glycans are at least one of the following: H4N4F1, H5N4F1, H5N4F1S1, H5N4F1S2, H5N4S2, and H6N5S3; or A reagent for detecting characteristic N-glycans of mouse brain tissue, wherein the characteristic N-glycans of brain tissue are at least one of the following: H4N3F1, H4N4F1S1, H4N6F1, H5N4F3, H5N5F2, H4N6F1S1, H5N5F2S1 and H6N5F3S1; or A reagent for detecting mouse serum characteristic N-glycan, wherein the mouse serum characteristic N-glycan is at least one of the following: H5N2, H6N5G3, H5N4G4, H5N4G3, H5N4F1G2, H4N5G2, H5N4F1G1, H5N4G1, H4N4G1, H5N3G1, H4N3G1 and H6N5G4.

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