Syndecan-1 as a marker in the prognostic evaluation of acute ischemic stroke patients treated with intravenous thrombolysis

Abstract

This invention discloses a biomarker for early diagnosis, assessment of disease progression, and prognostic evaluation in patients undergoing intravenous thrombolysis for acute ischemic stroke: Syndecan-1 in peripheral blood. This invention provides the application of a substance for detecting Syndecan-1 in peripheral blood in the preparation of products for early diagnosis of prognosis in patients undergoing intravenous thrombolysis for acute ischemic stroke; or in the preparation of products for assessing disease progression in patients undergoing intravenous thrombolysis for acute ischemic stroke; or in the preparation of products for assessing the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke. Research results indicate that plasma Syndecan-1 levels represent a novel and independent prognostic biomarker that can improve the current risk stratification of patients undergoing intravenous thrombolysis for acute ischemic stroke. Blood tests are easily accepted by patients and can serve as an effective means for early diagnosis, assessment of disease progression, and prognostic evaluation in patients undergoing intravenous thrombolysis for acute ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of Syndecan-1, a glycocalyx lysate, as a biomarker in the prognostic assessment of patients undergoing intravenous thrombolysis for acute ischemic stroke. Background Technology

[0002] The American Heart Association reports that ischemic stroke is a leading cause of disability and the third leading cause of death after heart disease and cancer, causing approximately 5.5 million deaths worldwide, two-thirds of which occur in developing countries. Furthermore, millions of ischemic stroke survivors each year must adapt to lives with limited daily activities, many relying on others for survival. Pathologically, tPA is associated with neurotoxicity, particularly cell damage caused by activation of excitatory amino acid receptors. Increasing evidence suggests that tPA mediates increased blood-brain barrier permeability caused by cerebral ischemia. Animal studies have also confirmed that tPA treatment induces Evans blue dye extravasation, a process associated with increased expression and activity of matrix metalloproteinase 9 (MMP-9), and may be involved in later neurotoxic processes.

[0003] An effective treatment for acute ischemic stroke (AIS) is to restore blood flow to the brain regions that are under-perfused. This approach, known as reperfusion therapy, must be initiated as soon as possible. Recombinant tissue plasminogen activator (rt-PA) is the only FDA-approved chemotherapy for ischemic stroke. In a study conducted at the National Institute of Neurological Disorders and Stroke (NINDS), treatment with tPA within 3 hours of the onset of AIS symptoms increased the number of patients with good prognoses by 11-13% and reduced the number of patients with disability and death at 3 months. However, rt-PA also has adverse effects, clinically manifested primarily as cerebral edema or intracranial hemorrhage after thrombolytic therapy. Increasing evidence suggests that tPA mediates increased blood-brain barrier permeability caused by cerebral ischemia. Animal studies have also confirmed that tPA treatment induces Evans dye extravasation, and this process is associated with increased MMP-9 expression and activity, and may be involved in the later neurotoxic process.

[0004] Recently, the endothelial glycocalyx (EG) has been defined as a component of the extended neurovascular unit, playing a crucial role in maintaining proper neuronal homeostasis. As a surface coating, the EG forms a barrier between the blood and blood vessels. Damage to the EG appears to be the first step in BBB dysfunction. The EG is primarily composed of proteoglycans (PGs) and their linked glycosaminoglycan (GAG) chains. Syndecans are members of the transmembrane protein heparan sulfate glycoprotein family, primarily expressed on the surface of cells, including vascular endothelial cells. Furthermore, their intact extracellular domains can detach into the extracellular environment. Syndecan-1 is considered an important component of the glycocalyx; therefore, alterations in its expression or distribution may affect glycocalyx integrity and endothelial barrier function. Elevated levels of Syndecan-1 in the blood may be a marker of glycocalyx degradation. Studies have found significantly reduced glycocalyx thickness in Syndecan-1- / - mice. Therefore, we selected Syndecan-1 as the primary biomarker for glycocalyx injury to investigate the status of glycocalyx injury after thrombolysis in acute ischemic stroke, in order to identify potential biomarkers that may be helpful in the early diagnosis and prediction of prognosis after thrombolysis in AIS. Summary of the Invention

[0005] The purpose of this invention is to provide a prognostic biomarker for early diagnosis and assessment of disease progression in patients undergoing intravenous thrombolysis for evaluating the prognosis of acute ischemic stroke.

[0006] The biomarker provided by this invention for early diagnosis and prognostic assessment of patients with acute ischemic stroke undergoing intravenous thrombolysis is Syndecan-1, a glycocalyx lysate in peripheral blood.

[0007] The present invention also provides the use of a substance for detecting Syndecan-1, a glycocalyx lysate in peripheral blood, in the preparation of products for early diagnosis and / or auxiliary diagnosis of the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke.

[0008] This invention also provides the use of a substance for detecting Syndecan-1, a glycocalyx lysate in peripheral blood, in the preparation of products for assessing and / or assisting in assessing the prognosis and disease progression of patients undergoing intravenous thrombolysis for acute ischemic stroke.

[0009] The present invention also provides the use of a substance for detecting Syndecan-1, a glycocalyx lysate in peripheral blood, in the preparation of products for assessing and / or assisting in the assessment of the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke.

[0010] The assessment and / or auxiliary assessment of the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke can further be used to assess and / or auxiliary assess the 3-month prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke.

[0011] The product in question is a reagent kit.

[0012] The kit also includes a carrier containing the following criteria: if the Syndecan-1 level in the plasma of patients undergoing intravenous thrombolysis for acute ischemic stroke is ≥102.82 ng / ml, then the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke within 3 months is considered poor.

[0013] This invention also protects a product comprising a substance for detecting the glycocalyx lysate Syndecan-1 in peripheral blood; said product having the function of (a), (b), or (c):

[0014] (a) Used for early diagnosis and / or auxiliary diagnosis of prognosis in patients with acute ischemic stroke undergoing intravenous thrombolysis;

[0015] (b) To assess and / or assist in assessing the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke;

[0016] (c) Assess and / or assist in assessing the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke.

[0017] The product in question is a reagent kit.

[0018] This invention also protects the application of Syndecan-1, a glycocalyx lysate in peripheral blood, as a biomarker; said biomarker is (I), (II), or (III) as follows:

[0019] (I) Biomarkers for early diagnosis and / or auxiliary diagnosis of prognosis in patients with acute ischemic stroke undergoing intravenous thrombolysis;

[0020] (II) Biomarkers for assessing and / or assisting in assessing the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke;

[0021] (III) Assess and / or assist in assessing prognostic biomarkers in patients undergoing intravenous thrombolysis for acute ischemic stroke.

[0022] This invention also protects a method for assessing and / or assisting in the assessment of the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke, comprising the following steps: detecting the concentration of glycocalyx lysate Syndecan-1 in the peripheral blood of the patient to be tested; patients with low concentrations of glycocalyx lysate Syndecan-1 in peripheral blood have a better prognosis than patients with high concentrations of glycocalyx lysate Syndecan-1 in peripheral blood.

[0023] This invention also protects a method for judging and / or assisting in judging the disease progression of patients undergoing intravenous thrombolysis for acute ischemic stroke, comprising the following steps: detecting the concentration of glycocalyx lysate Syndecan-1 in the peripheral blood of the patient to be tested; patients with high concentrations of glycocalyx lysate Syndecan-1 in peripheral blood have faster disease progression than patients with low concentrations of glycocalyx lysate Syndecan-1 in peripheral blood.

[0024] The substance used to detect Syndecan-1 in peripheral blood is the substance required to detect the protein content and / or abundance of Syndecan-1 in peripheral blood, such as a substance that can specifically recognize Syndecan-1 protein, such as an antibody.

[0025] This study found that plasma Syndecan-1 levels represent a novel and independent prognostic biomarker that can improve current risk stratification in patients undergoing thrombolysis for acute ischemic stroke. Our blood test method is easily accepted by patients and can serve as an effective means for early diagnosis, assessment of disease progression, and prognostic evaluation in patients undergoing intravenous thrombolysis for acute ischemic stroke. Our findings provide important clues for clinical applications and basic research. Attached Figure Description

[0026] Figure 1 This study compared plasma Syndecan-1 protein levels in different population subgroups and analyzed the correlation between Syndecan-1 levels and the neutrophil / lymphocyte ratio. Control group consisted of healthy controls, and AIS group consisted of patients with acute ischemic stroke undergoing intravenous thrombolysis. *P≤0.05, *P≤0.001 compared with the Control group.

[0027] Figure 2 The predictive model based on risk factors affecting poor prognosis in patients undergoing thrombolysis for acute ischemic stroke, as shown in the example nomogram, is used to illustrate this.

[0028] Figure 3 This is a DCA curve graph. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the raw materials are all available from publicly available commercial sources.

[0030] Example 1

[0031] I. Specific Experimental Methods and Data

[0032] 1. Research Methods

[0033] 1) Study Subjects: A total of 345 patients with acute ischemic stroke (AIS) who visited Xuanwu Hospital of Capital Medical University within 24 hours of onset between September 2018 and May 2019 were selected. Of these, 145 received rt-PA intravenous thrombolysis. 32 patients were excluded due to incomplete medical records or follow-up data, hemolysis, etc. 113 AIS patients undergoing thrombolysis were enrolled and underwent relevant testing. Four patients with abnormal plasma test values ​​were excluded, leaving 109 AIS patients for this study. After age and sex matching, 47 healthy individuals were also included in the study. Our study was approved by the Ethics Committee of Xuanwu Hospital of Capital Medical University.

[0034] 2) Inclusion criteria are as follows: 1) Patients meet the diagnostic criteria of the Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2010; 2) No obvious low-density changes on cranial CT; 3) Informed consent from the patient and their family and signed the informed consent form; 4) Clear neurological deficit; 5) Onset time <4.5h; 6) Complete case data and follow-up data.

[0035] 3) Exclusion criteria: 1) Previous cerebral infarction; 2) Hemorrhagic disease; 3) Comorbid malignant tumor, blood disease, severe infection, etc.; 4) Pregnancy, epilepsy, mental illness, coagulation dysfunction, liver and kidney dysfunction, etc.

[0036] 4) Clinical records and blood sample collection: For AIS patients, rt-PA was used for thrombolysis, administered at a dose of 0.9 mg / kg. 10% of the total dose was injected intravenously over 1 minute, and the remaining 90% was infused intravenously via a microinfusion pump over 1 hour, with a maximum dose <90 mg. General clinical data were collected, including demographic characteristics (age, sex, BMI), past medical history, smoking history, and post-admission blood pressure, blood glucose, serum c-reactive protein (CRP), serum homocysteine ​​(HCY), partial complete blood counts, and partial blood biochemical indicators. Stroke was simply classified into large vessel type, small vessel type, and other types. The severity of neurological deficits was assessed using the NIHSS (National Institutes of Health Stroke Scale); the 3-month clinical prognostic score was calculated using the mRS (modified Rankin Scale). An mRS score of 0-2 was defined as a good prognosis, while an mRS score of 3-5 and death were defined as a poor prognosis. Blood samples were collected from patients using EDTA tubes, and the separated plasma was stored at -80°C after centrifugation.

[0037] 5) Plasma Syndecan-1 Detection: Plasma Syndecan-1 detection was performed using the Human Syndecan-1 ELISA kit (catalog number: ab46506; batch number: GR3368280-5). The procedure was performed according to the kit instructions. The instrument used was a Bio-Tek Elx800 microplate reader. If the sample concentration exceeded the upper limit of the detection range, the sample was serially diluted before testing.

[0038] 6) Statistical Analysis: SPSS 22.0 software (IBM Corp., Armonk, NY, USA) and R software (version 4.1.1) were used for statistical analysis. Continuous variables that conform to a normal distribution were expressed as mean ± standard deviation and tested using t-tests or ANOVA. Non-normally distributed variables were expressed as median [interquartile range (IQR)] and tested using the Mann-Whitney U test. Correlation analysis used Spearman correlation coefficients. Categorical data were expressed as frequency (%), and chi-square tests were used for comparisons between groups. Multivariate prognostic analysis used binary logistic regression analysis. Model variables were selected using LASSO regression, primarily lambda.min and lambda.1se; variables with non-zero regression coefficients were included in the final model. p < 0.05 was considered statistically significant.

[0039] 2. Research Results

[0040] 1) Comparison of clinical baseline data between patients with acute ischemic stroke and healthy individuals.

[0041] We first compared the general clinical data of AIS patients and healthy individuals. There were 108 AIS patients and 47 healthy controls. There were no statistically significant differences between the two groups in terms of age, sex, and other demographic indicators. Peripheral plasma from AIS patients and healthy controls was isolated and analyzed by ELISA. The results showed that compared with healthy individuals, the level of Syndecan-1 protein in the plasma of AIS patients was significantly increased (t = 3.066, P = 0.0026). Figure 1 A).

[0042] 2) Analysis of clinical baseline data for thrombolysis in acute ischemic stroke with different prognoses.

[0043] Based on the mRS score at 3 months, 108 patients with AIS who underwent thrombolysis were divided into two groups: those with poor prognosis and those with good prognosis. Of the 108 patients, 39 (36.11%) had a poor prognosis, and 29 (74.36%) were male. Poor prognosis was mainly seen in elderly patients, patients with atrial fibrillation, those with high CRP levels, those undergoing mechanical thrombectomy, those with high blood glucose levels upon admission, high neutrophil counts, high neutrophil / lymphocyte ratios, high NIHSS scores, and high Syndecan-1 levels. Regarding stroke type, patients with large vessel disease had a worse prognosis, while patients with small vessel disease had a relatively better prognosis (p<0.10, Table 1).

[0044] Table 1. Analysis of general clinical data of patients with AIS who underwent intravenous thrombolysis

[0045]

[0046] IQR (interquartile range); HCY (homocysteine); CRP (C-reactive protein); HDL (high-density lipoprotein); LDL (low-density lipoprotein); TC (triglyceride); sICH (symptomatic intracranial hemorrhage).

[0047] 3) Correlation analysis of elevated plasma Syndecan-1 levels with prognosis and other related indicators in patients with acute ischemic stroke.

[0048] Subgroup analysis of peripheral plasma Syndecan-1 levels revealed that plasma Syndecan-1 levels in the poor prognosis group were significantly higher than those in the good prognosis group (t = 3.066, p = 0.003). Figure 1 B, Table 1), and the healthy individuals group had a significantly lower prognosis than the group with poor prognosis (t = 5.007, p < 0.001). Figure 1 B) The Syndecan-1 level in the mechanical thrombectomy group was significantly higher than that in the non-mechanical thrombectomy group (t = 2.335, p = 0.0214). Figure 1 C). Furthermore, correlation analysis showed that Syndecan-1 levels were correlated with the Neutrophil-to-lymphocyte ratio (p = 0.011). Figure 1 D) shows a positive correlation.

[0049] 4) Plasma Syndecan-1 levels can independently and in combination predict poor prognosis in patients undergoing thrombolysis for AIS.

[0050] After adjusting for age, NIHSS score, history of atrial fibrillation, mechanical thrombectomy, serum CRP, stroke type, and Syndecan-1 level as independent variables, a binary logistic regression analysis (Model 2) showed that Syndecan-1 level could still predict poor prognosis in AIS patients undergoing thrombolysis (p<0.001, Table 2). After multivariate adjustment, the odds ratio (OR) (95% CI) for Syndecan-1 level was 1.022 (1.009–1.035). Based on ROC curves, the optimal cutoff value for plasma Syndecan-1 level as a poor prognostic outcome was 102.82 ng / ml, with a sensitivity of 92.3% and a specificity of 49.3%. Syndecan-1 levels were divided into two groups based on the cutoff value: ≥102.82 ng / ml and <102.82 ng / ml. When Syndecan-1 was considered a continuous variable, Syndecan-1 ≥ 102.82 ng / ml (OR, 34.551; 95% CI, 5.408–220.732) was associated with poor prognosis in patients undergoing thrombolysis for acute ischemic stroke at 3 months (Table 2).

[0051] Table 2. Syndecan-1 alone and in combination predicts prognosis in patients undergoing thrombolysis for AIS.

[0052]

[0053] MODEL 2: Dependent variable, 3-month mRS>2; Adjusted factors include age, NIHSS score, history of atrial fibrillation, whether mechanical thrombectomy was performed, blood CRP, stroke type, and Syndecan-1 (variables included in the model were selected by lasso regression). MODEL 1: Dependent variable, 3-month mRS>2; Independent variable is Syndecan-1 (&, continuous variable; #, categorical variable).

[0054] 5) Syndecan-1 in peripheral plasma of AIS thrombolysis patients improves the predictive efficacy of traditional clinical models for risk.

[0055] With poor prognosis as the dependent variable, Model New was established using variables selected by LASSO regression (age, NIHSS score, history of atrial fibrillation, whether mechanical thrombectomy was performed, blood CRP, stroke type, and Syndecan-1) as independent variables. Model Old was established without the Syndecan-1 indicator variable. Binary logistic regression analysis was performed on both models. The results showed that in Model Old, NIHSS score, history of atrial fibrillation, and high CRP level were risk factors affecting prognosis (p<0.05, Table 3). In the new model, age, NIHSS score, history of atrial fibrillation, and high plasma Syndecan-1 level were risk factors affecting prognosis (p<0.05, Table 3).

[0056] Table 3. Syndecan-1 levels in peripheral plasma of AIS thrombolysis patients improve the predictive efficacy of traditional clinical models for risk.

[0057]

[0058] 6) Nomogram of prognosis after intravenous thrombolysis in acute ischemic stroke

[0059] Predictive models based on risk factors affecting poor prognosis in patients undergoing thrombolysis for acute ischemic stroke, as found in both the old and new models, are presented in the form of example nomograms. Figure 2 The Model New nomogram demonstrated good accuracy in assessing the prognosis of patients undergoing thrombolysis for AIS, with an uncorrected C-index of 0.935 (95% CI, 0.888–0.981) and a bootstrap-corrected C-index of 0.898. The Model Old nomogram showed an uncorrected C-index of 0.884 (95% CI, 0.815–0.954) and a bootstrap-corrected C-index of 0.846. This indicates that the Model New nomogram has better predictive power than the Model Old nomogram.

[0060] 7) Clinical Application

[0061] Decision curve analysis (DCA) determines the clinical applicability of prognostic nomograms by quantifying the net benefit at different threshold probabilities in a dataset. In the decision curve, the two curved lines represent Model New and Model Old, respectively. The decision curve shows that using the nomogram of Model New with the added Syndecan-1 index provides more benefit in predicting the prognosis of AIS thrombolysis patients than the traditional prediction model. Figure 3 The Model New nomogram suggests that it has high clinical application value.

[0062] Conclusion: The above findings indicate that the expression level of Syndecan-1 protein in peripheral plasma is significantly elevated in patients with acute ischemic stroke undergoing intravenous thrombolysis and those with poor prognosis. Furthermore, the expression level of Syndecan-1 in peripheral plasma of patients undergoing intravenous thrombolysis for acute ischemic stroke can predict the risk of adverse outcomes at 3 months, either alone or in combination. Compared with older clinical models, incorporating peripheral plasma Syndecan-1 protein expression significantly improves diagnostic efficacy. In conclusion, plasma Syndecan-1 level represents a novel and independent prognostic biomarker that can improve the current risk stratification of patients undergoing intravenous thrombolysis for acute ischemic stroke. Our blood test method is easily accepted by patients and can serve as an effective means for early diagnosis, assessment of disease progression, and prognostic evaluation in patients undergoing intravenous thrombolysis for acute ischemic stroke. Our findings provide important clues for clinical applications and basic research.

Claims

1. The use of a substance for detecting Syndecan-1, a glycocalyx lysate in peripheral blood, in the preparation of products for early prognostic diagnosis and / or auxiliary diagnosis of patients undergoing intravenous thrombolysis for acute ischemic stroke.

2. The use of substances for detecting Syndecan-1, a glycocalyx lysate in peripheral blood, in the preparation of products for assessing and / or assisting in the assessment of the prognosis of patients undergoing intravenous thrombolysis for acute ischemic stroke.

3. The use of substances for detecting Syndecan-1, a glycocalyx lysate in peripheral blood, in the preparation of products for assessing and / or assisting in assessing the prognosis and disease progression of patients undergoing intravenous thrombolysis for acute ischemic stroke.

4. The application according to any one of claims 1-3, characterized in that: The substance used to detect Syndecan-1 in peripheral blood is the substance required for detecting the protein content and / or protein abundance of Syndecan-1 in peripheral blood; the product is a kit.

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