Application of RCAN1 protein as AIS biomarker in acute ischemic stroke

Abstract

The invention belongs to the technical field of laboratory medicine, and particularly relates to application of RCAN1 protein as an AIS biomarker in acute ischemic stroke. It is found for the first time that RCAN1 is remarkably increased in blood plasma of an AIS patient, the level of RCAN1 and an onset time window have clear dynamic change characteristics, especially, the RCAN1 has the optimal diagnosis efficiency in the window period of 6-24 hours, and meanwhile, the RCAN1 can be used for diagnosis of the AIS patient. The invention also finds that the expression level of the RCAN1 protein in a plasma sample of an AIS patient with acute ischemic stroke is positively correlated with the volume of ischemic penumbra in the brain, and the discovery provides an important basis for predicting an AIS attack time window, namely a treatment target, by taking the RCAN1 as a blood marker.

Description

Technical Field

[0001] The present invention belongs to the technical field of laboratory medicine, and in particular relates to the use of RCAN1 protein as a biomarker for acute ischemic stroke (AIS). Background Art

[0002] Acute ischemic stroke (AIS) is one of the most common cerebrovascular diseases in clinical practice. Its pathological characteristics are irreversible damage to brain tissue due to ischemia (i.e., infarct core), accompanied by the presence of an ischemic penumbra with potential for rescue. In order to select appropriate treatment options, such as thrombolysis or mechanical thrombectomy, it is crucial to promptly and accurately determine the onset time window of AIS. However, current diagnostic methods mainly rely on patients' clinical symptoms, self-reported onset time, and imaging examinations, but these methods have limitations such as strong subjectivity and long detection time. Therefore, there is an urgent need to develop a rapid and objective biomarker to predict the onset time window of AIS to assist clinical diagnosis and treatment decisions.

[0003] The ischemic penumbra is a core concept in the management of AIS, which refers to a brain region with reduced blood flow but still with the potential for recovery, in contrast to the infarct core area that is no longer viable. Currently, advanced imaging techniques such as computed tomography perfusion (CTP) and magnetic resonance imaging (MRI) play a key role in identifying salvageable brain tissue and guiding treatment decisions. Specifically, diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) sequences of MRI are crucial for diagnosis and treatment decisions. High signal on DWI but no corresponding signal area on FLAIR usually indicates the presence of an ischemic penumbra, in which tissue may still be salvageable; while high signal on both DWI and FLAIR may represent the infarct core, indicating irreversible damage and reduced possibility of benefit from reperfusion therapy. However, these imaging techniques not only require patients to go to a designated location for examination, but may also require the injection of contrast agents (such as CTP), and the preparation process is complicated and time-consuming. In addition, the subjective nature of the examination and the need for specialized equipment and technology make it difficult to popularize in some clinical settings, especially in resource-poor areas.

[0004] Therefore, there is an urgent need to find a rapid and reliable biomarker that can reflect the evolving pathophysiology of AIS in real time. Such a biomarker can not only enhance clinical decision-making capabilities, especially within the critical 6-24 hours after stroke, but also enable timely and targeted interventions to rescue the ischemic penumbra and thus improve long-term neurological outcomes. Summary of the invention

[0005] The present invention aims to provide the use of RCAN1 protein as a biomarker for acute ischemic stroke AIS, and specifically relates to a blood marker for predicting the onset time window of acute ischemic stroke AIS or for evaluating the volume of the ischemic penumbra of acute ischemic stroke AIS patients. The present invention is the first to apply RCAN1 as a blood marker for the onset time window of acute ischemic stroke (AIS) in clinical practice. The detection method is rapid and non-invasive, which can significantly improve the diagnostic efficiency of AIS patients, provide an important reference for treatment decisions, and has broad application prospects.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to provide use of RCAN1 protein as a biomarker for acute ischemic stroke (AIS).

[0008] Preferably, the RCAN1 protein is a blood marker.

[0009] Preferably, the expression level of the RCAN1 protein in plasma samples of patients with acute ischemic stroke (AIS) presents specific dynamic changes: it reaches a peak value in the 6-24 hour window period; in the time period of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 decreases.

[0010] Preferably, the expression level of the RCAN1 protein in plasma samples of patients with acute ischemic stroke (AIS) is positively correlated with the volume of the ischemic penumbra in the brain.

[0011] The second object of the present invention is to provide the use of RCAN1 protein in preparing a product for predicting the onset time window of acute ischemic stroke (AIS) or for evaluating the volume of the ischemic penumbra of patients with acute ischemic stroke (AIS).

[0012] Preferably, the product is a detection kit, comprising reagents for quantitatively detecting the level of RCAN1 protein in blood and instructions for use.

[0013] Another object of the present invention is to provide a method for predicting the onset time window of acute ischemic stroke AIS or for evaluating the volume of the ischemic penumbra of acute ischemic stroke AIS patients, comprising the following steps:

[0014] a) Collect plasma samples from patients suspected of acute ischemic stroke (AIS);

[0015] b) detecting the concentration of RCAN1 protein in plasma samples;

[0016] c) predicting the onset time window of acute ischemic stroke (AIS) in patients or evaluating the volume of the ischemic penumbra in patients with acute ischemic stroke (AIS) based on the plasma level of RCAN1 protein; wherein:

[0017] The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) showed specific dynamic changes: it reached a peak value in the 6-24 hour window period; in the time period of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 decreased;

[0018] The expression level of the RCAN1 protein in plasma samples of patients with acute ischemic stroke (AIS) is positively correlated with the volume of the ischemic penumbra in the brain.

[0019] Preferably, the method for detecting the level of RCAN1 protein in the blood sample in step b) comprises enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay, protein blotting and mass spectrometry.

[0020] Preferably, the method further comprises step d) deciding whether to perform thrombolysis / thrombectomy on the acute ischemic stroke (AIS) patient according to the predicted onset time window or the assessed ischemic penumbra volume, and formulating a personalized treatment plan.

[0021] Another object of the present invention is to provide a system for predicting the onset time window of acute ischemic stroke (AIS) or for evaluating the volume of the ischemic penumbra of an acute ischemic stroke (AIS) patient, comprising:

[0022] a) A blood collection device for collecting blood samples from patients suspected of acute ischemic stroke (AIS);

[0023] b) RCAN1 protein detection module, detecting the concentration of RCAN1 protein in plasma samples;

[0024] c) A data processing module, which predicts the AIS onset time window of the patient or evaluates the volume of the ischemic penumbra of the acute ischemic stroke AIS patient according to the plasma level of RCAN1 protein; wherein:

[0025] The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) showed specific dynamic changes: it reached a peak value in the 6-24 hour window period; in the time period of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 decreased;

[0026] The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) is positively correlated with the volume of the ischemic penumbra in the brain;

[0027] d) Output module, which displays the prediction / evaluation results and treatment recommendations.

[0028] The inventive concept and principle of the present invention:

[0029] The present invention aims to evaluate the expression level of RCAN1 protein in the plasma of AIS patients and its potential as a blood biomarker, such as Figure 1 The figure shows the experimental design idea of ​​the present invention for determining RCAN1 protein as a blood biomarker for AIS patients. First, the present invention detects the level of RCAN1 protein in the patient's plasma by using enzyme-linked immunosorbent assay (ELISA) or other hematological analysis methods, and finds that: in the plasma samples of AIS patients, the expression of RCAN1 protein shows specific dynamic changes: it reaches a peak value in the 6-24 hour window period (AUC=0.920, sensitivity 94.74%, specificity 76.47%); in the time period of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 protein decreases significantly. It can be seen that based on the dynamic change law of RCAN1 protein, the onset time of the patient can be inferred by a single plasma test result or multiple consecutive test results, providing an important basis for clinical decision-making.

[0030] Secondly, the present invention further studied the correlation between RCAN1 protein level and ischemic penumbra in patients with acute ischemic stroke AIS. Through DWI-FLAIR mismatch and volume calculation, it was found that RCAN1 protein level was significantly positively correlated with the volume of ischemic penumbra (r=0.700, p=0.024), but had no significant correlation with the infarct core (r=0.294, p=0.103). Therefore, RCAN1 can not only predict the onset time window, but also indirectly reflect the range of ischemic penumbra, providing a basis for the selection of therapeutic targets.

[0031] Finally, the present invention verified through mouse experiments that blood RCAN1 protein can be used as a blood marker to predict the onset time window of acute ischemic stroke (AIS).

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention discovers for the first time that RCAN1 protein is significantly elevated in the plasma of AIS patients, and its level has clear dynamic change characteristics with the onset time window, especially with the best diagnostic efficacy in the 6-24 hour window period. At the same time, the present invention also finds that the expression level of RCAN1 protein in plasma samples of acute ischemic stroke AIS patients is positively correlated with the volume of the ischemic penumbra in the brain. This discovery provides an important basis for using RCAN1 as a blood marker to predict the onset time window and treatment target of AIS.

[0034] RCAN1 protein is used as a biomarker to detect the onset time window of acute ischemic stroke (AIS) or to evaluate the volume of the ischemic penumbra in the brain. The detection method is rapid and non-invasive, which can significantly improve the diagnostic efficiency of AIS patients and provide an important reference for treatment decisions. It has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The experimental design idea for the present invention to determine RCAN1 protein as a blood biomarker for AIS patients;

[0036] Figure 2 Information about the population studied for the present invention;

[0037] Figure 3 The results of detecting plasma RCAN1 protein levels in healthy controls and AIS patients using enzyme-linked immunosorbent assay (ELISA);

[0038] Figure 4 The comparison of plasma RCAN1 protein levels between the healthy control group and 35 AIS patients with onset time less than 6 hours;

[0039] Figure 5 The figure shows the comparison of plasma RCAN1 protein levels between the healthy control group and 19 AIS patients with onset time of 6 to 24 hours;

[0040] Figure 6 The figure shows the comparison of plasma RCAN1 protein levels between the healthy control group and 23 AIS patients with onset time ranging from 24 hours to 2 weeks;

[0041] Figure 7 The ROC curve shows the diagnostic efficacy of plasma RCAN1 protein for AIS in 77 AIS patients;

[0042] Figure 8 The ROC curve shows the diagnostic efficacy of plasma RCAN1 protein for AIS in 35 AIS patients with onset time less than 6 hours;

[0043] Fig. 9 The ROC curve shows the diagnostic efficacy of plasma RCAN1 protein for AIS in 19 AIS patients with onset time of 6 to 24 hours;

[0044] Fig.10 The ROC curve shows the diagnostic efficacy of plasma RCAN1 protein for AIS in 23 AIS patients with onset time of 24 hours to 2 weeks;

[0045] Fig.11 To analyze the volume maps of the ischemic penumbra (10 cases) and infarct core (32 cases) in patients with AIS using MRI imaging;

[0046] Fig.12 To evaluate the correlation between plasma RCAN1 protein level and ischemic penumbra volume in AIS patients (n=10) by Spearman correlation analysis; the results are presented in the form of scatter plots;

[0047] Fig.13 To evaluate the correlation between plasma RCAN1 protein level and infarct core volume in AIS patients (n=32) by Spearman correlation analysis; the results are presented in the form of scatter plots;

[0048] Fig.14 This is a scan of a mouse using MRA and T2WI;

[0049] Fig.15 This is the result of ELISA detection of RCAN1 protein level in the blood of MCAO mice. DETAILED DESCRIPTION

[0050] The above contents of the present invention are further described in detail below by means of specific implementation methods in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples.

[0051] 1. Study population

[0052] A total of 34 healthy controls (HCs) and 77 AIS patients were recruited. Figure 2 As shown, the study population of the present invention was collected in Qilu Hospital of Shandong University in July 2023 and May 2024. A total of 34 healthy controls (HCs) and 77 AIS patients were recruited. The age of the healthy control group was 53-83 years old, with an average age of 67.12±1.45 years old, and 22 males; the age of the AIS group was 40-91 years old, with an average age of 67.35±1.29 years old, and 48 males. There was no statistical difference in age and gender between the two groups. AIS patients were divided into the following three groups according to the time of symptom onset: <6 hours (n=35), 6-24 hours (n=19), and 24 hours to 2 weeks (n=23). Informed consent was obtained from all the above participants.

[0053] 2. Instruments, equipment and materials

[0054] Human calcineurin regulatory factor 1 (RCAN1) ELISA research kit was purchased from Jiangsu Enzyme Immunity Industry Co., Ltd., model MM-51332H1;

[0055] The mice used in the experiment were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and all were male KM mice (25-30 g, 4-6 weeks old).

[0056] Example 1 Detection and statistical analysis of RCAN1 protein plasma levels

[0057] The study population was divided into a healthy control group and an acute ischemic stroke group. The acute ischemic stroke group was divided into a <6-hour group, a 6-24-hour group, and a 24-hour to 2-week group according to the onset time. The plasma RCAN1 protein level of HCs and AIS patients was detected by enzyme-linked immunosorbent assay (ELISA), and further grouped by onset time for analysis. Specifically,

[0058] (1) The human calcineurin regulatory factor 1 (RCAN1) ELISA research kit was used to detect the plasma RCAN1 protein level in the healthy control group and acute ischemic stroke group according to the instructions. The data are expressed as mean ± standard error (mean ± SEM). Among them, the healthy control group n = 34, the total acute ischemic stroke group n = 77, the <6 hour group n = 35, the 6-24 hour group n = 19, and the 24 hour to 2 week group n = 23. The Student's t test was used to statistically analyze the plasma levels of RCAN1 protein in the healthy control group and the acute ischemic stroke group and in different time windows. The experimental results are shown in Figure 2. Figures 3 to 6 shown.

[0059] Figure 3 The plasma RCAN1 protein levels in the healthy control group and 77 patients with acute ischemic stroke were shown in Figure 2. The experimental results showed that the plasma RCAN1 protein level in AIS patients was significantly increased compared with that in the healthy control group.

[0060] Figure 4 Comparison of plasma RCAN1 protein levels between the healthy control group and 35 AIS patients with onset time less than 6 hours. The experimental results showed that the plasma RCAN1 protein level was significantly increased in AIS patients with onset time less than 6 hours.

[0061] Figure 5 The plasma RCAN1 protein level was compared between the healthy control group and 19 AIS patients with onset time of 6 to 24 hours. The experimental results showed that the plasma RCAN1 protein level was significantly increased in AIS patients with onset time of 6 to 24 hours.

[0062] Figure 6 The plasma RCAN1 protein level was compared between the healthy control group and 23 AIS patients with onset time between 24 hours and 2 weeks. The experimental results showed that the plasma RCAN1 protein level was significantly increased in AIS patients with onset time between 24 hours and 2 weeks.

[0063] In summary, compared with healthy controls (HCs), the plasma RCAN1 protein levels in patients with acute ischemic stroke (AIS) at different onset time periods were significantly increased.

[0064] (2) Furthermore, the receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of RCAN1 protein levels in all AIS patients and in different onset time groups.

[0065] The experimental results are as follows Figures 7-10 As shown, where: Figure 7 In 77 AIS patients, the ROC curve showed the diagnostic efficacy of plasma RCAN1 protein for AIS. The experimental results showed that the area under the curve (AUC) was 0.770 (95% confidence interval: 0.68-0.86) and the optimal cutoff value of plasma RCAN1 was 174.5 ng / L, with a sensitivity of 67.53% and a specificity of 76.47%, indicating that RCAN1 has good diagnostic potential.

[0066] Figure 8 The ROC curve shows the diagnostic efficacy of plasma RCAN1 protein for AIS in 35 AIS patients with onset time less than 6 hours. The experimental results show that when the onset time of AIS is less than 6 hours, the AUC is 0.693, with high specificity (97.06%) and low sensitivity (34.29%).

[0067] Fig. 9 The ROC curve showed the diagnostic efficacy of plasma RCAN1 protein for AIS in 19 AIS patients with onset time of 6 to 24 hours. The experimental results showed that the AIS onset time was 24 hours to 24 hours, with an AUC of 0.920, and a sensitivity and specificity of 94.74% and 76.47%, respectively. This suggests that the diagnostic efficacy of plasma RCAN1 protein is optimal at this stage.

[0068] Fig.10 The ROC curve showed the diagnostic efficacy of plasma RCAN1 protein for AIS in 23 AIS patients with onset time of 24 hours to 2 weeks. The experimental results showed that when the onset time of AIS was between 24 hours and 2 weeks, the AUC was 0.762, and the sensitivity and specificity were 69.57% and 76.47%, respectively.

[0069] In summary, the expression of RCAN1 protein in the plasma samples of AIS patients showed specific dynamic changes: it reached a peak in the 6-24 hour window period (AUC=0.920, sensitivity 94.74%, specificity 76.47%); in the time periods of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 protein decreased significantly.

[0070] It can be seen that based on the dynamic changes of RCAN1 protein, the onset time of the patient can be inferred through a single plasma test result or multiple consecutive test results, providing an important basis for clinical decision-making.

[0071] Example 2 Correlation analysis between RCAN1 protein level and ischemic penumbra volume

[0072] MRI testing, namely magnetic resonance imaging (Magnetic Resonance Imaging), is an advanced medical imaging technology. The MRI devices used in the present invention to collect patient images are all 3T magnetic resonance imaging systems, and the models are uMR 880 (Shanghai United Imaging Medical Technology Co., Ltd., China), Prisma (Siemens Medical Systems, Germany) and SIGNAtm Architect (GE Healthcare, USA).

[0073] like Fig.11 As shown, the present invention uses MRI segmentation method to identify ischemic penumbra and infarct core, and uses ITK-SNAP software to analyze and calculate the ischemic penumbra (10 cases) and infarct core (32 cases) volumes of AIS patients for subsequent statistical analysis. The ischemic penumbra is identified by FLAIR-DWI mismatched area, and the infarct core is determined by FLAIR-DWI matched area.

[0074] like Fig.12 As shown, the correlation between plasma RCAN1 protein level and ischemic penumbra volume in AIS patients (n=10) was evaluated by Spearman correlation analysis, and the results are presented in the form of scatter plots. The experimental results show that in AIS patients, the plasma RCAN1 protein level is significantly correlated with the ischemic penumbra volume in the brain.

[0075] like Fig.13 As shown, the correlation between plasma RCAN1 protein level and infarct core volume in AIS patients (n=32) was evaluated by Spearman correlation analysis, and the results are presented in the form of scatter plots. The experimental results showed that in AIS patients, the plasma RCAN1 protein level was not correlated with the infarct core volume in the brain.

[0076] In summary, RCAN1 level was significantly positively correlated with the volume of the ischemic penumbra (r=0.700, p=0.024), but not significantly correlated with the volume of the infarct core (r=0.294, p=0.103). Therefore, RCAN1 protein can indirectly reflect the extent of the ischemic penumbra and provide a basis for the selection of therapeutic targets.

[0077] Example 3 Verification of Blood RCAN1 Protein as a Blood Marker for Acute Ischemic Stroke (AIS)

[0078] The mouse MCAO (middle cerebral artery occlusion) model is a commonly used animal model in ischemic stroke research, and its representative data mainly include behavioral scores, infarct volume, pathological histological analysis, etc. The mice were divided into a sham operation group (Sham) and a MCAO group: the MCAO group dissected and exposed the right carotid artery, external carotid artery and internal carotid artery, and a silicon-coated nylon filament (MSMC23B120PK50, Reward, China) was inserted into the right middle cerebral artery (MCA) to induce local ischemia, and the laser speckle blood flow imaging system (LSCI, Reward, China) was used to repeat cerebral blood flow detection 10 minutes and 24 hours after surgery. Compared with the baseline, mice with a cerebral blood flow reduction of more than 70% were included in the study; the sham operation group was only exposed and sutured.

[0079] The present invention uses a 9.4T magnetic resonance imaging system (BioSpec 94 / 20USR, Bruker, Germany) to perform magnetic resonance angiography (MRA) and T2-weighted imaging (T2WI) scans on mice, and measures the cerebrovascular structure and brain tissue status of mice in the sham operation group (Sham) and middle cerebral artery occlusion (MCAO) group to verify the relationship between RCAN1 expression changes and cerebral infarction.

[0080] (1) Representative data of the mouse MCAO (middle cerebral artery occlusion) model

[0081] The experimental results are as follows Fig.14 As shown, the experimental results showed that the model of the middle cerebral artery occlusion (MCAO) group was successfully established. After 24 hours, MRA of the MCAO group mice showed no visualization of the right middle cerebral artery, and a large area of ​​infarction occurred in the right cerebral hemisphere.

[0082] (2) Determination of RCAN1 protein level in blood of MCAO mice

[0083] The level of RCAN1 protein in the blood of MCAO mice was detected using a mouse ELISA kit (purchased from Jiangsu Enzyme Immunity Industry Co., Ltd., model MM-46718M1), and the data are expressed as mean ± standard error (mean ± SEM) (n = 11-12). Statistical analysis was performed using the Student's t test, with significance levels of: *p < 0.05, **p < 0.01, ***p < 0.001.

[0084] The experimental results are as follows Fig.15 As shown, the experimental results showed that compared with the sham operation group, the plasma RCAN1 protein level of mice in the middle cerebral artery occlusion (MCAO) group was significantly increased 24 hours after surgery.

[0085] In summary, the present invention verified through MRI examination and Elisa experiment that plasma RCAN1 protein can be used as a blood marker for predicting the onset time window of acute ischemic stroke (AIS).

[0086] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Use of RCAN1 protein as a biomarker for acute ischemic stroke (AIS).

2. The use according to claim 1, characterized in that: The RCAN1 protein is a blood marker.

3. The use according to claim 1, characterized in that: The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) showed specific dynamic changes: it reached a peak value in the 6-24 hour window period; in the time periods of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 decreased.

4. The use according to claim 1, characterized in that: The expression level of the RCAN1 protein in plasma samples of patients with acute ischemic stroke (AIS) is positively correlated with the volume of the ischemic penumbra in the brain.

5. Use of RCAN1 protein in the preparation of products for predicting the onset time window of acute ischemic stroke (AIS) or for evaluating the volume of the ischemic penumbra in patients with acute ischemic stroke (AIS).

6. The use according to claim 5, characterized in that: The product is a detection kit, including reagents for quantitatively detecting the level of RCAN1 protein in the blood and instructions for use.

7. A method for predicting the onset time window of acute ischemic stroke (AIS) or for evaluating the volume of the ischemic penumbra of patients with acute ischemic stroke (AIS), characterized in that: The following steps are involved: a) Collect plasma samples from patients suspected of acute ischemic stroke (AIS); b) detecting the concentration of RCAN1 protein in plasma samples; c) predicting the onset time window of acute ischemic stroke (AIS) in patients or evaluating the volume of the ischemic penumbra in patients with acute ischemic stroke (AIS) based on the plasma level of RCAN1 protein; wherein: The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) showed specific dynamic changes: it reached a peak value in the 6-24 hour window period; in the time period of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 decreased; The expression level of the RCAN1 protein in plasma samples of patients with acute ischemic stroke (AIS) is positively correlated with the volume of the ischemic penumbra in the brain.

8. The method according to claim 7, characterized in that The methods for detecting the level of RCAN1 protein in the blood sample in step b) include enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay, protein blotting and mass spectrometry.

9. The method according to claim 7, characterized in that: The method also includes step d) deciding whether to perform thrombolysis / thrombectomy on patients with acute ischemic stroke (AIS) based on the predicted onset time window or the assessed ischemic penumbra volume, and formulating a personalized treatment plan.

10. A system for predicting the onset time window of acute ischemic stroke (AIS) or for evaluating the volume of the ischemic penumbra of acute ischemic stroke (AIS) patients, characterized in that: include: a) A blood collection device for collecting blood samples from patients suspected of acute ischemic stroke (AIS); b) RCAN1 protein detection module, detecting the concentration of RCAN1 protein in plasma samples; c) A data processing module, which predicts the AIS onset time window of the patient or evaluates the volume of the ischemic penumbra of the acute ischemic stroke AIS patient according to the plasma level of RCAN1 protein; wherein: The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) showed specific dynamic changes: it reached a peak value in the 6-24 hour window period; in the time period of <6 hours and >24 hours, the diagnostic efficacy of RCAN1 decreased; The expression level of the RCAN1 protein in the plasma samples of patients with acute ischemic stroke (AIS) is positively correlated with the volume of the ischemic penumbra in the brain; d) Output module, which displays the prediction / evaluation results and treatment recommendations.

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