Application of biomarker in preparation of product for diagnosing and / or evaluating T2MD post-stroke cognitive impairment

Abstract

The invention relates to the technical field of biomedicine, in particular to application of a biomarker in preparation of a product for diagnosing and / or evaluating T2MD post-stroke cognitive impairment. The biomarker SLC1A2 and / or NPTX1 can accurately diagnose the acute-stage cognitive impairment of the type 2 diabetes stroke, evaluate the acute-stage, subacute / chronic-stage continuous cognitive impairment after the type 2 diabetes stroke and aggravation of the cognitive impairment, and provide a more timely clinical strategy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of biomarkers in preparing products for diagnosing and / or evaluating T2MD post-stroke cognitive impairment. Background Art

[0002] Ischemic stroke is the leading cause of death and disability worldwide and an important cause of poststroke cognitive impairment (PSCI). PSCI is characterized by cognitive deficits that usually appear 3 to 6 months after overt stroke, with a prevalence of approximately 50% in the first year after stroke. This directly affects function and quality of life after stroke and is associated with poor functional prognosis.

[0003] Astrocytes are the most abundant cell type in the brain, each of which contacts thousands of synapses, and they play a crucial role in promoting synaptic transmission and plasticity. Reactive astrocytes can promote synaptogenesis and functional recovery after ischemic stroke. Synaptic dysfunction has been identified as a major cause of cognitive impairment. Excitatory amino acid transporter 2 (EAAT2, SLC1A2) is responsible for most of the glutamate uptake and plays a crucial role in neurodevelopment, synaptic plasticity, learning and memory. Low levels of EAAT2 are associated with cognitive impairment in patients with Parkinson's disease. Neuronal pentraxin 1 (NPTX1) encodes a long neuronal pentraxin called NP1, which is essential for synaptic plasticity. NPTX1 levels are reduced in cerebrospinal fluid (CSF) of patients with sporadic cerebral amyloid angiopathy (sCAA) and AD compared with controls. A recent meta-analysis of PSCI found that diabetes is one of the most important vascular risk factors. Diabetes aggravates the severity of PSCI, which may be due to hyperglycemia exacerbating synaptic plasticity defects and cognitive impairment.

[14] .

[0004] Exosomes are an important class of extracellular vesicles (EVs) that can transport proteins, nucleic acids, and lipids and are considered to be important mediators of intercellular communication. Exosomes containing central nervous system (CNS) components can be detected in peripheral blood through the blood-brain barrier (BBB). However, there is still a lack of specific evidence on the relationship between synaptic proteins in serum astrocyte-derived exosomes (ADEs) and PSCI. Summary of the invention

[0005] The present invention provides the use of biomarkers in the preparation of products for diagnosing and / or evaluating cognitive impairment after T2MD stroke. The biomarkers SLC1A2 and / or NPTX1 can accurately diagnose cognitive impairment in the acute phase of type 2 diabetes stroke, evaluate persistent cognitive impairment in the acute phase, subacute phase / chronic phase, and aggravation of cognitive impairment after type 2 diabetes stroke, and solve the problems existing in the prior art.

[0006] The technical solution adopted by the present invention is:

[0007] The use of biomarkers in the preparation of products for diagnosing and / or evaluating cognitive impairment after stroke in type 2 diabetes, characterized in that the biomarkers are SLC1A2 and / or NPTX1.

[0008] Furthermore, the biomarker is a combination of SLC1A2 and NPTX1.

[0009] Furthermore, the source of the biomarker is synaptic protein in astrocyte-derived exosomes ADEs in peripheral blood serum.

[0010] Furthermore, by detecting the expression level of the biomarker, it is possible to diagnose or evaluate whether the type 2 diabetic post-stroke patient has acute cognitive impairment, persistent cognitive impairment or aggravated cognitive impairment after ischemic stroke.

[0011] Furthermore, the expression levels of SLC1A2 and NPTX1 in serum ADE of patients with cognitive impairment in the acute phase of ischemic stroke were significantly lower than those of patients without cognitive impairment; the low expression levels of SLC1A2 and NPTX1 in serum ADE of patients existed in both the acute and subacute / chronic phases of ischemic stroke, indicating that the patients had persistent cognitive impairment; the low NPTX1 levels in serum ADE of patients without cognitive impairment in the acute phase existed in the subacute / chronic phase, indicating that the risk of worsening cognitive impairment after stroke was reduced.

[0012] Furthermore, low expression levels of SLC1A2 and NPTX1 in serum ADE indicated

[0013] 78.33~156.22pg / mL, 707.57~1217.20pg / mL.

[0014] Furthermore, the acute phase refers to within 3 months after ischemic stroke; the subacute / chronic phase refers to 3-12 months after ischemic stroke.

[0015] Furthermore, in the type 2 diabetes post-stroke acute cognitive impairment group, compared with the group without cognitive impairment, the SLC1A2 level was 119.07 (78.33-156.22) pg / mL vs. 153.71 (106.51-221.31) pg / mL (p=0.001); the NPTX1 level was 936.01 (707.57-1217.20) pg / mL vs. 1325.12 (887.13-19400.03) pg / mL (p<0.001), indicating that the significantly low level expression of SLC1A2 and NPTX1 is the key marker for the diagnosis of acute cognitive impairment.

[0016] Furthermore, the above application, through the simultaneous detection of SLC1A2 and NPTX1, performed ROC curve analysis, and the diagnostic accuracy of acute cognitive impairment in patients with type 2 diabetes after stroke was 79.8%.

[0017] Furthermore, there was no cognitive impairment in the acute phase after stroke in type 2 diabetes, but low SLC1A2 and NPTX1 levels in the subacute / chronic phase, suggesting that cognitive impairment in patients worsened.

[0018] Furthermore, the absence of cognitive impairment in the acute phase after stroke in patients with type 2 diabetes and low NPTX1 levels in serum ADE in the subacute / chronic phase suggest that the risk of cognitive impairment after stroke in patients is reduced.

[0019] Furthermore, the product includes a chip or a kit.

[0020] Furthermore, the kit is a PCR kit for detecting SLC1A2 and NPTX1.

[0021] Beneficial effects of the present invention:

[0022] The present invention prospectively studies the relationship between synaptic proteins SLC1A2, NPTX1 and PSCI in serum ADEs of patients with type 2 diabetes mellitus (T2DM) in the acute phase of ischemic stroke. It was determined that SLC1A2, NPTX1 and PSCI are associated, and the significantly low-level expression of the two markers can accurately diagnose cognitive dysfunction in patients in the acute phase after stroke. The combination of the two biomarkers provides assistance for the early diagnosis of cognitive impairment after stroke in type 2 diabetes, and can be used as an effective basis for diagnosis and prediction, facilitating early and effective therapeutic intervention, and providing more timely clinical strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Comparison of the concentrations of synaptic proteins SLC1A2 and NPTX1 in astrocyte-derived exosomes between the two groups of the present invention; CI is the cognitive impairment group, NCI is the non-cognitive impairment group, *p<0.05, **p<0.01, ***p<0.001;

[0024] Figure 2 This is the receiver operating characteristic (ROC) curve of the present invention. DETAILED DESCRIPTION

[0025] In order to clearly illustrate the technical features of the present invention, the present invention is described in detail below through specific implementation methods in conjunction with the accompanying drawings.

[0026] 1. Clinical Trials

[0027] The present invention recruited T2DM patients with mild to moderate AIS for the first time from August 2020 to March 2022 and a National Institutes of Health Stroke Scale (NIHSS) ≤ 10. These patients were hospitalized in the Department of Neurology of Weihai Municipal Hospital within 72 hours of symptom onset. The diagnosis of AIS was based on the 1989 World Health Organization criteria and confirmed by magnetic resonance imaging (MRI).

[0028] Exclusion criteria were as follows: hemorrhagic stroke, transient ischemic stroke, stroke caused by trauma or invasive surgery, dementia or cognitive impairment (score ≥3.19 on the Elderly Cognitive Decline Questionnaire), neurological diseases that may affect cognition (such as Parkinson's disease), taking medications that may affect cognitive function, severe heart, liver, and kidney diseases, and inability to complete neuropsychological assessment. Neurological assessment included verification of stroke pathogenesis and NIHSS. Cardiovascular risk factors were determined by medical history and laboratory results. All subjects signed informed consent before enrollment. This study followed the Declaration of Helsinki and was approved by the Ethics Committee of Weihai Hospital.

[0029] 1.1 Neuropsychological assessment

[0030] The Beijing version of the Montreal Cognitive Assessment (MoCA) was used to assess cognitive function and cognitive subdomains (including visual executive function, naming, attention, language, and abstraction) of all participants in the acute phase and subacute / chronic phase (3, 6, and 12 months after stroke) of ischemic stroke. In the acute phase of ischemic stroke, a MoCA score < 25 was considered acute cognitive impairment.

[16] In the subacute / chronic phase after ischemic stroke, a MoCA score of <25 was classified as PSCI (post-stroke cognitive impairment), and a MoCA score of ≥25 was classified as no post-stroke cognitive impairment. Patients with the same cognitive impairment in the acute and subacute / chronic phases were classified as persistent cognitive impairment. Patients with no cognitive impairment in the acute phase but cognitive impairment in the subacute / chronic phase were classified as worsening cognitive impairment.

[0031] Hamilton Depression Rating Scale-17 (HAMD-17) was used to assess depressive symptoms, and HAMD-17 scores ≥ 17 were considered depressive symptoms. The STOP-Bang questionnaire was used to assess the risk of obstructive sleep apnea (OSA), and STOP-Bang scores ≥ 3 were considered high risk of OSA.

[0032] 1.2 Magnetic resonance data acquisition

[0033] Whole-brain MRI and brain magnetic resonance angiography (MRA) were obtained on the same 3.0T Skyra MRI scanner (Siemens, Erlangen, Germany). T1-weighted and T2-weighted fluid-attenuated inversion recovery (FLAIR) images, apparent diffusion coefficient (ADC) images, and diffusion-weighted imaging (DWI) images were acquired. Image acquisition parameters were: repeat time (TR) 4040 ms, echo time (TE) 64 ms, field of view (FOV) 220 × 220, matrix size = 512 × 512, slice thickness = 5 mm. Three-dimensional TOF-MRA scanning parameters were: TR 21 ms, TE 3.43 ms, FOV 200 mm × 180 mm, matrix 313 × 384, signal excitation number 1, and slice thickness 0.7 mm. Infarct volumes were calculated on DWI images using a semi-automated free open-source 3D slicer software (http: / / www.slicer.org). The infarct area was determined using the “Segment Editor” in 3D Slicer. Characteristic MRI was used to evaluate small vessel disease (SVD), including lacunar infarcts, white matter hyperintensities (WMHs), cerebral microbleeds (CMBs), enlarged perivascular spaces (PVS), and brain atrophy. White matter hyperintensities were defined as the presence of areas of high intensity in the white matter on FLAIR images. WMHs in periventricular and deep white matter were visually assessed using the Fazekas score. We used a visual rating scale to assess PVS and calculated the PVS visible on MRI of the basal ganglia (BGPVS) on one side. SVD was graded from 0 to 4 based on a Fazekas score ≥2 for deep WMHs or a periventricular score ≥3, the presence of ≥1 lacunes, ≥1 CMB, and a BGPVS score ≥2.

[17] In this study, AIS was divided into three types according to imaging data: cortical infarction, subcortical infarction, and infratentorial infarction.

[0034] 1.3 Extraction of exosomes and quantification of synaptic proteins

[0035] After fasting for 12 hours, peripheral blood was collected from the patient's forearm vein. The blood samples were centrifuged (4000g, centrifugation for 10 minutes) to separate the cells from the cell debris, and then 500μl of the supernatant was transferred to a sterile centrifuge tube. 126μl / tube of the kit method exosome precipitation solution (SystemBiosciences, EXOQ20A-1, CA, USA) was added to each tube, mixed to a cloudy state, and incubated at 4°C for 60 minutes. After centrifugation at 1500×g for 30 minutes at 4°C, total exosomes were isolated from each sample.

[0036] To extract astrocyte-enriched exosomes, total exosomes were resuspended in 250 μl of Dulbecco's phosphate-buffered saline, vortexed, and incubated with 2.3 μl of glutamine aspartate transporter (ACSA-1) biotinylated antibody (Miltenyi Biotec, 130-118-984, Auburn, CA) in 50 μl of 3% bovine serum albumin for 60 min at 20°C. Then, 25 μl of streptavidin-agarose resin (Thermo Fisher Scientific, 53117, Waltham, MA, USA) containing 50 μl of 3% bovine serum albumin was added and incubated at 4°C for 30 min. 4℃, 800×g centrifugation for 10 minutes, discard the supernatant, be careful not to suck away the resin below, place in 100μl 0.05M glycine-HCl (pH 3.0) vortex shake for 10 minutes, centrifuge at 4000×g for 10 minutes (4℃). Then transfer the supernatant to a pre-cooled Eppendorf tube containing 100μl 10% bovine serum albumin and 20μl 1M Tris-HCl (pH 8.6), and then add 550μl mammalian protein extraction reagent (M-PER) (Thermo Fisher Scientific, 78505, Waltham, MA, USA), and store the astrocyte-derived exosome lysate at -80℃.

[0037] Exosomes were identified using transmission electron microscopy. A small portion of the exosomes was resuspended in 50 μl of 1× phosphate buffered saline solution and mixed with 50 μl of 4% paraformaldehyde solution. Then 10 μl of the exosome suspension was dropped onto the sealing film, and the Formvar-carbon copper mesh was immersed in the exosome suspension with the Formvar membrane facing down and fixed for 30 minutes. 50 μl of 1× phosphate buffered saline solution was dropped onto the sealing film and rinsed for a total of 40 minutes, 10 minutes each time. The other side of the copper mesh was kept dry and the Formvar membrane was facing down. After rinsing, 50 μl of 1% glutaraldehyde was dropped onto the sealing film, immersed in 5 μl, and rinsed again with phosphate buffered saline for 40 minutes, the method was the same as above. After rinsing, it was immersed in 3% tungsten phosphate solution for negative staining for 3 minutes, and the negative staining solution was blotted with filter paper and dried at room temperature. Exosomes were identified morphologically using a Hitachi H7500 electron microscope.

[0038] After confirming that the exosomes were successfully extracted, the exosomes were quantified: 250 μl of M-PER containing EDTA and a mixture of protease inhibitors was added, and the mixture was shaken and centrifuged. 2 μl of supernatant was used for BCA exosome total protein concentration determination. The protein sample was diluted 1:25, and the protein standard was diluted with 1× phosphate buffered saline solution to prepare standard protein solutions of 500 μg / ml, 400 μg / ml, 300 μg / ml, 200 μg / ml, 100 μg / ml, 50 μg / ml, 25 μg / ml, and 0 μg / ml concentrations. 25 μl of protein solution / standard was added to the 96-well plate. BCA reagent (Servicebio, G2026, Wuhan) A solution and B solution (50:1) were prepared into a mixture and 200 μl was added to each well. Incubate in the dark for 30 minutes (37°C). Place in the full wavelength microplate reader-Varioskan LUX 3020 instrument (Thermo Fisher Scientific Oy, Finland), wavelength 562nm, determine the concentration and prepare a standard curve. Then use protein blotting to detect exosome protein markers: mix the separated exosomes with the loading buffer and put them in a 100℃ water bath for 5 minutes, centrifuge, place on ice, and wait for loading. Prepare SDS-PAGE separation gel and gradually add each solution with a concentration of 12%, mix and add to two glass plates for gel making, and seal with isopropanol. After the lower layer of gel solidifies, pour out the isopropanol, rinse with deionized water and dry, and prepare the concentrated gel. Add each solution with a concentration of 5% and mix, add to two glass plates for gel making, immediately insert the comb, and pull out the comb after solidification. Place the gel tank on ice and pour in the electrophoresis buffer, load 10-35μl into each well, and record the order of loading. Prepare electrophoresis buffer [Tris. hydrochloric acid 3.028g (25mM), Gly 14.413g (192mM), 10% SDS 10ml (0.1% SDS, dilute to 1000ml with deionized water] in advance, precool at 4℃; transfer buffer [Tris. hydrochloric acid 3.028g (25mM), Gly 14.413g (192mM), methanol 100ml, dilute to 1000ml with deionized water], precool at 4℃. Upper gel 60V, 30 minutes; lower gel 110V, 120 minutes. After the electrophoresis, the gel needs to be rinsed in phosphate buffer solution for 2 minutes, and then soaked in transfer buffer for equilibrium. Soak the cut PVDF membrane (Millipore, IPVH00010) in methanol for 2 minutes, then soak in deionized water for 2 minutes, and finally soak in transfer buffer for equilibrium for 15 minutes. Transfer on ice, 100V constant voltage transfer for 50 minutes. Placement of mat, membrane and gel: red, (+) white rack, mat, filter paper, membrane, gel, filter paper, mat, black rack (-), black. The transfer band is from - to +. After the transfer is completed, soak the membrane in phosphate buffered saline for 3-5 minutes and cut the membrane.Block with 5% skim milk (0.75g milk powder + 15ml phosphate buffered saline) at 37°C for 1-2 hours. Wash the membrane with 1× phosphate buffered saline-T (1000ml 1× phosphate buffered saline + 1ml Tween-20) for 5-10 minutes, 3 times. Dilute the primary antibody according to the antibody instructions [CD63 antibody: 1:500; TSG101 antibody (PROTEINTECH, 28283-1-AP, USA): 1:1000; HSP70 antibody (PROTEINTECH, 10995-1-AP, USA): 1:2000; Calnexin antibody (PROTEINTECH, 10427-2-AP, USA): 1:2000] and incubate overnight on a shaker at 4°C. Take the shaker out of the ice storage and wash the membrane with 1× phosphate buffered saline-T for 5-10 minutes, 3 times. Secondary antibodies [rabbit secondary antibody (Jackson Immuno Research, 111-035-003, USA) 1:5000; mouse secondary antibody (Jackson Immuno Research, 115-035-003, USA) 1:10000] were added and incubated on a shaker at 37°C for 2 h. The membrane was washed with 1× phosphate buffered saline-T for 5 min, 5 times, and developed by chemiluminescence.

[0039] Enzyme-linked immunosorbent assay (ELISA) kits were used to quantify the four-span exosome markers CD63 (RayBio, ELH-CD63, Norcross, GA 30092, USA), human excitatory amino acid transporter 2 / EAAT2 (SLC1A2) (Abbexa Ltd, abx151369, Cambridge, UK), and human neuronal Pentraxin 1 (NPTX1) (Abbexa Ltd, abx152501, Cambridge, UK). The ADEs level of CD63 protein was determined to normalize the exosome content. The intra-group coefficient of variation was less than 10%, and the inter-group coefficient of variation was less than 10%-12%. The ELISA microplates were read using a V Arioskan LUX 3020 instrument (Thermo Fisher Scientific Oy, Ratastie 2, Vantaa, Finland). Serum samples from all subjects were analyzed by the same technician who was blinded to the clinical data.

[0040] 1.4 Statistical analysis

[0041] Statistical analysis was performed using R software version 4.4.1 and SPSS software version 27.0, and graphics were processed using GraphPad Prism 10.0. The Kolmogorov-Smirnov test was used to determine the normality of continuous data. Normally distributed variables were analyzed using the t-test, and nonparametric variables were analyzed using the Mann-Whitney U test. Categorical variables were compared using the χ2 test. All tests were two-tailed, and differences were considered statistically significant at P < 0.05. Mediation models were established to determine whether synaptophysin in serum ADEs mediates the effects of OH, OSA, depression, infarct size, and SVD on cognitive function and its subdomains in the acute phase of ischemic stroke. Binary logistic regression and multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% CIs of PSCI (persistent cognitive impairment, worsening cognitive impairment) of SLC1A2 and NPTX1, with no cognitive impairment as the reference. Covariates included age, sex, education, BMI, hypertension, current smoking, and current drinking. After adjusting for the above covariates, the effects of serum SLC1A2 and NPTX1 levels on PSCI were evaluated.

[0042] 2. Results

[0043] 2.1 Baseline demographics and biomarker characteristics

[0044] A total of 244 patients were included in the acute phase of this study. The participants were divided into a cognitive impairment group and a non-cognitive impairment group, of which 181 patients (74.18%) had cognitive impairment. In the acute phase of ischemic stroke, the levels of SLC1A2 and NPTX1 in serum ADE in the cognitive impairment group were significantly lower than those in the non-cognitive impairment group. Specifically, the SLC1A2 level was 119.07 (78.33-156.22) pg / mL vs. 153.71 (106.51-221.31) pg / mL (p=0.001); the NPTX1 level was 936.01 (707.57-1217.20) pg / mL vs. 1325.12 (887.13-19400.03) pg / mL (p<0.001), see Figure 1 .

[0045] 2.2 Predictive value of SLC1A2 and NPTX1 in serum ADEs for cognitive impairment after stroke in type 2 diabetes

[0046] To explore the potential of SLC1A2 and NPTX1 in serum ADEs as prognostic biomarkers for PSCI in patients with T2DM, binary logistic regression and multivariate logistic regression models were used, with cognitive function as the dependent variable and SLC1A2 and NPTX1 as independent variables.

[0047] At 3 months follow-up after stroke, after adjusting for age, sex, education, BMI, hypertension, current smoking and drinking, low serum SLC1A2 levels in ADE were associated with PSCI (OR 0.903, 95% CI 0.826-0.986) and increased risk of persistent cognitive impairment after stroke (OR 0.878, 95% CI 0.797-0.966). Low serum NPTX1 levels in ADE (OR 0.873, 95% CI 0.768-0.993) were also associated with an increased risk of persistent cognitive impairment after stroke. In contrast, low NPTX1 levels were associated with a reduced risk of worsening cognitive impairment after stroke (OR 1.170, 95% CI 1.012-1.352). There was no significant interaction between NPTX1 levels and PSCI (p>0.05). There was no significant interaction between SLC1A2 level and the worsening of cognitive impairment after stroke (all p>0.05) (Table 1).

[0048] Table 1 ORs and 95% CIs of cognitive impairment 3 months after ischemic stroke

[0049]

[0050]

[0051] Note: Model 0: Unadjusted. Model 1: Adjusted for age, gender, and years of education. Model 2: Adjusted for age, gender, years of education, BMI, hypertension, smoking, and drinking. OR, odds ratio. CI, confidence interval.

[0052] Similar results were observed at 6-month follow-up after stroke. After adjusting for covariates, T2DM patients with lower SLC1A2 levels in serum ADE had a higher incidence of PSCI (OR 0.895, 95% CI 0.817-0.979) and persistent cognitive impairment after stroke (OR 0.877, 95% CI 0.795-0.967). In addition, lower NPTX1 levels (OR 0.871, 95% CI 0.764-0.992) were associated with a higher incidence of persistent cognitive impairment after stroke. In contrast, patients with lower NPTX1 levels (OR1.175, 95% CI 1.019-1.355) had a lower incidence of worsening cognitive impairment after stroke. The interaction between NPTX1 levels and PSCI was not significant (p<0.05). In addition, there was no significant interaction between SLC1A2 levels and worsening cognitive impairment after stroke (all p>0.05) (Table 2).

[0053] Table 2 ORs and 95% CIs for cognitive impairment 6 months after ischemic stroke

[0054]

[0055] Note: Model 0: Unadjusted. Model 1: Adjusted for age, gender, and years of education. Model 2: Adjusted for age, gender, years of education, BMI, hypertension, smoking, and drinking. OR, odds ratio. CI, confidence interval.

[0056] After 12 months of follow-up, similar results were observed. Low serum ADE levels of SLC1A2 were associated with an increased risk of PSCI (unadjusted OR 0.908, 95% CI 0.839-0.983) and persistent cognitive impairment after stroke (unadjusted OR 0.884, 95% CI 0.810-0.965). In addition, low serum NPTX1 levels in ADE (unadjusted OR 0.865, 95% CI 0.770-0.972) were associated with an increased risk of persistent cognitive impairment after stroke. In contrast, low NPTX1 levels (unadjusted OR 1.238, 95% CI 1.074-1.428) were associated with a reduced risk of worsening cognitive impairment after stroke. There was no significant interaction between NPTX1 levels and PSCI (p>0.05), and there was no significant interaction between SLC1A2 levels and worsening cognitive impairment after stroke (p>0.05). However, after adjusting for covariates, no significant interaction was observed between SLC1A2 levels in serum ADE and PSCI (p>0.05), nor between NPTX1 levels and persistent cognitive impairment after stroke (p>0.05) (Table 3).

[0057] Table 3 ORs and 95% CIs for cognitive impairment 12 months after ischemic stroke

[0058]

[0059] Note: Model 0: Unadjusted. Model 1: Adjusted for age, gender, and years of education. Model 2: Adjusted for age, gender, years of education, BMI, hypertension, smoking, and drinking. OR, odds ratio. CI, confidence interval.

[0060] 2.3 Diagnostic value of serum astrocyte-derived exosome synaptophysin for cognitive dysfunction in acute stroke in type 2 diabetic patients

[0061] Receiver operating characteristic (ROC) curve analysis was performed to verify the diagnostic value of serum astrocyte-derived exosomal mitochondrial proteins (SLC1A2 and NPTX1) for cognitive dysfunction in acute stroke in type 2 diabetes. The diagnostic accuracy of serum astrocyte-derived exosomal complement protein SLC1A2 for cognitive dysfunction in acute stroke in type 2 diabetes was 72.3%; the diagnostic accuracy of serum astrocyte-derived exosomal complement protein NPTX1 was 76.4%; and the diagnostic accuracy of combined PPARGC1 and Nrf2 for cognitive dysfunction in acute stroke in type 2 diabetes was 79.8%.

[0062] 3. Discussion and Conclusion

[0063] The present invention explored the relationship between SLC1A2 and NPTX1 in serum ADE and PSCI at three time points in patients with T2DM and ischemic stroke. The results showed that: (1) Reduced levels of SLC1A2 in serum ADE were associated with an increased risk of PSCI and persistent cognitive impairment after stroke. (2) Low levels of NPTX1 in serum ADE were associated with an increased risk of persistent cognitive impairment after stroke and a reduced risk of worsening cognitive impairment after stroke. (3) Serum astrocyte-derived exosomal synaptic proteins SLC1A2 and NPTX1 as a biomarker combination have significant diagnostic value for cognitive dysfunction in the acute phase of type 2 diabetic stroke.

[0064] Low levels of SLC1A2 in serum ADEs may be a risk factor for PSCI, suggesting that it has the potential to be used as a biomarker for the acute diagnosis of T2DM patients with PSCI and for predicting the persistence of cognitive impairment; its combination with NPTX1 as a biomarker can improve the accuracy of predicting acute cognitive impairment and the persistence of cognitive impairment. The low expression of NPTX1 in the subacute / chronic phase of T2DM patients with PSCI also suggests that patients without cognitive impairment in the acute phase have a reduced risk of worsening cognitive impairment. In addition, synaptic dysfunction may be a potential pathophysiological mechanism and new therapeutic target for PSCI.

[0065] The above specific implementation manner cannot be used as a limitation on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation manner of the present invention falls within the protection scope of the present invention.

[0066] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. Use of a biomarker in the preparation of a product for diagnosing and / or evaluating cognitive impairment after stroke in T2MD, characterized in that: The biomarkers are SLC1A2 and / or NPTX1.

2. The use according to claim 1, characterized in that: The biomarkers are a combination of SLC1A2 and NPTX1.

3. The use according to claim 1, characterized in that: The biomarkers SLC1A2 and NPTX1 are derived from synaptic proteins in astrocyte-derived exosomes ADEs in peripheral blood serum.

4. The use according to claim 1, characterized in that: By detecting the expression level of the biomarker, it is possible to diagnose or evaluate whether type 2 diabetic post-stroke patients have acute cognitive impairment, persistent cognitive impairment or aggravated cognitive impairment after ischemic stroke.

5. The use according to claim 4, characterized in that: The expression levels of SLC1A2 and NPTX1 in serum ADE of patients with cognitive impairment in the acute phase of ischemic stroke were significantly lower than those of patients without cognitive impairment; the low expression levels of SLC1A2 and NPTX1 in serum ADE of patients existed in both the acute phase and subacute / chronic phase of ischemic stroke, indicating that the patients had persistent cognitive impairment; the low NPTX1 level in serum ADE of patients without cognitive impairment in the acute phase existed in the subacute / chronic phase, indicating that the risk of worsening cognitive impairment after stroke was reduced.

6. The use according to claim 4, characterized in that: The low expression levels of SLC1A2 and NPTX1 in serum ADE were 78.33-156.22 pg / mL and 707.57-1217.20 pg / mL respectively.

7. The use according to claim 1, characterized in that: The product includes a chip or a kit.

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