A detection method for assisting diagnosis of sepsis-related encephalopathy

Abstract

The present application relates to the technical field of biomarkers of sepsis-associated encephalopathy, and particularly relates to a detection method for assisting diagnosis of sepsis-associated encephalopathy, which is not used for disease diagnosis purposes, comprising the following steps: collecting a peripheral blood sample of a subject and separating plasma; separating exosomes from the plasma; detecting the expression level or concentration of carboxylesterase 1 protein in the separated exosomes; comparing the detected carboxylesterase 1 protein level with a preset reference value to obtain a comparison result. The present application has the beneficial effect that the carboxylesterase 1 protein in plasma exosomes is established as a specific biomarker strongly related to SAE for the first time.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology for sepsis-associated encephalopathy, and in particular to a detection method for the auxiliary diagnosis of sepsis-associated encephalopathy, a method for detecting the expression level of carboxylesterase 1 protein in plasma exosomes, and a kit. Background Technology

[0002] Sepsis-associated encephalopathy (SAE) is one of the most common and serious complications of sepsis, clinically manifested as delirium, altered consciousness, and other widespread brain dysfunctions. It is not only a marker of severe illness but also an independent risk factor for increased mortality and long-term cognitive impairment. However, the clinical evaluation of SAE currently faces significant challenges.

[0003] First, the evaluation of SAE lacks a universally accepted "gold standard." It primarily relies on neurological function assessment using tools such as the Glasgow Coma Scale, assuming a confirmed diagnosis of sepsis, while ruling out other possible causes of altered consciousness, such as central nervous system infections, structural brain injury, and severe metabolic disorders. This "exclusionary" evaluation approach is extremely difficult to implement in the complex environment of the intensive care unit (ICU) (where patients are often deeply sedated and mechanically ventilated), highly subjective, and easily influenced by external factors. This leads to significant variability in SAE incidence data across different studies, resulting in high rates of missed and misdiagnosed cases in clinical practice. Second, current technologies for biomarker research have significant shortcomings.

[0004] Although some studies have explored the application of traditional serum biomarkers such as S100β protein and neuron-specific enolase (NSE) in SAE, these proteins can be nonspecifically elevated in various conditions such as systemic inflammatory responses, traumatic brain injury, and liver and kidney dysfunction. This results in a serious lack of specificity and accuracy in evaluating SAE, limiting their clinical value. The core reason for this is that the existence of the blood-brain barrier prevents most proteins in the peripheral circulation from directly and specifically reflecting pathological changes within the brain parenchyma.

[0005] Therefore, how to screen highly specific protein detection indicators from plasma exosomes remains a major technical challenge. Summary of the Invention

[0006] This invention provides a detection method for the auxiliary diagnosis of sepsis-related encephalopathy, a method for detecting the expression level of carboxylesterase 1 protein in plasma exosomes, and a kit to solve the above-mentioned problems.

[0007] This invention discloses a detection method for the auxiliary diagnosis of sepsis-related encephalopathy, wherein the method is not used for disease evaluation purposes, and includes the following steps: Peripheral blood samples were collected from the subjects and plasma was separated; Exosomes were isolated from the plasma; Detect the expression level or concentration of carboxylesterase 1 protein in the isolated exosomes; In the step of isolating exosomes from the plasma, the separation of exosomes is performed using ultracentrifugation, size exclusion chromatography, polymer precipitation, or microfluidic chip technology. In the step of detecting the expression level or concentration of carboxylesterase 1 protein in the isolated exosomes, the detection of carboxylesterase 1 protein can be performed using Western blotting, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or mass spectrometry.

[0008] Optionally, the step of separating exosomes from the plasma includes: thawing the frozen plasma at 4°C and centrifuging it at 4°C and 12,000×g for 30 minutes to thoroughly remove cell debris, platelets and apoptotic bodies.

[0009] Optionally, the step of detecting the expression level or concentration of carboxylesterase 1 protein in the isolated exosomes specifically involves: adding RIPA lysis buffer containing a protease inhibitor to the exosome suspension and lysing on ice for 30 minutes. Subsequently, centrifuging at 4°C and 12,000×g for 15 minutes, and collecting the supernatant as the total exosome protein. The protein concentration is determined using a BCA protein quantification kit.

[0010] Optionally, the step of separating exosomes from the plasma includes filtering the supernatant through a 0.22 μm polyethersulfone filter membrane to further remove impurities.

[0011] Optionally, the purified exosomes can be resuspended in 100-200 μL of 1× phosphate buffer and stored at -80°C or used directly for subsequent analysis.

[0012] Optionally, the collection of peripheral blood samples and separation of plasma from the subject includes the following steps: using an EDTA anticoagulant vacuum blood collection tube, collect 5-10 mL of peripheral venous blood from a patient suspected or confirmed to have sepsis. Within 2 hours after collection, centrifuge at 1500×g for 10 minutes at 4°C to remove blood cells, carefully aspirate the supernatant plasma, and transfer it to a new centrifuge tube.

[0013] This invention also discloses a method for detecting the expression level of carboxylesterase 1 protein in plasma exosomes, comprising the following steps: Peripheral blood samples were collected from the subjects and plasma was separated; Exosomes were isolated from the plasma; The expression level or concentration of carboxylesterase 1 protein in the isolated exosomes was detected.

[0014] The present invention also discloses a kit for detecting SAE, the kit comprising an exosome separation component, a protein lysis buffer, and a detection reagent; the exosome separation component comprising a pre-treatment centrifuge tube for removing large particles, a filter, and / or an exosome extraction column or magnetic beads based on polymer precipitation or membrane affinity methods.

[0015] The beneficial effects of the detection method provided in this invention are as follows: For the first time, carboxylesterase 1 protein in plasma exosomes has been established as a specific biomarker strongly associated with SAE. Compared with existing technologies, it has the following outstanding advantages and positive effects: First, high specificity. This invention utilizes the characteristic that exosomes can cross the blood-brain barrier and carry specific signals from their source cells. It is the first to discover and confirm that the level of exosome-derived CES1 in plasma is specifically elevated in some SAE patients. This association is independent of confounding factors such as liver function damage, thus overcoming the deficiency of poor specificity in traditional serum biomarkers. Second, it provides an objective basis for judgment. This invention provides an objective method based on biomolecular detection. Carboxylesterase 1 can indicate the presence of organ dysfunction and has a strong correlation with SAE. Its evaluation efficacy (area under the curve) has been experimentally verified to be significantly superior to the clinically commonly used SOFA (Sequential Organ Failure Assessment) and APACHE II (Acute Physiology and Chronic Health Evaluation II) scores. It overcomes the deficiency of insufficient specificity in plasma exosome protein detection in existing technologies and can provide a highly specific method for detecting plasma exosome carboxylesterase 1 protein. Attached Figure Description

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. In the accompanying drawings: Figure 1A A schematic diagram of the workflow for plasma exosome isolation, identification, and proteomics analysis; Figure 1B Principal component analysis score plots show the separation trend of exosomal protein profiles between non-SAE patients and SAE patients; Figure 1C The bar chart shows the number of proteins upregulated and downregulated in SAE patients compared to non-SAE controls; Figure 2A Physicochemical properties of plasma exosome samples; Figure 2B A representative transmission electron microscope image of plasma exosomes; Figure 2C Western blot analysis of exosome marker proteins; Figure 2D Size distribution of plasma exosomes; Figure 3A Western blot analysis of CES1 protein expression in plasma exosomes of non-SAE and SAE patients; Figure 3B Quantitative statistical analysis of CES1 protein expression levels in exosomes; Figure 4A The proportion of patients with low and high plasma exosomal CES1 levels in the SAE and non-SAE patient groups; Figure 4B Receiver operating characteristic curve analysis was used to compare the efficacy of plasma exosome CES1, SOFA score, and APACHE II score in assessing organ dysfunction including SAE. Figure 4C To compare the ICU stay length between the low and high CES1 expression groups; Figure 4D To compare the duration of mechanical ventilation between the low and high expression groups of CES1; Figure 4E Kaplan-Meier curves for 28-day survival rates in the low-expression and high-expression groups of CES1; Figure 5 Correlation analysis of plasma exosome CES1 levels and liver function indicators; Figure 6A Western blot analysis of CES1 protein expression in brain tissue of SAE model mice induced by intraperitoneal injection of different doses of LPS or CLP. Figure 6B Quantitative analysis of CES1 protein levels detected by Western blotting; Figure 7A -D indicates Nissl staining to show the morphology of hippocampal and dentate gyrus neurons in SAE model mice induced by different doses of LPS; Figure 7E Morphology of hippocampal and dentate gyrus neurons in CLP-induced SAE model mice; Figure 7F Statistical analysis of the survival rate of neurons in the dentate gyrus of the hippocampus in each group. Detailed Implementation

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0018] The applicant's research has found that exosomes, as cell-secreted nanovesicles carrying parental cell-specific molecular information, can provide a novel approach for the non-invasive evaluation of central nervous system diseases. Exosomes can cross the blood-brain barrier for bidirectional transport, potentially allowing brain-derived molecules (such as proteins and RNA) to enter the peripheral circulation. Therefore, theoretically, analyzing exosomes in plasma holds promise for achieving an indirect, specific "liquid biopsy" of intracranial pathological states. This offers a potential solution to the problem of the lack of specificity in SAE biomarkers. However, prior to this, no publicly available technology or research has clearly indicated which specific proteins in plasma exosomes can serve as effective biomarkers for SAE.

[0019] Carboxylesterase 1 (CES1) is a metabolic enzyme primarily expressed in the liver and is traditionally known for its involvement in drug and lipid metabolism. The applicant's research has found that CES1 is also highly expressed in immune cells such as monocytes and macrophages, playing a crucial immunomodulatory role and negatively regulating inflammatory responses. Given that neuroinflammation is a core pathogenesis of SAE, CES1 may participate in the pathological process of SAE through its immunomodulatory function.

[0020] However, to date, no existing technology has linked "plasma exosome-derived CES1" to the evaluation or pathophysiology of SAE. In other words, existing technologies have neither provided a highly specific peripheral blood biomarker to solve the evaluation dilemma of SAE, nor have they revealed the potential value of exosome CES1 in SAE.

[0021] Therefore, the present invention aims to overcome the above-mentioned defects of the prior art and provide a novel biomarker that can non-invasively, objectively, and with high specificity assist in the evaluation of SAE and reflect its neuroinflammatory pathological process.

[0022] To address the aforementioned problems, this invention provides a novel technical solution. This invention first proposes a method and kit for detecting the level of carboxylesterase 1 protein in plasma exosomes. It should be noted that all technical solutions and experimental results of this invention are only used to verify the correlation between CES1 and SAE pathology, and are not directly used for human evaluation or disease diagnosis.

[0023] Specifically, this invention discloses a detection method for the auxiliary diagnosis of sepsis-related encephalopathy, wherein the method is not for disease diagnosis purposes, and includes the following steps: Peripheral blood samples were collected from the subjects and plasma was separated; Exosomes were isolated from the plasma. The expression level or concentration of carboxylesterase 1 protein in the isolated exosomes was detected; The detected carboxylesterase 1 protein level was compared with a preset reference value to obtain the comparison results.

[0024] The separation of exosomes can be achieved using techniques such as ultracentrifugation, size exclusion chromatography, polymer precipitation, or microfluidic chips; the detection of carboxylesterase 1 protein can be achieved using methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or mass spectrometry. The reference value is determined by detecting the plasma exosomal carboxylesterase 1 levels in a group of healthy controls. Furthermore, a reference range is established beforehand by detecting the plasma exosomal carboxylesterase 1 levels in a certain number of healthy controls.

[0025] The beneficial effects of this invention are that it establishes carboxylesterase 1 protein in plasma exosomes as a specific biomarker strongly associated with SAE for the first time. Compared with existing technologies, it has the following outstanding advantages and positive effects: First, high evaluation specificity. This invention utilizes the characteristic that exosomes can cross the blood-brain barrier and carry specific signals from their source cells to discover and confirm for the first time that plasma exosome-derived CES1 levels are specifically elevated in SAE patients. This association is independent of confounding factors such as liver function impairment, thus overcoming the deficiency of poor specificity of traditional serum biomarkers. Second, it provides objective evaluation criteria. This invention provides an objective method based on biomolecular detection. Carboxylesterase 1 can indicate the presence of organ dysfunction and has a strong correlation with SAE. Its evaluation efficacy (area under the curve) has been experimentally proven to be significantly superior to the commonly used clinical SOFA (Sequential Organ Failure Assessment) and APACHE II (Acute Physiology and Chronic Health Evaluation II) scores. It overcomes the deficiency of insufficient specificity in plasma exosome protein detection in existing technologies and can provide a highly specific method for detecting plasma exosome carboxylesterase 1 protein.

[0026] The detected carboxylesterase 1 protein level is compared with a preset reference value. If the level is higher than the reference value, it indicates the risk of sepsis-associated encephalopathy. Specifically, based on the present invention, a kit or detection system for in vitro evaluation can be developed. Its application involves the quantitative detection of plasma exosome CES1 levels in sepsis patients, providing an objective and specific laboratory indicator to assist in the rapid evaluation of sepsis-associated encephalopathy, effectively compensating for the shortcomings of current subjective assessments and improving the accuracy and timeliness of evaluation.

[0027] In the fields of drug development and translational medicine, the present invention also holds significant value. For pharmaceutical companies and research institutions, plasma exosome CES1 can serve as a key pharmacodynamic biomarker. In new drug development, it can be used to validate the rationale for targeting CES1 or its related pathways, and in trials, it serves as a crucial objective indicator for evaluating whether candidate drugs can effectively regulate SAE-related neuroinflammation.

[0028] In the fields of basic and translational science research, this invention provides new tools and perspectives for in-depth exploration of the pathological mechanisms of sepsis-related encephalopathy. Researchers can utilize this invention to investigate the specific role of exosome CES1 in mediating the dialogue between the peripheral immune system and the central nervous system, elucidating its role in key aspects such as blood-brain barrier dysfunction and abnormal microglial activation. This is expected to advance more precise molecular subtyping of the disease and the discovery of new intervention targets.

[0029] With the further portability and cost reduction of detection technologies, this invention also has potential applications in health management. For example, it can be developed into a rapid detection device.

[0030] This invention also discloses a method for detecting the expression level of carboxylesterase 1 protein in plasma exosomes, comprising the following steps: Peripheral blood samples were collected from the subjects and plasma was separated; Exosomes were isolated from the plasma. The expression level or concentration of carboxylesterase 1 protein in the isolated exosomes was detected.

[0031] The present invention also discloses a kit for detecting SAE, the kit comprising an exosome separation component, a protein lysis buffer, and a detection reagent; the exosome separation component comprising a pre-treatment centrifuge tube for removing large particles, a filter, and / or an exosome extraction column or magnetic beads based on polymer precipitation or membrane affinity methods.

[0032] The present invention will be further described in detail through the following embodiments.

[0033] Example 1 This embodiment provides a detection method for the auxiliary diagnosis of sepsis-related encephalopathy, the procedure of which can be found in [reference needed]. Figure 1A and Figure 1C .in Figure 1A This is a schematic diagram of the workflow for plasma exosome isolation, identification, and proteomics analysis. Figure 1B Principal component analysis score plots show the separation trend of exosomal protein profiles between non-SAE patients and SAE patients. Figure 1C The bar chart shows the number of proteins upregulated and downregulated in SAE patients compared to non-SAE controls.

[0034] To systematically screen potential plasma exosomal protein biomarkers associated with SAE, we first used ultracentrifugation combined with microfluidic chip (UC / EXODUS) technology to isolate exosomes from plasma samples of 8 non-SAE sepsis patients and 8 SAE patients. Figure 1A After verifying the integrity of the isolated exosomes using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting (Figure 2), we performed data-independent acquisition (DIA) proteomics analysis. Principal component analysis showed significant clustering differences in the exosome proteomes between the two groups (principal component 1: 43.676%, principal component 2: 10.788%). Figure 1B A total of 60 differentially expressed proteins were identified. Compared with the non-SAE control group, 41 proteins were upregulated and 19 proteins were downregulated in SAE patients. Figure 1C Among the top 10 most differentially expressed proteins, members of the immunoglobulin V family (e.g., IG c919 heavy chain_IGHV3-15_IGHD3-10_IGHJ4, log2FC=1.61, P = 0.0004) accounted for the highest proportion (6 / 10), suggesting that abnormal humoral immune responses may be involved in the pathological process of SAE. More importantly, several differentially expressed proteins with high research potential were identified. Among them, proteins related to metabolism and signal transduction, including carboxylesterase 1 (CES1) (log2FC=1.26, P = 0.0028) and Ras-associated protein Ral-B (log2FC=1.33, P = 0.0128), were significantly upregulated in plasma exosomes of SAE patients (Figure 2 table). Given the key role of CES1 in lipid metabolism and immune regulation, we selected it as a core candidate biomarker for subsequent research.

[0035] Step S101: Sample Collection and Plasma Separation. Collect 10 mL of peripheral venous blood from a suspected or confirmed sepsis patient using an EDTA-anticoagulated vacuum blood collection tube. Within 2 hours of collection, centrifuge at 1500×g for 10 minutes at 4°C to remove blood cells. Carefully aspirate the supernatant plasma and transfer it to a new centrifuge tube. It can be used immediately for exosome separation or aliquoted and stored long-term in an ultra-low temperature freezer at -80°C.

[0036] Step S102: Isolation and purification of plasma exosomes. Isolating high-purity exosomes from plasma is a key step in this method, which can be achieved using ultracentrifugation, size exclusion chromatography, or commercially available exosome extraction kits. This embodiment preferably employs an automated method combining ultracentrifugation and a microfluidic chip, as detailed below: Thaw the frozen plasma at 4°C and centrifuge it at 4°C and 12,000×g for 30 minutes to thoroughly remove large particles such as cell debris, platelets, and apoptotic bodies.

[0037] The supernatant was filtered through a 0.22 μm polyethersulfone filter membrane to further remove impurities.

[0038] The filtered plasma supernatant was injected as follows: Figure 1C The EXODUS (Exosome Obtaining Device Using Sonication, a fully automated exosome extraction system) and other automated exosome separation systems, as shown, operate according to the device's preset program within their dedicated nanofluidic chip. Based on microfluidics and acoustic wave technologies, this system can efficiently and with high purity separate exosomes.

[0039] The purified exosomes were resuspended in 100-200 μL of 1× phosphate buffer and stored at -80°C or used directly for subsequent analysis.

[0040] The isolated exosomes can be obtained through Figure 2B The transmission electron microscope image shows its typical cup-shaped or round vesicle morphology. Figure 2C The Western blot results shown validated the expression of exosome marker proteins (CD9, CD63, TSG101) and the absence of negative markers (Calnexin) to ensure separation quality. Figure 2D The nanoparticle tracking analysis shown confirms that the main peak of its particle size is located in the 100-150 nm range.

[0041] in, Figure 2A The physicochemical properties of plasma exosome samples were analyzed. Quantitative parameters included exosome particle concentration, average diameter, number of particles per microgram of protein, and separation and recovery rate. Data showed that both independently separated samples exhibited typical nanoscale size, high purity, and good recovery efficiency.

[0042] Figure 2B These are representative transmission electron microscopy images of plasma exosomes. Both Sample 1 and Sample 2 show round or cup-shaped vesicle structures with intact and clear membrane morphology. Scale bar: 100 nm.

[0043] Figure 2C Western blotting analysis of exosome marker proteins. Both Sample 1 and Sample 2 expressed exosome-specific positive markers CD9, CD81, and TSG101, while the negative marker Calnexin protein was not detected.

[0044] Figure 2DThe size distribution of plasma exosomes is shown. Nanoparticle tracking analysis revealed that the main peaks of both Sample 1 and Sample 2 were located between 100 and 120 nanometers, and were uniformly distributed.

[0045] Step S103: Detection of CES1 protein in exosomes. This step aims to quantitatively detect the expression level of CES1 protein in the isolated exosomes. This example uses Western blotting: Protein extraction and quantification: RIPA lysis buffer containing protease inhibitors was added to the exosome suspension obtained in the previous step, and the mixture was lysed on ice for 30 minutes. Subsequently, the mixture was centrifuged at 4°C and 12,000×g for 15 minutes, and the supernatant was collected as total exosome protein. Protein concentration was determined using a BCA protein quantification kit.

[0046] Western Blot assay: Take 20-30 μg of total exosomal protein, separate by SDS-PAGE electrophoresis, and then transfer the protein to a PVDF membrane. Block with 5% skim milk at room temperature for 1 hour, then incubate overnight at 4°C with a primary antibody that specifically recognizes CES1 protein (e.g., rabbit anti-human CES1 monoclonal antibody, recommended dilution 1:1000). After washing with TBST, incubate with the corresponding horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour. Finally, develop and expose using ECL chemiluminescent substrate. To correct the sample loading volume, simultaneous detection should be performed on the same membrane using exosomal membrane proteins CD9 or CD81 as internal controls.

[0047] Step S104: Result analysis and interpretation.

[0048] Quantitative analysis: Image analysis software such as ImageJ was used to quantify the gray values ​​of the bands in the Western Blot results. The relative expression level of CES1 was obtained by dividing the gray value of the target band (CES1) by the gray value of the internal reference band (CD9).

[0049] Reference value establishment: In clinical applications, a reference range needs to be established in advance by measuring the plasma exosome CES1 levels of a certain number (e.g., no fewer than 30) of patients with sepsis who have been clearly evaluated as non-SAE (control group). This reference value can be the mean ± 2 standard deviations, or the optimal cutoff value determined by the receiver operating characteristic curve.

[0050] Clinical interpretation: such as Figure 3A , 3B As shown, when the relative expression level of plasma exosomal CES1 in a patient is significantly higher than the pre-defined reference or threshold value, it indicates an extremely high risk of SAE, providing important objective laboratory evidence for clinical evaluation. Its evaluation efficacy is as follows: Figure 4B As shown, it outperforms traditional scoring methods.

[0051] in, Figure 3A Western blot analysis of CES1 protein expression in plasma exosomes from non-SAE and SAE patients. CD9 was used as an exosome marker for sample loading standardization. Figure 3B Quantitative statistical analysis of CES1 protein expression levels in exosomal tissues. Figure 4A -E represents the correlation analysis between CES1 expression level and clinical prognosis. Figure 4A The proportions of patients with low and high plasma exosomal CES1 levels were defined in the SAE and non-SAE patient groups. The low and high CES1 expression groups were defined based on the median plasma exosomal CES1 level. Chi-square tests were used for comparison. Figure 4B Receiver operating characteristic (ROC) curve analysis was used to compare the efficacy of plasma exosome CES1, SOFA score, and APACHE II score in evaluating SAE. Figure 4C To compare the ICU length of stay between the low and high CES1 expression groups. Figure 4D To compare the duration of mechanical ventilation between the low and high expression groups of CES1. Figure 4E Kaplan-Meier curves for 28-day survival rates in the CES1 low-expression and high-expression groups are shown. Time-series tests were used for comparison.

[0052] To validate the proteomics screening results, this application examined the expression level of CES1 in plasma exosomes from an independent validation cohort. Western blot analysis showed that, compared with non-SAE patients, CES1 protein expression in plasma exosomes of SAE patients was significantly upregulated (…). Figure 3A The band intensity was quantitatively analyzed using ImageJ software, and normalized using the exosome marker CD9 as an internal reference. The results confirmed that the relative expression level of CES1 was significantly higher in the SAE group (P = 0.0291). Figure 3B This finding is consistent with previous proteomics results and further supports the reliability of plasma exosome CES1 as a potential biomarker for SAE.

[0053] Example 2 This embodiment provides a kit for detecting SAE.

[0054] This embodiment provides a specific implementation of a reagent kit based on the method of Embodiment 1 above, which includes the following core components: Exosome separation assembly: includes pretreatment centrifuge tubes for removing large particles, a 0.22 μm filter, and / or exosome extraction columns / magnetic beads based on polymer precipitation or membrane affinity methods.

[0055] Protein lysis buffer: Used to lyse exosomes and release total protein.

[0056] Test reagents: Primary antibody: Rabbit anti-human CES1 monoclonal antibody, used to specifically recognize and bind to CES1 protein in exosomes, at a dilution ratio of 1:1000.

[0057] Secondary antibody: HRP-conjugated anti-ribbit IgG, used to bind with the primary antibody and then be detected by chemiluminescence.

[0058] Protein lysis buffer: RIPA lysis buffer containing protease inhibitors, used to lyse exosomes to release total protein, lyse on ice for 30 minutes.

[0059] Protein quantification reagent: BCA protein quantification kit, used to determine the total protein concentration of exosomal tissues.

[0060] Electrophoresis-related reagents: precast gel, loading buffer, and running buffer.

[0061] Transfer material: PVDF membrane, pre-soaked in ethanol before transfer, and then equilibrated in deionized water and transfer buffer.

[0062] Blocking solution: Skim milk blocking solution, used to block non-specific binding sites.

[0063] Washing buffer: PBST buffer, used for washing the membrane.

[0064] Chemiluminescent substrate: ECL chemiluminescent solution, used for development and exposure.

[0065] Reference: A series of recombinant human CES1 protein solutions of known concentrations were used to prepare a standard curve.

[0066] Positive and negative controls: quality control products containing high concentrations of CES1 protein and those without CES1 protein, respectively.

[0067] Detailed instruction manual: This includes sample pretreatment methods, testing procedures, result calculation methods, and reference ranges and result interpretations based on clinical research data.

[0068] Application example: Clinical sample testing and validation Using the method of Example 1 and the kit of Example 2, plasma samples from an independent validation cohort consisting of 12 patients with SAE and 12 patients with non-SAE sepsis were tested.

[0069] The results are as follows Figure 3A , 3BAs shown, the plasma exosome CES1 level in the SAE group was significantly higher than that in the non-SAE group. ROC curves were plotted using the detection data, and the area under the curve was 0.722, demonstrating good accuracy in evaluation.

[0070] Example 3: Animal model validation of the correlation between CES1 and SAE pathology This embodiment provides important implementation reference for understanding the biological basis of the present invention, and its core findings are presented in Figure 5 The animal experiment results in this embodiment are only used to verify the correlation between CES1 and SAE pathology, and are not directly used for human evaluation.

[0071] Figure 5 The correlation analysis between plasma exosome CES1 levels and liver function indicators is shown in the figure, comparing plasma liver function indicators between the SAE group and the non-SAE group. Data are expressed as mean ± standard error.

[0072] Model establishment: SAE mouse models were established by intraperitoneal injection of lipopolysaccharide (LPS) or cecal ligation and perforation (CLP), and saline injection or sham operation groups were set up as controls.

[0073] Sample acquisition: 48 hours after modeling, mice were sacrificed and brain tissue was collected.

[0074] Pathological and molecular validation: Nissl staining (results can be seen in Figure 7) confirmed hippocampal neuronal damage in the SAE model group mice. Simultaneously, total protein was extracted from brain tissue, and CES1 expression was detected by Western blotting. Results are as follows... Figure 6A , 6B As shown, compared with the control group, the expression level of CES1 protein in the brain tissue of SAE model mice was significantly upregulated. This confirms the strong correlation between CES1 and the pathological process of SAE at the in vivo level, and supports its rationale as a biomarker in the peripheral blood of patients.

[0075] in, Figure 6A -B represents the analysis of CES1 protein expression levels in the brain tissue of the SAE mouse model. Figure 6A This study aimed to perform Western blot analysis on the expression of CES1 protein in the brain tissue of SAE model mice induced by intraperitoneal injection of different doses of LPS or CLP. Figure 6B Quantitative analysis of CES1 protein levels detected by Western blotting.

[0076] Figure 7A -F represents the analysis of morphological changes and survival rate of hippocampal neurons in the SAE mouse model. Figure 7A -D indicates Nissl staining to show the morphology of hippocampal and dentate gyrus neurons in SAE model mice induced by different doses of LPS. Figure 7EMorphology of hippocampal and dentate gyrus neurons in CLP-induced SAE model mice. Figure 7F Statistical analysis of the survival rate of hippocampal dentate gyrus neurons in each group. Data are expressed as mean ± standard error; P<0.05, P<0.01, P<0.001.

[0077] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Those skilled in the art can modify the technical solutions described in the above embodiments, or make equivalent substitutions for some of the technical features; and all such modifications and substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. A detection method for the auxiliary diagnosis of sepsis-related encephalopathy, wherein the method is not used for disease diagnosis, characterized in that, Including the following steps: Peripheral blood samples were collected from the subjects and plasma was separated; Exosomes were isolated from the plasma; The expression level or concentration of carboxylesterase 1 protein in the isolated exosomes was detected; The detected carboxylesterase 1 protein level was compared with a preset reference value to obtain the comparison results.

2. The detection method according to claim 1, characterized in that, In the step of separating exosomes from the plasma, the separation of exosomes is performed using ultracentrifugation, size exclusion chromatography, polymer precipitation, or microfluidic chip technology.

3. The detection method according to claim 1, characterized in that, The detection of the expression level or concentration of carboxylesterase 1 protein in the isolated exosomes can be performed using Western blotting, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or mass spectrometry.

4. The detection method according to claim 1, characterized in that, The step of separating exosomes from the plasma includes: Thaw the frozen plasma at 4°C and centrifuge at 12,000×g for 30 minutes at 4°C to thoroughly remove cell debris, platelets and apoptotic bodies.

5. The detection method according to claim 1, characterized in that, The specific steps for detecting the expression level or concentration of carboxylesterase 1 protein in the isolated exosomes are as follows: Exosome suspension was lysed with RIPA lysis buffer containing protease inhibitors for 30 minutes on ice. Subsequently, it was centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatant was collected as total exosome protein. Protein concentration was determined using a BCA protein quantification kit.

6. The detection method according to claim 4, characterized in that, The step of separating exosomes from the plasma includes: The supernatant was filtered through a 0.22 μm polyethersulfone filter membrane to further remove impurities.

7. The detection method according to claim 6, characterized in that, The purified exosomes were resuspended in 100-200 μL of 1× phosphate buffer and stored at -80°C or used directly for subsequent analysis.

8. The detection method according to claim 1, characterized in that, The process of collecting peripheral blood samples from subjects and separating plasma includes the following steps: Collect 5-10 mL of peripheral venous blood from the subject using an EDTA-anticoagulated vacuum blood collection tube. Within 2 hours after collection, centrifuge at 1500×g for 10 minutes at 4°C to remove blood cells, carefully aspirate the supernatant plasma, and transfer it to a new centrifuge tube.

9. A method for detecting the expression level of carboxylesterase 1 protein in plasma exosomes, characterized in that, Including the following steps: Peripheral blood samples were collected from the subjects and plasma was separated; Exosomes were isolated from the plasma. The expression level or concentration of carboxylesterase 1 protein in the isolated exosomes was detected.

10. A kit for detecting SAE, characterized in that, The kit includes an exosome separation component, a protein lysis buffer, and a detection reagent; the exosome separation component includes pretreated centrifuge tubes for removing large particles, a filter, and / or an exosome extraction column or magnetic beads based on polymer precipitation or membrane affinity methods.

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