Application of ZBTB7A as a target in the preparation of products for the diagnosis or treatment of sepsis-related encephalopathy

Abstract

This invention discloses the application of ZBTB7A as a target in the preparation of products for the diagnosis or treatment of sepsis-associated encephalopathy (SAE), belonging to the field of biomedical technology. This invention confirms that ZBTB7A can serve as a diagnostic biomarker and therapeutic target for SAE, providing strong technical support for the early diagnosis and treatment of SAE. Clinical sample validation shows that the expression level of ZBTB7A in peripheral blood astrocyte-derived exosomes of SAE patients is significantly downregulated, enabling early, non-invasive auxiliary diagnosis of SAE with high clinical diagnostic efficacy. This invention clarifies that high expression of ZBTB7A can effectively inhibit the release of pro-inflammatory factors, reduce lipid peroxidation and iron overload, improve the structural and functional integrity of mitochondria, protect neuronal and synaptic structures, and significantly reverse cognitive impairment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of ZBTB7A as a target in the preparation of products for the diagnosis or treatment of sepsis-related encephalopathy. Background Technology

[0002] Sepsis-associated encephalopathy (SAE), a diffuse dysfunction of the central nervous system caused by sepsis and septic shock, is the main manifestation of sepsis affecting the nervous system. Its clinical features are primarily delirium, seizures, cognitive impairment, and even coma. Epidemiological data shows that approximately 48.9 million new sepsis patients are diagnosed globally each year, and more than 11 million die from it, accounting for 19.7% of all deaths worldwide. Among sepsis patients, the proportion of those with SAE is as high as 50-70%. The mortality rate for sepsis complicated by SAE can reach 70%, making it one of the leading causes of death in intensive care units. Despite significant advancements in basic theories, anti-infective treatment strategies, fluid resuscitation, life support, and neurorehabilitation techniques, the incidence and mortality rates of SAE remain high, and effective prevention and treatment methods are still lacking. Even after SAE patients recover and are discharged from the hospital, they will still suffer from long-term sequelae such as varying degrees of memory loss and cognitive impairment, which will have adverse effects on their families, society, medical care, and the economy.

[0003] Neurons are the core cells of the brain responsible for cognitive functions, and their synaptic plasticity is the molecular basis for learning and memory formation. The dynamic homeostasis of neuronal mitochondria and the energy supply at synapses directly determine the signal transmission efficiency of neural circuits. The integrity of the mitochondrial cristae structure in the synaptic body is crucial for maintaining neurotransmitter release and postsynaptic potential conduction. Damage to the mitochondrial cristae can lead to a local energy crisis, inducing synaptic retraction and neuronal apoptosis, which is a core link in the development of cognitive impairment in sepsis-related encephalopathy. Energy metabolism disorders are pathological events that appear early in sepsis-related brain injury; therefore, maintaining the structure and function of neuronal mitochondria and optimizing brain energy metabolism may be important targets for improving post-sepsis cognitive function. Currently, the main treatment measures for sepsis-related encephalopathy and its resulting cognitive impairment include: control of the primary infection and anti-infective therapy, organ function support (such as mechanical ventilation and renal replacement therapy), fine-tuning of sedative and analgesic drugs, and anti-inflammatory therapy targeting excessive inflammatory responses. However, current interventions primarily focus on systemic support rather than specific neuroprotection, resulting in unsatisfactory long-term cognitive recovery from this complication and severely impacting patients' quality of life. Therefore, finding new treatments to reduce mitochondrial damage in neurons may be an important strategy for improving sepsis-related encephalopathy. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ZBTB7A as a target in the preparation of products for the diagnosis or treatment of sepsis-associated encephalopathy, thereby addressing the problems existing in the prior art. This invention has discovered that ZBTB7A can serve as a diagnostic biomarker and therapeutic target for sepsis-associated encephalopathy, providing strong technical support for the early diagnosis and treatment of this condition.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a diagnostic biomarker for sepsis-associated encephalopathy, wherein the diagnostic biomarker is ZBTB7A protein or ZBTB7A gene.

[0006] Furthermore, the lower the expression level of the diagnostic marker, the higher the risk of sepsis-associated encephalopathy.

[0007] The present invention also provides the application of reagents for detecting the expression levels of the above-mentioned diagnostic markers in the preparation of diagnostic products for sepsis-associated encephalopathy.

[0008] Furthermore, the diagnostic product is a reagent kit.

[0009] The present invention also provides a diagnostic product for sepsis-associated encephalopathy, comprising reagents for detecting the expression levels of the aforementioned diagnostic markers.

[0010] Furthermore, the diagnostic product is a reagent kit.

[0011] The present invention also provides the use of reagents for increasing the expression level of the diagnostic markers in the preparation of medicaments for the prevention and / or treatment of sepsis-associated encephalopathy.

[0012] Furthermore, the reagent is any one of (1)-(3): (1) Gene expression cassette containing the ZBTB7A gene; (2) A recombinant expression vector containing the gene expression cassette described in (1); (3) A host cell containing the recombinant expression vector described in (2).

[0013] Furthermore, the recombinant expression vector is a recombinant expression vector of adeno-associated virus.

[0014] The present invention also provides a medicament for the prevention and / or treatment of sepsis-associated encephalopathy, wherein the active ingredient comprises a reagent for increasing the expression level of the aforementioned diagnostic markers.

[0015] The present invention discloses the following technical effects: This invention confirms that ZBTB7A can serve as a diagnostic biomarker and therapeutic target for sepsis-associated encephalopathy (SAE), providing strong technical support for the early diagnosis and treatment of SAE. Clinical sample validation shows that the expression level of ZBTB7A in peripheral blood astrocyte-derived exosomes of SAE patients is significantly downregulated, enabling early, non-invasive auxiliary diagnosis of SAE with high clinical diagnostic efficacy. This invention clarifies the core regulatory role of ZBTB7A in inhibiting neuroinflammation, blocking neuronal ferroptosis, and protecting mitochondrial function. It can effectively inhibit the release of pro-inflammatory factors, reduce lipid peroxidation and iron overload, improve the structural and functional integrity of mitochondria, protect neuronal and synaptic structures, and significantly reverse cognitive dysfunction. This invention provides pharmaceutical applications for ZBTB7A-related drugs, which can be used to prepare drugs for the prevention or treatment of SAE, providing a novel and efficient molecular target and drug development direction for the clinical diagnosis and treatment of SAE, possessing significant clinical translational value and broad application prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a cell clustering diagram based on single-cell sequencing. Figure 2 This is an analysis diagram of marker genes corresponding to the cell clustering map; Figure 3 A volcano diagram of differentially expressed genes in astrocytes; Figure 4 The results of multicolor immunofluorescence detection of ZBTB7A expression localization are shown in the figure. Figure 5 Figure 1 shows the detection results of ZBTB7A expression level; where A is the detection results of ZBTB7A protein level in hippocampal tissue of mice with sepsis-associated encephalopathy; B is the detection results of ZBTB7A protein expression level in primary astrocytes; and C is the detection results of ZBTB7A transcription level in primary astrocytes. Figure 6Figure 1 shows the expression of ZBTB7A in patients with sepsis-associated encephalopathy and its clinical relevance analysis. Specifically, A represents the statistical graph of ZBTB7A expression levels in astrocyte-derived exosomes in the peripheral blood of patients; B represents the receiver operating characteristic (ROC) curve of ZBTB7A in sepsis-associated encephalopathy; C represents the statistical graph of HMGB1 secretion levels in serum; D represents the statistical graph of IL-1β secretion levels in serum; E represents the statistical graph of IL-6 secretion levels in serum; and F represents the statistical graph of TNF-α secretion levels in serum. Figure 7 A vector map of adeno-associated virus; Figure 8 A schematic diagram illustrating the construction of mice with ZBTB7A gene-specific overexpression by stereotactic injection of adeno-associated virus into the hippocampus and the identification of expression. Figure 9 The graph shows the survival rate and sepsis score of mice; where A is a schematic diagram of grouping; B is the sepsis score of mice; C is the 7-day survival rate of mice; and D is a statistical graph of the secretion level of pro-inflammatory cytokines; ZBTB7A-cOE represents the AAV-GFAP-ZBTB7A group; and CLP+ZBTB7A-cOE represents the CLP+AAV-GFAP-ZBTB7A group. Figure 10 The images show the results of ferroptosis detection in mouse hippocampal tissue. A represents the detection results of ZBTB7A and 4HNE protein expression levels; B represents the statistical graph of MDA (lipid peroxide) levels; C represents the statistical graph of iron content levels; E and D represent the multicolor immunofluorescence detection and quantitative analysis of 4HNE in neurons, respectively; F represents the transmission electron microscopy observation of mitochondrial changes in hippocampal neurons; ZBTB7A-cOE represents the AAV-GFAP-ZBTB7A group; CLP+ZBTB7A-cOE represents the CLP+AAV-GFAP-ZBTB7A group. Figure 11The images show the results of neuronal damage and synaptic level detection in the mouse hippocampus. A shows HE and Nissl staining observations of neuronal death; B shows the count of Nissl-stained positive neurons; C shows the count of HE-stained positive neurons; F shows Golgi staining observations of dendritic spine density changes on the main branch and branches; D and E show quantitative analysis of dendritic spine density changes on the main branch and branches; G shows transmission electron microscopy observations of the hippocampal synaptic level, where SV represents synaptic vesicles, SC represents the synaptic cleft, PSD represents the postsynaptic density, and AZ represents the presynaptic membrane active zone; HK represent the width of the synaptic cleft, the thickness of the PSD, the length of the active zone, and the number of SVs per unit area, respectively. Statistical chart of area; ZBTB7A-cOE represents AAV-GFAP-ZBTB7A group; CLP+ZBTB7A-cOE represents CLP+AAV-GFAP-ZBTB7A group; Figure 12 The results of the water maze experiment and their quantitative analysis are shown in the following graphs: A is the mouse movement trajectory graph in the water maze experiment; B is the escape latency curve graph; C is the quantitative statistical graph of the number of platform crossings; and D is the quantitative statistical graph of the time spent in the target quadrant. Figure 13 The results of the open field experiment and their quantitative analysis are shown in the following graphs: A is the mouse movement trajectory graph of the open field experiment; B is the quantitative statistical graph of the number of times crossing the central area; and C is the quantitative statistical graph of the duration in the central area. Figure 14 The results of the Y-maze experiment and its quantitative analysis are shown in the following graphs: A is the mouse movement trajectory graph of the Y-maze experiment; B is the quantitative statistical graph of the number of entries in the novel arm; and C is the quantitative statistical graph of the duration in the novel arm. Figure 15 This is a graph showing the detection results of ferroptosis levels in primary neurons; where A is the LPO fluorescence intensity peak graph; B is the quantitative statistical graph of LPO levels; and C is the Fe... 2+ Fluorescence intensity peak diagram; D represents Fe 2+Quantitative statistical graph of ROS levels; E is the ROS fluorescence intensity peak diagram; F is the quantitative statistical graph of ROS levels; Figure 16 This is a Western blot image showing the expression level of LCN2 protein in primary astrocytes. Figure 17 Western blot analysis of LCN2, 4HNE, and DRP1 protein expression levels in primary neurons; Figure 18 Immunofluorescence staining detection (A) and quantitative statistical graph (B) of 4HNE level in primary neurons; Figure 19 The image shows the morphological length of mitochondria in primary neurons (A) and the quantitative statistical graph (B). Figure 20 Immunofluorescence detection and co-localization fluorescence intensity analysis of LCN2 and DRP1 in primary neurons; Figure 21 A statistical graph showing the expression levels of the MTTL1 (A) and ND1 (B) genes in the mitochondria of primary neurons; Figure 22 Fluorescence intensity peaks (A) and quantitative statistical graph (B) of mitochondrial membrane potential (TMRE) detected by flow cytometry. Figure 23 The graph shows the results of mitochondrial function and cell viability in human neuronal cells. In the graph, A represents grouping information; B and D are statistical graphs of the secretion levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, respectively; E is a statistical graph of LCN2 secretion level; F and G are statistical graphs of the expression levels of mitochondrial MTTL1 (F) and ND1 (G); and H is a statistical graph of cell viability in human SH-SY5Y neuronal cells. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Terminology Explanation: The ZBTB7A involved in this invention refers to a zinc finger and BTB domain protein 7A, which is a transcription factor encoded by a gene located on chromosome 19p13.3. It belongs to the POK / ZBTB protein family. It specifically binds to DNA through its C-terminal zinc finger domain and recruits co-repressive complexes (such as NCoR and HDAC) through its N-terminal BTB / POZ domain to regulate the transcription of target genes.

[0024] This invention, for the first time, utilizes single-cell transcriptome sequencing technology to discover a specific downregulation of the transcription factor ZBTB7A expression in hippocampal astrocytes of sepsis-associated encephalopathy (SAE) mice, and that this expression change is significantly correlated with neuronal ferroptosis-related pathways. Clinical validation in clinical samples also revealed a significant decrease in ZBTB7A expression in peripheral blood astrocyte-derived exosomes from SAE patients. This finding establishes a link between ZBTB7A and the pathological mechanism of SAE for the first time, clarifying that ZBTB7A is a key upstream molecule regulating astrocyte-neuronal pathological crosstalk, providing a novel target for the diagnosis and treatment of SAE.

[0025] Example 1 This embodiment uses single-cell transcriptome sequencing and molecular biology techniques to analyze the expression changes of ZBTB7A in the hippocampus of wild-type mice and SAE model mice, especially the expression characteristics of ZBTB7A in astrocytes, providing a basis for ZBTB7A as a diagnostic biomarker and key regulatory target of SAE.

[0026] 1. Experimental Materials Experimental animals: Male C57BL / 6J mice aged 8 weeks and weighing between 20-25g were selected as experimental subjects.

[0027] Housing environment: All experimental mice were housed in the Specific Pathogen Free (SPF) Laboratory Animal Center of Wuhan University. Housing conditions: room temperature between 22±2℃, humidity between 50±10%, alternating light and dark lighting for 12 hours, and free access to water and food.

[0028] 2. Experimental Methods 2.1 Grouping of experimental animals and construction of SAE model Eight-week-old male C57BL / 6J wild-type mice were randomly divided into two groups (n=5 per group): SHAM group (sham surgery group): only laparotomy was performed, without cecal ligation and perforation; CLP group (SAE model group): the SAE model was constructed by cecal ligation and perforation (CLP).

[0029] SAE Model Construction Method: Eight-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (45-50 mg / kg). A longitudinal incision of approximately 1.0-1.5 cm was made along the midline of the abdomen to separate and expose the cecum. The cecum was ligated at 50%-75% of its distal end with 4-0 sterile silk sutures. A 21G sterile injection needle was used to puncture the ligated segment of the cecum once, gently squeezing out a small amount of intestinal contents into the abdominal cavity to confirm patency of the perforation. The cecum was then returned to the abdominal cavity, and the abdominal wall was sutured layer by layer. The sham-operated group underwent the same procedures as the mice, except that cecal ligation and perforation were omitted.

[0030] Twenty-four hours after modeling, the mice were decapitated and their brains were removed under deep anesthesia, and the bilateral hippocampal tissues were separated on ice.

[0031] 2.2 Preparation of single-cell suspension Hippocampal tissue was rapidly transferred to centrifuge tubes containing enzymatic digestion solution (1 mg / mL papain, 100 U / mL DNase I, and 0.5 mg / mL collagenase IV in Hanks' balanced salt solution). Mechanical dissociation and enzymatic digestion were performed using a tissue dissociator in a 37°C water bath for 15-20 minutes. After digestion, the cell suspension was sequentially passed through 70 μm and 40 μm cell sieves to remove undigested tissue fragments. Percoll density gradient centrifugation was used to remove debris and dead cells, obtaining a high-viability single-cell suspension (viability >85%), with the cell concentration adjusted to 700-1200 cells / μL.

[0032] 2.3 Single-cell library construction and sequencing Single-cell libraries were constructed using the 10× Genomics Chromium Next GEM Single Cell 3' Reagent Kits (v3.1). Single-cell suspensions were co-encapsulated with gel beads in oil droplets (GEMs) to complete cell lysis, mRNA capture, and reverse transcription, constructing strip-shaped cDNA libraries. After purification, amplification, and fragment selection, the libraries were sequenced using the Illumina NovaSeq 6000 sequencing platform with paired ends, aiming for a sequencing depth of 50,000-100,000 reads per cell.

[0033] 2.4 Single-cell sequencing data analysis The sequencing data was deconvolved, barcoded, and counted using Cell Ranger software (version 6.0.0), and aligned to a mouse reference genome (mm10) for gene quantification. Subsequent bioinformatics analysis was performed in the R environment using the Seurat package (version 4.3.0). (1) Cell quality control filtration: remove cells with an excessively high proportion of mitochondrial genes (>20%) and an excessively low number of genes; (2) Data standardization and screening of highly variable genes; (3) Principal component analysis and cell clustering: a graph-based clustering method; (4) Nonlinear dimensionality reduction visualization: using t-SNE or UMAP algorithms; (5) Cell type annotation: Annotated with known cell markers, including astrocytes (Gfap, Aqp4), neurons (Map2, Syp), microglia (Tmem119, Cx3cr1), and oligodendrocytes (Olig2, Mbp). (6) Differential gene expression analysis: Using the FindMarkers function, we compared the specific transcriptional changes of the CLP group and the SHAM group in different cell types.

[0034] 2.5 Validation of Single-Cell Sequencing Results To verify the single-cell sequencing results, hippocampal tissues from mice in the SHAM and CLP groups (n=8 per group) were collected and subjected to the following tests: (1) Immunofluorescence co-staining Paraffin sections were dewaxed, hydrated, and retrievald with sodium citrate antigen. They were then blocked with 5% BSA and incubated overnight at 4°C with GFAP antibody (1:500, an astrocyte marker) and ZBTB7A antibody (1:200). The next day, secondary antibodies labeled with Alexa Fluor488 and 594 were added, and the nuclei were counterstained with DAPI. The colocalization and fluorescence intensity of GFAP and ZBTB7A were observed under a laser confocal microscope, and the fluorescence intensity of ZBTB7A in astrocytes was calculated.

[0035] (2) Western blot detection Hippocampal tissue was harvested, and total protein was extracted using RIPA lysis buffer and quantified using the BCA method. 30 μg of protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk, and then incubated overnight at 4°C with ZBTB7A antibody (1:1000) and β-actin antibody (1:5000). The next day, HRP-labeled secondary antibody was added, and the membrane was developed using ECL. The band gray values ​​were analyzed using ImageJ software.

[0036] (3) Real-time quantitative PCR (RT-qPCR) detection Hippocampal tissue was harvested, total RNA was extracted using the TRIzol method, and cDNA was synthesized by reverse transcription. ZBTB7A mRNA expression levels were detected by RT-qPCR using the SYBR Green assay, with β-actin as an internal control. ^-ΔΔCt The relative expression level is calculated using this method.

[0037] 3. Experimental Results like Figures 1-5 As shown, ZBTB7A expression was specifically downregulated in hippocampal astrocytes of SAE mice and was significantly associated with neuronal ferroptosis-related processes, suggesting that ZBTB7A is a key regulator and potential intervention target for astrocyte functional changes in SAE.

[0038] Example 2 1. Clinical Samples Peripheral venous blood samples were collected from 80 healthy volunteers and 80 patients with sepsis-associated encephalopathy meeting the diagnostic criteria for Sepsis-3.0. The peripheral venous blood samples were placed in EDTA anticoagulant tubes, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. GFAP-positive astrocyte-derived exosomes were further sorted using immunomagnetic beads. The origin of the exosomes was confirmed by Western blot detection of exosome markers CD63 and CD81, and the astrocyte-specific marker GFAP.

[0039] 2. Detection of pro-inflammatory cytokines Peripheral blood serum was extracted, and the secretion levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and HMGB1) in the serum were detected using an ELISA kit. The results are shown in the figure. Figure 6 CF (Chinese CF)

[0040] 3. Detection of ZBTB7A expression and its correlation with sepsis-associated encephalopathy Total RNA was extracted from exosomal tissues, and the expression level of ZBTB7A mRNA was detected by real-time quantitative PCR (RT-qPCR), with β-actin as an internal control. The association between ZBTB7A and sepsis-associated encephalopathy was analyzed.

[0041] like Figure 6 As shown in Figure A, compared with healthy volunteers, the expression level of ZBTB7A mRNA in peripheral blood astrocyte-derived exosomes of SAE patients was significantly reduced. P <0.001). Pearson correlation analysis suggested that downregulation of ZBTB7A expression was closely related to the pathogenesis of SAE (Table 1).

[0042] Table 1. Correlation between ZBTB7A mRNA levels and SAE diagnosis 4. Diagnostic efficacy verification Another 80 samples (40 from healthy volunteers and 40 from patients with sepsis-associated encephalopathy meeting the diagnostic criteria for Sepsis-3.0) were collected to detect ZBTB7A mRNA expression levels, and receiver operating characteristic (ROC) curves were plotted. The results are shown below. Figure 6 The results showed that ZBTB7A has high diagnostic value for sepsis-related encephalopathy, with an AUC value of 0.881.

[0043] Example 3 1. Experimental Materials Experimental animals: Male C57BL / 6J mice aged 8 weeks and weighing between 20-25g were selected as experimental subjects.

[0044] Housing environment: All experimental mice were housed in the Specific Pathogen Free (SPF) level experimental animal center of Wuhan University Experimental Animal Center. Housing conditions: room temperature between 22±2℃, humidity between 50±10%, light and dark alternation lighting time of 12 hours, and free access to water and food.

[0045] Adeno-associated virus: (1) Construction of adeno-associated virus vector The full-length CDS sequence (SEQ ID NO.1) of mouse ZBTB7A was codon-optimized and cloned into the pAAV-GFAP-EGFP vector (purchased from Hanheng Biotechnology Co., Ltd.), replacing the EGFP gene, to obtain the pAAV-GFAP-ZBTB7A recombinant plasmid. Figure 7 The vector elements include: a 5' ITR, a glial fibrillary acidic protein (GFAP) promoter (approximately 0.68 kb in length), a ZBTB7A coding sequence, an SV40 polyA signal, and a 3' ITR. The GFAP promoter is used to drive the specific expression of ZBTB7A in astrocytes. Simultaneously, an AAV9-GFAP-EGFP control virus was constructed (expressing only EGFP fluorescent protein, without expressing ZBTB7A).

[0046] (2) Virus packaging and purification AAV virus packaging was performed using a three-plasmid co-transfection method in HEK293T cells: the recombinant plasmid pAAV-GFAP-ZBTB7A, the pAAV-RC vector (providing AAV9 Rep / Cap protein) (purchased from Miaoling Plasmid Platform), and the pHelper vector (providing adenovirus helper function) (purchased from Miaoling Plasmid Platform) were co-transfected into logarithmically growing HEK293T cells at a 1:1:1 molar ratio. Cells and supernatant were collected 72 hours after transfection, purified by freeze-thaw cycles, nuclease treatment, and iodixanol density gradient centrifugation, and finally concentrated by dialysis with PBS to obtain high-purity AAV9-GFAP-ZBTB7A virus. The viral titer was detected by real-time quantitative PCR and adjusted to 2 × 10⁻⁶. 12 vg / mL, aliquoted and stored at -80℃ for later use.

[0047] 2. Experimental Methods 2.1 Experimental Grouping The experimental groups are as follows (8 animals per group): SHAM group: sham surgery group, without SAE modeling; CLP group: SAE model group, no intervention was performed after SAE modeling; AAV-GFAP-ZBTB7A group: only injected with the virus for treatment, without SAE modeling; CLP+AAV-GFAP-ZBTB7A group: experimental group, AAV9-GFAP-ZBTB7A virus was injected into the hippocampus 4 weeks before SAE modeling.

[0048] Method for injecting AAV9-GFAP-ZBTB7A virus into the hippocampus: Eight-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg) and fixed on a stereotaxic instrument (Reward, China). Using the anterior fontanelle as the origin, the injection coordinates for the CA1 region of the hippocampus were determined as follows: 2.0 mm posterior to the anterior fontanelle, ±1.8 mm lateral to the midline, and 1.8 mm subdurally. 500 nL of virus solution was slowly injected at a constant rate of 100 nL / min using a microsyringe. The needle was left in place for 10 minutes after injection, and then slowly withdrawn. The same procedure was repeated on the contralateral hippocampus. The virus expression period was 4 weeks. After the virus expression period, hippocampal tissue was collected from the mice, and immunofluorescence staining (GFAP and ZBTB7A co-localization) was used to detect the infection efficiency. Figure 8 ).

[0049] The SAE modeling method was the same as in Example 1. Twenty-four hours after modeling, the mice's mental state, activity level, and sepsis clinical score were observed. Hippocampal tissue was harvested, and the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α were detected using ELISA to verify the successful model construction.

[0050] 2.2 Behavioral experiments to detect cognitive function in mice The Morris water maze, Y maze, and open field experiments were used to systematically evaluate the effect of ZBTB7A overexpression on cognitive function in SAE mice.

[0051] 2.2.1 Morris Water Maze Experiment The water maze is a circular pool with a diameter of 120 cm, a height of 60 cm, a water depth of 40 cm, and a water temperature of (23±1)℃. The pool is divided into four quadrants, and a hidden platform (10 cm in diameter) is placed 1.5 cm below the water surface in the target quadrant.

[0052] Orientation and navigation experiment: For 5 consecutive days, the mice were trained 4 times a day. Each time, they were placed in the water facing the pool wall from different entry points. The time it took to find the platform within 90 seconds was recorded (escape latency). If the mice did not find the platform within 90 seconds, they were guided to the platform by the experimenters and stayed there for 15 seconds. The latency was recorded as 90 seconds.

[0053] Space exploration experiment: On day 6, the platform was removed, and the mice were put into the water from the quadrant opposite to the original platform. The number of times the mice crossed the original platform position and the time spent in the target quadrant were recorded within 60 seconds.

[0054] 2.2.2 Y-maze experiment The Y-maze consists of three arms, each 40 cm long, 10 cm wide, and 20 cm high, with each pair of arms forming a 120° angle. Mice are placed in the starting arm and allowed to explore freely for 8 minutes. The total number of arm entries and the number of spontaneous alternations (entering three different arms consecutively) are recorded. Spontaneous alternation rate (%) = (actual number of alternations / maximum possible number of alternations) × 100%.

[0055] 2.2.3 Open Field Experiment The open area was a black, opaque box measuring 50 cm × 50 cm × 40 cm, with its bottom divided into 16 squares of equal area, the four central squares being the central area. Mice were gently placed in the central area, and the total distance traveled, the time spent in the central area, and the number of times mice entered the central area were recorded over 10 minutes.

[0056] 2.2.4 Survival Rate Analysis Record the mortality of mice in each group within 7 days after CLP modeling and calculate the 7-day survival rate.

[0057] 2.3 Pathological and molecular biological detection of sepsis-associated encephalopathy model mice 2.3.1 Tissue Sample Collection After the behavioral tests, the mice were euthanized under deep anesthesia, and hippocampal tissue was isolated. Part of the hippocampal tissue was used for Western blot, ELISA, and biochemical assays; another part was fixed in 4% paraformaldehyde and used for HE staining, Nissl staining, and immunofluorescence staining; and yet another part was fixed in glutaraldehyde and used for transmission electron microscopy.

[0058] 2.3.2 Detection of neuronal ferroptosis markers (1) Observation of mitochondrial morphology by transmission electron microscopy Take 1 mm 3 Hippocampal tissue blocks were fixed with 2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, dehydrated with graded ethanol, and embedded in epoxy resin to prepare 70 nm ultrathin sections. The sections were stained with uranium acetate and lead citrate, and the morphology of hippocampal neuronal mitochondria was observed under a Hitachi HT-7800 transmission electron microscope.

[0059] (2) MDA content detection Hippocampal tissue homogenate was collected and processed according to the Beyotime MDA assay kit instructions. The detection wavelength was 532 nm, and the reference wavelength was 600 nm. The MDA content was calculated using a standard curve and expressed as nmol / mg protein.

[0060] (3) Detection of iron content in tissue Hippocampal tissue homogenate was collected and processed according to the instructions of the Nanjing Jiancheng Tissue Iron Detection Kit. The detection wavelength was 520 nm, and the reference wavelength was 620 nm. Iron content was calculated using a standard curve and expressed as μmol / g protein.

[0061] (4) Immunofluorescence co-staining Paraffin sections were dewaxed, hydrated, and retrievald with sodium citrate antigen. They were then blocked with 5% BSA, and NeuN antibody (1:500) and 4HNE antibody (1:200) were added. The sections were incubated overnight at 4°C. The next day, secondary antibodies labeled with Alexa Fluor 488 and 594 were added, and the nuclei were counterstained with DAPI. The sections were observed and the colocalization coefficients were calculated under a laser confocal microscope.

[0062] 2.3.3 Validation of ZBTB7A expression in astrocytes (1) Immunofluorescence co-staining After dewaxing, hydration, and antigen retrieval, paraffin sections were incubated overnight at 4°C with GFAP antibody (1:500) and ZBTB7A antibody (1:200) and added. The next day, fluorescent secondary antibody was added and DAPI was used for counterstaining. The colocalization and fluorescence intensity of GFAP and ZBTB7A were observed under a laser confocal microscope.

[0063] (2) Western blot detection Hippocampal tissue was harvested, and total protein was extracted using RIPA lysis buffer and quantified using the BCA method. 30 μg of protein was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk, and then incubated overnight at 4°C with ZBTB7A antibody (1:1000) and β-actin antibody (1:5000). The next day, HRP-labeled secondary antibody was added, and the membrane was developed using ECL. The band gray values ​​were analyzed using ImageJ software.

[0064] 2.3.4 Evaluation of neuronal survival and synaptic damage (1) HE staining After dewaxing and hydration, paraffin sections were stained with hematoxylin for 8-10 minutes, differentiated with 1% hydrochloric acid ethanol for a few seconds, blued with tap water for 15 minutes, stained with eosin for 2 minutes, dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and the morphology of neurons in the CA3 region of the hippocampus was observed under an optical microscope.

[0065] (2) Nissl staining After dewaxing and hydration, paraffin sections were stained with 0.1% tar violet solution at 37°C for 20 minutes, differentiated with Nissl differentiation solution for 30-90 seconds, dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and the number of Nissl bodies in neurons of the CA3 region of the hippocampus was observed under an optical microscope.

[0066] (3) Golgi staining Whole brain tissue was collected, soaked in Golgi staining solution for 14 days, and then vibrated sections were prepared at a thickness of 120 μm. After the color reaction, the sections were dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and the density of dendritic spines of hippocampal neurons was observed under an optical microscope.

[0067] 3. Experimental Results 3.1 Model Validation Results Model validation results are shown below Figure 9 The results showed that the CLP group mice exhibited typical sepsis symptoms such as lethargy, piloerection, arched back, and reduced activity, and their clinical sepsis scores were significantly higher than those of the SHAM group (P<0.001). The levels of IL-1β, IL-6, and TNF-α in the hippocampus were significantly elevated (P<0.001), indicating that the SAE mouse model was successfully established.

[0068] 3.2 Pathological and molecular biological results of the sepsis-associated encephalopathy model mouse like Figures 10-11 As shown, compared with the CLP group, the CLP+AAV-ZBTB7A group (experimental group) mice showed significantly improved ultrastructural changes of typical ferroptosis in hippocampal neurons, such as mitochondrial shrinkage, increased membrane density, and reduced cristae; the MDA and iron contents in hippocampal tissues were significantly reduced; the intensity of 4HNE fluorescence signal in neurons was significantly weakened, and the colocalization coefficient with NeuN was reduced; HE staining showed that neurons in the CA3 region of the hippocampus were neatly arranged, and nuclear condensation was significantly reduced; Nissl staining showed that the number and staining depth of Nissl bodies were significantly restored; Golgi staining showed that the density of dendritic spines was significantly increased.

[0069] The above results indicate that ZBTB7A overexpression can effectively inhibit ferroptosis in hippocampal neurons of SAE mice and protect neuronal structure and synaptic integrity.

[0070] 3.3 Results of the behavioral experiment The results of the behavioral experiments are shown in Figures 12-14 The results showed that, compared with the CLP group, the CLP+AAV-GFAP-ZBTB7A group mice had significantly shorter escape latency in the Morris water maze, significantly increased time spent in the target quadrant and number of platform crossings; significantly increased spontaneous alternation rate in the Y maze; significantly increased time spent in the central area of ​​the open field, while the total movement distance was not significantly different, excluding the interference of movement ability; and significantly improved 7-day survival rate.

[0071] The above results indicate that ZBTB7A overexpression can significantly improve cognitive dysfunction and increase survival rate in SAE mice.

[0072] Example 4 This embodiment uses an in vitro mouse primary astrocyte-neuron co-culture model to verify the protective effect of astrocyte ZBTB7A overexpression on neuronal ferroptosis and mitochondrial function.

[0073] 1. Experimental Methods 1.1 Extraction and culture of primary astrocytes Hippocampal tissue from C57BL / 6J neonatal rats within 24 hours of birth was collected, minced, and digested with 0.25% trypsin for 15-20 minutes. After digestion was terminated, a single-cell suspension was prepared by pipetting and filtering through a 70 μm cell sieve and seeded into poly-L-lysine-coated culture flasks. After 7-10 days of culture, when the cell confluence reached 80%-90%, the cells were placed in a 37°C constant-temperature shaker at 200-250 rpm for 18-24 hours to remove microglia, obtaining high-purity astrocytes (GFAP immunofluorescence purity >95%).

[0074] 1.2 Extraction and Culture of Primary Neurons Hippocampal tissue from C57BL / 6J fetal rats at 16-18 days of gestation was collected, minced, and digested with 0.25% trypsin for 15-20 minutes. After digestion was stopped, the tissue was pipetted to prepare a single-cell suspension, filtered through a 40 μm cell sieve, and seeded into poly-L-lysine-coated culture plates. The cells were cultured in Neurobasal medium (containing 2% B27 and 0.5 mM GlutaMAX), with half the medium changed every 3 days. The cells were cultured until they matured in 7-9 days and then used for experiments.

[0075] 1.3 Astrocyte processing and conditioned medium collection Primary astrocytes were divided into the following 4 groups: Control group: Normal culture; LPS group: LPS (1 μg / mL) stimulation for 24 hours; OE-ZBTB7A group: 48 hours after transfection with AAV9-GFAP-ZBTB7A virus (MOI=100), the group was stimulated with an equal amount of blank solvent for 24 hours. LPS+OE-ZBTB7A group: 48 hours after transfection with AAV9-GFAP-EGFP control virus (MOI=100), LPS (1 μg / mL) was administered for 24 hours.

[0076] Collect conditioned medium (ACM) from each group of astrocytes, centrifuge to remove cell debris, and store at -80℃ for later use.

[0077] 1.4 Neuron processing and ferroptosis marker detection After treating primary neurons with the above ACM for 24 hours, the following tests were performed: (1) Flow cytometry detection of Fe 2+ ROS and LPO levels: Fe 2+Detection: Neurons were incubated with FerroOrange probe (1 μM) at 37°C in the dark for 30 minutes, washed with PBS, and the fluorescence intensity was detected by the PE channel of flow cytometry; ROS detection: Neurons were incubated with DCFH-DA probe (10 μM) at 37°C in the dark for 30 minutes, washed with PBS, and the fluorescence intensity was detected by the FITC channel of flow cytometry; LPO detection: Neurons were incubated with Liperfluo probe (1 μM) at 37°C in the dark for 30 minutes, washed with PBS, and the fluorescence intensity was detected by the FITC channel of flow cytometry.

[0078] (2) Western blot detection of 4HNE protein expression Total protein was extracted from neurons using RIPA lysis buffer, quantified by BCA method, and the expression level of 4HNE protein was detected by Western blot, with β-actin as an internal control.

[0079] (3) Immunofluorescence detection of co-localization of MAP2 and 4HNE Neurons were fixed with 4% paraformaldehyde, then MAP2 antibody (1:500) and 4HNE antibody (1:200) were added and incubated overnight at 4°C. The next day, fluorescent secondary antibody was added, DAPI was counterstained, and colocalization coefficients were observed and calculated using laser confocal microscopy.

[0080] 1.5 Mitochondrial Function Detection (1) Detection of mitochondrial DNA (mtDNA) Genomic DNA was extracted from neurons, and mtDNA copy number was detected using real-time quantitative PCR, with nDNA as an internal reference and relative mtDNA copy number = 2. -ΔCt , ΔCt = Ct(mtDNA) - Ct(nDNA).

[0081] (2) Detection of mitochondrial membrane potential (TMRE) Neurons were incubated with a TMRE probe (200 nM) at 37°C in the dark for 30 minutes, washed with PBS, and the fluorescence intensity was detected by the PE channel of a flow cytometer.

[0082] (3) Immunofluorescence detection of TOM20 mitochondrial morphology Neurons were fixed with 4% paraformaldehyde, then MAP2 antibody (1:500) and TOM20 antibody (1:200) were added and incubated overnight at 4°C. The next day, fluorescent secondary antibody was added, DAPI was used for counterstaining, and the mitochondrial network morphology was observed by laser confocal microscopy.

[0083] (4) Immunofluorescence detection of co-localization of LCN2 and DRP1 Neurons were fixed with 4% paraformaldehyde, then LCN2 antibody (1:200) and DRP1 antibody (1:200) were added and incubated overnight at 4°C. The next day, fluorescent secondary antibody was added, DAPI was counterstained, and colocalization coefficients were observed and calculated using laser confocal microscopy.

[0084] 2. Experimental Results like Figures 15-22 As shown, compared with the LPS group, the LPS+OE-ZBTB7A group (overexpressing ZBTB7A) showed the following after ACM treatment of neurons: intraneuronal Fe 2+ The levels of ROS and LPO were significantly reduced; the expression level of 4HNE protein was significantly reduced, and the co-localization signal of MAP2 and 4HNE was weakened; the mitochondrial DNA copy number was significantly increased, and the fluorescence intensity of mitochondrial membrane potential (TMRE) was significantly increased; TOM20 immunofluorescence showed that the mitochondrial network morphology tended to be more complete and fragmentation was reduced; the co-localization coefficient of LCN2 and DRP1 was significantly reduced.

[0085] The above results indicate that overexpression of ZBTB7A in astrocytes can reduce neuronal ferroptosis and mitochondrial dysfunction by inhibiting LCN2 expression and secretion.

[0086] Example 5 This embodiment uses an in vitro human astrocyte (SVG p12 cell line)-neuron (SH-SY5Y cell line) co-culture model to verify the protective effect of human astrocyte ZBTB7A overexpression on neuronal death and mitochondrial function.

[0087] 1. Experimental Materials Co-cultured cells: The supernatant of SVG p12 cell culture was collected and used to culture SH-SY5Y cells to construct an in vitro human astrocyte-neuron cell model.

[0088] Adeno-associated virus: An adeno-associated virus overexpressing the human ZBTB7A gene (SEQ ID NO.2) was constructed. The construction method was the same as in Example 3, except that the nucleotide sequence of the ZBTB7A gene was different.

[0089] 2. Experimental Methods 2.1 Experimental Grouping The co-cultured human astrocyte-neuron cells were divided into the following 4 groups: Control group: Normal culture; LPS group: LPS (1 μg / mL) stimulation for 24 hours; OE-ZBTB7A group: 48 hours after transfection with AAV9-GFAP-ZBTB7A virus (MOI=100), the group was stimulated with an equal amount of blank solvent for 24 hours. LPS+OE-ZBTB7A group: 48 hours after transfection with AAV9-GFAP-EGFP control virus (MOI=100), LPS (1 μg / mL) was administered for 24 hours.

[0090] 2.2 Mitochondrial DNA (mtDNA) Detection Collect cell culture supernatant and centrifuge at 3000 rpm for 10 minutes at 4°C to remove cell debris and suspended impurities. After centrifugation, aliquot the supernatant into sterile EP tubes and store them immediately at -80°C for later use, avoiding repeated freeze-thaw cycles. Use an ELISA kit to detect the concentrations of IL-1β, IL-6, TNF-α, and LCN2 in the samples. The final results are expressed in pg / mL. Three replicates are set for each sample to ensure the accuracy and reproducibility of the results.

[0091] 2.3 Mitochondrial DNA (mtDNA) Detection Genomic DNA was extracted from neurons, and mtDNA copy number was detected using real-time quantitative PCR, with nDNA as an internal reference and relative mtDNA copy number = 2. -ΔCt , ΔCt = Ct(mtDNA) - Ct(nDNA).

[0092] 2.4 Cell viability detection Cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, which was then used for cell counting and the density was adjusted to 5 × 10⁶ cells / cells. 4 Cells / mL, at 100 μL per well (i.e., 5 × 10⁶ cells / mL, i.e., 5 × 10⁶ cells / well). 3 (Number of cells) were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours until the cells were fully adherent. According to the experimental groups, the old culture medium was discarded, and 100 μL of fresh culture medium containing the corresponding treatment factor (e.g., LPS, ZBTB7A overexpression virus) was added to each well. Incubation continued for 48 hours until the treatment endpoint. Upon reaching the detection time point, 10 μL of CCK-8 reagent was added to each well (avoiding air bubbles). The plate was gently shaken to thoroughly mix the reagent and culture medium, and the plate was incubated in the dark for 1-4 hours (the specific time needs to be determined based on cell type and cell density in preliminary experiments; generally, the OD value of the untreated control group should reach 1.0-1.5). After incubation, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader. 450Meanwhile, 630 nm was set as the reference wavelength for background correction; each treatment group had 6 replicates, and the experiment was independently repeated 3 times. Cell viability was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%, where the blank group consisted of wells with only culture medium and no cells, and the control group consisted of normal cultured cells without any treatment.

[0093] 3. Experimental Results like Figure 23 As shown, compared with the LPS group, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the ACM of the LPS+OE-ZBTB7A group (overexpressing ZBTB7A) decreased, and the secretion level of LCN2 also decreased. However, after treating neurons with adeno-associated virus overexpressing ZBTB7A, the copy number of mitochondrial DNA in neurons increased significantly, and the survival rate of neurons increased significantly.

[0094] The above results indicate that overexpression of ZBTB7A in human astrocytes can reduce neuronal death and mitochondrial dysfunction by inhibiting LCN2 secretion, thereby achieving the therapeutic effect of improving cognitive impairment and increasing survival rate in SAE.

[0095]

[0096]

[0097] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A diagnostic biomarker for sepsis-associated encephalopathy, characterized in that, The diagnostic marker is the ZBTB7A protein or the ZBTB7A gene.

2. The diagnostic biomarker according to claim 1, characterized in that, The lower the expression level of the diagnostic marker, the higher the risk of sepsis-associated encephalopathy.

3. The use of a reagent for detecting the expression level of the diagnostic marker of claim 1 in the preparation of diagnostic products for sepsis-associated encephalopathy.

4. The application according to claim 3, characterized in that, The diagnostic product is a reagent kit.

5. A diagnostic product for sepsis-associated encephalopathy, characterized in that, Includes reagents for detecting the expression level of the diagnostic biomarker of claim 1.

6. The diagnostic product according to claim 5, characterized in that, The diagnostic product is a reagent kit.

7. The use of the reagent for increasing the expression level of the diagnostic marker of claim 1 in the preparation of a medicament for the prevention and / or treatment of sepsis-associated encephalopathy.

8. The application according to claim 7, characterized in that, The reagent is any one of (1)-(3): (1) Gene expression cassette containing the ZBTB7A gene; (2) A recombinant expression vector containing the gene expression cassette described in (1); (3) A host cell containing the recombinant expression vector described in (2).

9. The application according to claim 8, characterized in that, The recombinant expression vector is a recombinant expression vector of adeno-associated virus.

10. A drug for the prevention and / or treatment of sepsis-associated encephalopathy, characterized in that, The active ingredient includes a reagent for increasing the expression level of the diagnostic biomarker as described in claim 1.

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