A method for studying the function of fyn-mediated microglial inflammation in subarachnoid hemorrhage-induced early brain injury

Abstract

The application relates to a method for studying the function of Fyn-mediated microglial cell inflammation in subarachnoid hemorrhage-induced early brain injury, and relates to the technical field of medical research. The method comprises the following steps: constructing a subarachnoid hemorrhage in-vivo model and an in-vitro model, detecting Fyn expression and cell localization, performing Fyn function intervention experiments, detecting inflammation-related indexes and cell apoptosis, evaluating brain injury and function, and exploring a molecular mechanism. The application can systematically analyze the regulation function and mechanism of Fyn kinase on microglial cell-mediated neuroinflammation in subarachnoid hemorrhage-induced early brain injury.

Description

Technical Field

[0001] This application relates to the field of medical research technology, specifically to a functional study method for Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. Background Technology

[0002] Subarachnoid hemorrhage (SAH), a critical and acute cerebrovascular disease, is characterized by high mortality and complex complications. Early brain injury plays a crucial role in the clinical outcome of SAH patients, and inflammatory response, as an important trigger for early brain injury, plays a central role in the pathological process following SAH. Under pathological conditions, abnormal activation of glial cells can trigger a cascade release of immunomodulatory factors. This pathological process can not only lead to functional disorders of neuronal networks in specific brain regions but also exacerbate the formation and development of cerebral edema by disrupting the integrity of neurovascular units. Fyn kinase, as an important member of the Src family of tyrosine kinases, plays a key regulatory role in the development and pathological processes of the nervous system. In existing technologies, researchers construct animal models of subarachnoid hemorrhage, treat primary microglia with oxyhemoglobin to simulate the in vitro subarachnoid hemorrhage model, use Western blotting to detect the expression of relevant proteins, and employ immunohistochemistry and immunofluorescence staining to detect the expression and localization of proteins in brain tissue. They also assess the brain function status of the animals through neurological function scoring and behavioral experiments, and use in vivo and in vitro reverse genetics techniques such as siRNA interference, overexpression plasmid transfection, immunoprecipitation, and AAV-siFyn stereotactic injection to study the pathological mechanisms following subarachnoid hemorrhage.

[0003] However, a systematic analysis of the spatiotemporal dynamic regulatory network of neuroinflammation associated with subarachnoid hemorrhage is lacking, and the key regulatory molecular mechanisms remain unclear. Summary of the Invention

[0004] This application provides a method for studying the function of Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage, which can solve the technical problem of how to systematically analyze the regulatory function and mechanism of Fyn kinase on microglial-mediated neuroinflammation in early brain injury induced by subarachnoid hemorrhage. To achieve the above objective, this application provides the following technical solution: This application provides a method for functional research on Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage, including the following steps: Constructing an in vivo subarachnoid hemorrhage model: Rat and mouse models of subarachnoid hemorrhage are constructed using vascular interventional techniques. A sham-operated group and a subarachnoid hemorrhage group are set up. The subarachnoid hemorrhage group is divided into subgroups at different time points. The sham-operated group undergoes the same surgical procedure but without vascular puncture. Constructing an in vitro model: Primary microglia and primary neurons are extracted. Primary microglia are treated with hemoglobin to simulate an in vitro subarachnoid hemorrhage model. A co-culture of the BV-2 microglia cell line and the HT22 neuronal cell line is constructed. The model was designed to simulate an in vitro subarachnoid hemorrhage (SAH) model. Fyn expression and cellular localization were detected: Western blotting was used to detect Fyn protein expression levels at different time points in the subarachnoid hemorrhage model; immunohistochemistry was used to detect Fyn expression in the hippocampus and cortex; immunofluorescence staining was used to detect the co-localization of Fyn with microglia, neurons, and astrocytes. Fyn functional intervention experiments were conducted: an adeno-associated virus carrying a Fyn interference sequence was constructed and injected into the mouse hippocampus using stereotactic localization technology. An AAV-ZsGreen control group was set up. In vitro and in vivo models were used to interfere with or overexpress Fyn expression using siRNA. Inflammation-related markers were detected: [The text abruptly ends here, likely due to an incomplete translation or a missing section.] White matter immunoblotting was used to detect the expression of NLRP3 inflammasome-related proteins such as NLRP3 and Caspase-1p20; immunofluorescence was used to detect ASC oligomerization; and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α. Apoptosis was detected by TUNEL staining and flow cytometry was used to detect the apoptosis rate of HT22 cells or primary neurons in co-culture systems. Brain injury and functional assessment were performed by assessing the degree of brain injury through neurological function scoring and brain water content measurement, and by evaluating animal cognitive, learning, and exploratory functions using behavioral experiments such as the Y-maze, elevated maze, open field test, and Morris water maze. Molecular mechanisms were explored. Research: Downstream regulatory genes of Fyn were screened through proteomics analysis, and gene transcription levels were detected using real-time quantitative PCR. Protein-protein interactions were verified by co-immunoprecipitation, and ubiquitination modification and degradation pathways of Sirt1 protein were detected. Target reverse screening verification: Xol, a small molecule compound that targets and inhibits Fyn, was screened through molecular docking. The binding ability of Fyn and Xol was detected by cell thermal displacement analysis (CETSA) and drug affinity target stability assay (DARTS). The protective effect of Xol against SAH was evaluated using in vivo and in vitro models. AAV-siFyn reverse genetics was used to assess whether Xol depends on Fyn to exert a protective effect against SAH.

[0005] In one optional embodiment, the specific procedures for constructing the SAH model include: after anesthetizing the experimental animals with an intraperitoneal injection of 100 mg / kg ketamine, a midline incision is made in the neck to separate the bifurcation region of the internal carotid artery, the external carotid artery is double-ligated and the blood flow between the internal carotid artery and the left common carotid artery is temporarily blocked, a puncture is made 3 mm below the ligation point at the distal end of the external carotid artery and a microcatheter is inserted retrogradely to the origin of the middle cerebral artery, the catheter is withdrawn after penetrating the vascular intima to simulate bleeding; the time groups of the SAH rat model include 3h, 6h, 12h, 24h, 48h, and 72h, and 24h is selected as the key time point for subsequent experiments.

[0006] In one optional embodiment, the specific operations for primary cell extraction and culture include: taking cortical tissue from newborn mice, washing it with pre-cooled PBS, digesting it with 0.125% EDTA-free trypsin at 37°C for 7-10 minutes, filtering it through a cell sieve to obtain a single-cell suspension, culturing primary neuronal cells in DMEM medium containing 2% B27, 1% L-glutamine and 1% penicillin antibiotics, and culturing primary microglia in DMEM high-glucose medium containing 10% FBS and 1% penicillin antibiotics, identifying the morphology and purity of primary neurons by NeuN, and identifying the morphology and purity of primary microglia by Iba1 staining.

[0007] In one optional embodiment, the Western blot experiment uses Fyn target antibody and GAPDH internal reference protein to calculate the relative expression level of Fyn; the immunohistochemical experiment uses Fyn target antibody, DAB staining, and microscopic observation of Fyn positive cells; the immunofluorescence staining uses Fyn target antibody and Iba1 / Neun / GFAP neural markers, staining with FITC-labeled goat anti-rabbit secondary antibody and CY3-labeled goat anti-mouse secondary antibody, and DAPI staining of the nucleus, and confocal microscopy to observe the colocalization of Fyn with different nerve cells.

[0008] In one optional embodiment, the operational parameters for virus injection and cell transfection include: during AAV injection, a mouse brain stereotaxic instrument is used to accurately locate the mouse hippocampus, with an injection volume of 0.5 μL per mouse, and the interference efficiency is detected after 4 weeks; during in vitro cell transfection, 100 pmol siRNA per well is mixed with 5 μL of liposome transfection reagent, incubated in serum-free DMEM medium for 5 minutes to form a complex, which is then added to the cell culture system. After 4 hours of transfection, the medium is replaced with complete medium containing 10% FBS, and the interference or overexpression efficiency is detected after 3-5 days of culture.

[0009] In one optional embodiment, the specific conditions for detecting inflammatory markers include: Western blot detection of a 118 kDa NLRP3 target band, Caspase-1p20 detection of a 20 kDa target band; immunofluorescence detection of punctate aggregated fluorescence signals as ASC oligomerization signals; and ELISA detection of OD values ​​of IL-1β, IL-6, and TNF-α at an OD wavelength of 450 nm.

[0010] In one optional embodiment, the specific procedure for apoptosis detection includes: during TUNEL staining, frozen sections are fixed with 4% paraformaldehyde, incubated with the reaction solution according to the kit instructions, and apoptotic cells are counted under a fluorescence microscope; during flow cytometry detection, Annexin V-FITC / PI double staining is used, cells are washed with PBS, resuspended in Binding Buffer, incubated with the fluorescent probe, and detection is completed within 1 hour, and the proportion of early and late apoptotic cells is analyzed using FlowJo software.

[0011] In one optional embodiment, the specific criteria for brain injury and functional assessment include: neurological function scoring using a modified Garcia scale, with scores ranging from 0 to 3 points across six dimensions, including spontaneous activity, forelimb abduction, and climbing ability, for a total score of 0-18 points; brain water content determination, after drying brain tissue in a 70°C oven for 48 hours, weighing the tissue and calculating the content using the formula [(wet weight - dry weight) / wet weight] × 100%; and behavioral experiments, using the Y-maze to record the time it takes for mice to enter the new arm, the open field test to analyze the dwell time and movement distance in the central region, and the Morris water maze to record the time it takes to reach the platform and the dwell time in the target quadrant.

[0012] In one optional embodiment, the specific methods for exploring the molecular mechanism include: in proteomics analysis, LC-MS / MS technology is used to identify changes in oxyhemoglobin and proteins in microglia treated with siFyn; GSEA and KEGG analysis are used to enrich related pathways; in the qPCR experiment, β-actin is used as an internal reference gene, and 2... -ΔΔCt The method calculates the relative expression level of genes; in the COIP experiment, after cell lysis, Flag or Myc specific antibodies are added and incubated overnight, then agarose beads are added to capture immune complexes, and Western blot is used to detect interacting proteins.

[0013] In an optional embodiment, the experiment further includes a xanthotoxin intervention experiment, the specific steps of which are: screening xanthotoxin targeting Fyn through molecular docking, verifying the binding stability of the two by cell thermal displacement analysis and drug affinity target stability experiment; setting up different concentrations of xanthotoxin treatment groups in in vivo and in vitro models, detecting the effects of xanthotoxin on Fyn expression, NLRP3 inflammasome activation, cell apoptosis and animal brain function, and clarifying the mechanism of action of xanthotoxin targeting Fyn.

[0014] This application provides a method for studying the function of Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. This method involves constructing multi-level in vivo and in vitro models of subarachnoid hemorrhage, establishing a dynamic expression profile and spatial localization map of Fyn, implementing Fyn functional intervention, multi-dimensional inflammation detection, bimodal apoptosis assessment, integrated brain injury and functional evaluation, and in-depth mechanism mining. Spatiotemporal control modeling reveals that Fyn is temporally upregulated after subarachnoid hemorrhage. Immunofluorescence staining to detect the co-localization of Fyn and microglia confirms that Fyn is mainly enriched in microglia. By constructing an adeno-associated virus carrying Fyn interference sequences and performing stereotactic injection into the brain, Fyn functional intervention is achieved, significantly reducing NLRP3 inflammasome activation, decreasing neuronal apoptosis, and improving behavioral performance. Furthermore, proteomic analysis was used to screen downstream regulatory genes of Fyn, and real-time quantitative PCR was employed to detect gene transcription levels. Immunoprecipitation was used to verify protein-protein interactions, and ubiquitination modification and degradation pathways of Sirt1 protein were detected. This confirmed that Fyn degrades Sirt1 through K48 ubiquitination, thereby relieving Sirt1's negative regulation of NLRP3 and driving the amplification of the neuroinflammatory cascade. This research method establishes for the first time the causal chain of the Fyn-Sirt1-NLRP3 axis in subarachnoid hemorrhage-early brain injury, providing a complete research paradigm that is quantifiable, reproducible, and cross-validated at multiple levels, laying a methodological foundation for neuroprotective strategies targeting Fyn. By constructing in vivo and in vitro models of subarachnoid hemorrhage, this study systematically elucidates the expression characteristics and functional roles of Fyn in the pathological process of subarachnoid hemorrhage. Fyn expression and cellular localization detection clarify the spatiotemporal distribution of Fyn in brain tissue. Fyn functional intervention experiments verify the regulatory effects of Fyn on neuroinflammation and apoptosis. Inflammation-related marker detection and apoptosis detection assess the impact of Fyn intervention on neuroinflammation and neuronal damage. Brain injury and functional assessment correlates molecular-level changes with overall brain function improvement. Molecular mechanism exploration elucidates the molecular pathways through which Fyn regulates neuroinflammation. This method is interconnected and logically rigorous, with all techniques serving the core objective of "elucidating Fyn function," providing a novel perspective for understanding the pathological mechanisms of subarachnoid hemorrhage and confirming that Fyn can serve as a potential key therapeutic target for subarachnoid hemorrhage. Detailed Implementation

[0015] The technical solutions in the embodiments of this invention / invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention / invention, and not all embodiments. Based on the embodiments of this invention / invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention / invention.

[0016] To achieve the above-mentioned objectives, this invention provides a method for functionally studying Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The overall technical solution is as described in the embodiments of this application, mainly including the following core technical elements: constructing a time-resolution in vivo SAH model and an OxyHb-induced in vitro microglial / neuron co-culture model; establishing a dynamic expression profile of Fyn protein and a microglial-specific spatial localization map; achieving Fyn functional intervention through AAV-siFyn brain region targeted delivery and in vitro siRNA interference; multi-level verification of NLRP3 inflammasome activation status (NLRP3 protein expression, Caspase-1 p20 cleavage, ASC oligomerization, and pro-inflammatory factor secretion); and simultaneously conducting in vivo TUNEL staining and in vitro Annexin staining. V-FITC / PI double staining flow cytometry was used to detect neuronal apoptosis; neurological function scores, brain water content measurement, and behavioral methods such as the Y maze / open field / Morris water maze were integrated to assess the degree of brain injury and cognitive function; combined with LC-MS / MS proteomics screening, qPCR transcriptional verification, COIP interaction analysis, and K48-specific ubiquitination detection, the molecular axis of Fyn→C-cbl phosphorylation→Sirt1 K48 ubiquitination degradation→NLRP3 deinhibition was systematically elucidated; further, xanthocyanin (Xol) was introduced as a natural small molecule probe targeting Fyn, and a closed-loop study from mechanism discovery to drug validation was completed through molecular docking, CETSA / DARTS binding verification, and gradient dose efficacy evaluation. The above technical elements work together to constitute the overall technical solution of this invention.

[0017] The first aspect of this invention provides a method for studying the functional role of Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The method includes: constructing an in vivo subarachnoid hemorrhage (SAH) model: SAH rat and mouse models were constructed using vascular interventional techniques. A sham-operated group and an SAH group were established, with the SAH group divided into subgroups at different time points. The sham-operated group underwent the same surgical procedure but without vascular wall penetration. In vitro model construction: Primary microglia and primary neurons were extracted. Primary microglia were treated with hemoglobin (OxyHb) to simulate the in vitro SAH model. A co-culture system of BV-2 microglia and HT22 neuronal cell lines was constructed. Fyn expression and cell localization detection: Fyn expression at different time points in the SAH model was detected using Western blotting. The expression level of Fyn protein was assessed using immunohistochemistry in the hippocampus and cortex. Immunofluorescence staining was used to detect the co-localization of Fyn with microglia (Iba1), neurons (NeuN), and astrocytes (GFAP). Fyn functional intervention experiments were conducted: an adeno-associated virus (AAV-siFyn) carrying an interference sequence was constructed and injected into the mouse hippocampus using stereotaxic techniques. An AAV-ZsGreen control group was included. In vivo and in vitro models, siRNA was used to interfere with or overexpress Fyn. Inflammation-related markers were detected using Western blotting for NLRP3 and Caspase-1. Expression of NLRP3 inflammasome-related proteins such as p20 was assessed; ASC oligomerization was detected by immunofluorescence; and the levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). Apoptosis was detected in vivo using TUNEL staining, and the apoptosis rate of HT22 cells or primary neurons in co-culture systems was detected by flow cytometry. Brain injury and functional assessment were performed: the degree of brain injury was assessed through neurological function scoring and brain water content measurement; and behavioral experiments such as the Y-maze, elevated maze, open field test, and Morris water maze were used to assess animal cognitive, learning, and exploratory functions. Molecular mechanism investigation was conducted: Fyn was screened through proteomics analysis. Downstream regulatory genes were identified using quantitative real-time PCR (QPCR) to detect gene transcription levels. Co-immunoprecipitation (COIP) was used to verify protein-protein interactions, and ubiquitination modification and degradation pathways of Sirt1 protein were detected. Target reverse screening was performed: molecular docking was used to screen for the small molecule compound xanthotoxin (Xol) that targets and inhibits Fyn. Cell thermal displacement analysis (CETSA) and drug affinity target stability assay (DARTS) were used to detect the binding ability of Fyn and Xol. In vivo and in vitro models were used to evaluate the protective effect of Xol against SAH. AAV-siFyn reverse genetics was used to assess whether Xol depends on Fyn to exert its protective effect against SAH.In this invention, a dual-modal pathological simulation system of "in vivo SAH model + in vitro OxyHb co-culture model" was constructed to accurately capture the dynamic changes of Fyn in the spatiotemporal dimensions. The biological basis for the specific enrichment of Fyn in microglia was clarified by jointly analyzing Fyn expression levels, tissue distribution, and cellular sublocalization using Western blot / IHC / IF triple technology. A genetic intervention platform consisting of AAV-siFyn brain region targeted delivery and in vitro siRNA interference ensured the spatial precision and molecular specificity of Fyn functional perturbation. Furthermore, NLRP3 protein expression, Caspase-1 p20 cleavage, ASC oligomerization morphology, and changes in IL-1β / IL-6 / TNF-α concentrations were used as four-dimensional verification indicators to elevate the "presence" of the NLRP3 inflammasome to the "functional activation" level. Simultaneously, TUNEL (DNA break end marker) and Annexin were employed. V-FITC / PI double staining (phosphatidylserine eversion + loss of membrane integrity) characterizes late apoptosis in vivo and early / late apoptosis in vitro, forming a cross-scale apoptosis evidence chain. Ultimately, it integrates neurological function scores (0–18 points, continuous variable), brain water content ([(wet weight − dry weight) / wet weight] × 100% quantitative formula), and three behavioral paradigms: the Y maze (working memory), the open field (anxiety and exploratory motivation), and the Morris water maze (spatial learning and long-term memory), grounding the molecular intervention effect in the overall recovery of animal neurological function. This technology system identifies Sirt1 as a key downstream target through unbiased proteomics screening, and excludes transcriptional regulation via qPCR and confirms Fyn−C-cbl−Sir via COIP. The formation of the t1 ternary complex and the direct verification of ubiquitination modification type by K48-linkage specific antibodies reveal, at the molecular mechanism level, the causal chain in which Fyn reshapes its E3 ligase substrate recognition spectrum through phosphorylation of C-cbl, drives K48-linked ubiquitination modification of Sirt1 and triggers its proteasome degradation, relieves Sirt1's negative regulation of NLRP3, and ultimately leads to the amplification of the neuroinflammatory cascade. All technical modules are interconnected and logically rigorous around the core scientific question of the "Fyn-Sirt1-NLRP3 axis", providing a standardized research paradigm that is quantifiable, reproducible, and cross-validated at multiple levels for systematically analyzing the neuroinflammatory regulatory network of early brain injury after SAH.

[0018] As a specific implementation method, this invention provides a method for functional study of Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The specific procedures for constructing the SAH model in step 1) include: after anesthetizing experimental animals with an intraperitoneal injection of 100 mg / kg ketamine, a midline incision is made in the neck to separate the bifurcation region of the internal carotid artery. The external carotid artery is double-ligated, and blood flow between the internal carotid artery and the left common carotid artery is temporarily blocked. A small incision is made 3 mm below the ligation point at the distal end of the external carotid artery, and a microcatheter is inserted retrogradely to the origin of the middle cerebral artery. After penetrating the vascular intima, the catheter is withdrawn to simulate hemorrhage. The time groups for the SAH rat model include 3h, 6h, 12h, 24h, 48h, and 72h, with 24h selected as the critical time point for subsequent experiments. In this invention, by limiting the ketamine dose (100 mg / kg), puncture site (3 mm below the distal ligation point of the external carotid artery), catheter insertion depth (to the origin of the middle cerebral artery), and time gradient (3–72 h), the high consistency of the SAH model in terms of hemodynamic impact intensity, degree of vascular wall damage, and pathological progression was ensured. Among them, 24 h was established as a critical time point because it corresponds to the peak of Fyn protein expression (Western blot showed an increase of about 3.2 times compared to the sham-operated group), the peak of NLRP3 inflammasome activation (Caspase-1 p20 band intensity increased by 2.8 times), and the significant initiation period of neuronal apoptosis (TUNEL positive cell number increased by 4.5 times compared to the sham-operated group). This ensures that all subsequent molecular detection and functional assessment are anchored to the pathological window with the most significant biological significance, thereby guaranteeing the timeliness and reliability of experimental conclusions.

[0019] As a specific implementation method, this invention provides a method for studying the function of Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The specific operations for primary cell extraction and culture in step 2) include: taking cortical tissue from newborn mice, washing it with pre-cooled PBS, incubating it with 0.125% EDTA-free trypsin at 37°C for 7–10 minutes for digestion, filtering the cell suspension to obtain a single-cell suspension, centrifuging it, and then seeding it with primary neurons and primary microglia, respectively; primary neurons are cultured in DMEM medium containing 2% B27, 1% L-glutamine, and 1% penicillin antibiotics, while primary microglia are cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin antibiotics; the purity of primary neurons is verified by NeuN immunofluorescence staining, and the purity of primary microglia is verified by Iba1 immunofluorescence staining. In this invention, by employing EDTA-free trypsin to avoid the disruption of cell membrane integrity caused by calcium chelation, combining B27 and L-glutamine to synergistically support neuronal synergistic development and long-term survival, and using a combination of high-glucose DMEM and 10% FBS to promote microglia adhesion and homeostasis, the stable acquisition of primary neurons with >95% purity and primary microglia with >90% purity was achieved. NeuN / Iba1 dual-label immunofluorescence verification excluded astrocyte and oligodendrocyte contamination, ensuring that the co-culture system contained only the two target cell types. This provides a high-fidelity in vitro pathological microenvironment model for detecting OxyHb-induced microglia activation, Fyn interference-induced inflammatory factor release, and HT22 neuron apoptosis.

[0020] As a specific implementation method, this invention provides a method for functionally studying Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. Specific parameters for Fyn detection in step 3) include: using GAPDH or β-actin as internal control proteins in Western blot experiments; using DAB staining in immunohistochemistry experiments; observing at least three different fields of view under a microscope; using FITC-labeled goat anti-rabbit secondary antibody and CY3-labeled goat anti-mouse secondary antibody in immunofluorescence staining; staining the nuclei with DAPI; and observing co-localization under a confocal microscope. In this invention, by uniformly using GAPDH or β-actin as housekeeping protein internal controls, the influence of sample loading differences on Fyn protein quantification results is eliminated; the DAB colorimetric intensity and reaction time are strictly controllable, ensuring the comparability of immunohistochemical semi-quantitative results between different batches; the FITC / CY3 dual-channel excitation wavelength separation combined with DAPI nuclear localization calibration enables the precise resolution of the subcellular colocalization relationship of Fyn and Iba1 / NeuN / GFAP under confocal imaging (such as the co-enrichment of Fyn and Iba1 in the cytoplasm and cell membrane), confirming the dominant expression characteristics of Fyn in microglia (Fyn / Iba1 colocalization coefficient is 0.82±0.05, significantly higher than Fyn / NeuN 0.31±0.04), providing direct spatial evidence for its role as a microglia-specific regulatory target.

[0021] As a specific implementation method, this invention provides a method for functionally studying Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The operational parameters for virus injection and cell transfection in step 4) include: during AAV injection, a mouse stereotaxic instrument is used to precisely locate the mouse hippocampus, and the injection dose per mouse is a preset standard dose; during in vitro cell transfection, 100 pmol siRNA per well is mixed with 5 μL of liposome transfection reagent, incubated in serum-free DMEM medium for 5 minutes to form a complex, which is then added to the cell culture system. Four hours after transfection, the medium is replaced with complete medium containing 10% FBS, and the interference or overexpression efficiency is detected after 3–5 days of culture. In this invention, AAV-siFyn is precisely delivered to the CA1 region of the hippocampus (coordinates: AP −2.0 mm, ML ±1.5 mm, DV −1.8 mm) using a stereotaxic instrument, ensuring that the intervention is limited to the core damaged area of ​​Fyn high expression and early brain injury, avoiding diffuse interference throughout the brain. The standardized siRNA transfection complex ratio (100 pmol : 5 μL) and the timing of medium change (4 h later) maximize the Fyn protein knockdown efficiency (Western blot showed a reduction of 73.2% ± 4.1% compared to the control group) while controlling liposome cytotoxicity within an acceptable range (cell viability >92% as detected by CCK-8 assay), thereby ensuring that the Fyn functional intervention effect is attributed to the target gene itself rather than a side effect of the operation.

[0022] As a specific implementation method, this invention provides a method for functionally studying Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. Specific conditions for detecting inflammatory markers in step 5) include: when detecting NLRP3 using Western blot, a band corresponding to a molecular weight of 118 kDa is selected; when detecting ASC oligomerization using immunofluorescence, ASC is labeled with red fluorescence in brain tissue samples and green fluorescence in cell samples; the ELISA experiment is performed strictly according to the kit instructions, with the detection wavelength set to the recommended wavelength, and three replicates are set for each sample. In this invention, molecular weight anchoring (118 kDa corresponds to the mature NLRP3 monomer, and 20 kDa corresponds to the active cleavage body p20 of Caspase-1) eliminates non-specific band interference, ensuring that the Western blot results reflect the true assembly and activation state of the NLRP3 inflammasome. Different fluorescent labels are used for brain tissue and cell samples (red for tissue sections to avoid autofluorescence, and green for cell smears to improve the signal-to-noise ratio), making ASC oligomerized spots (diameter > 0.5 μm) clearly identifiable in confocal images. The triple-well design of ELISA and standard wavelength detection minimize the intra-assay coefficient of variation (CV < 8.5%), ensuring that the concentration data of IL-1β, IL-6, and TNF-α have statistical power, thus forming a three-dimensional mutual verification with protein and morphological data, confirming that the NLRP3 inflammasome pathway is systematically blocked after Fyn inhibition.

[0023] As a specific implementation method, this invention provides a method for functionally studying Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The specific procedure for apoptosis detection in step 6) includes: for TUNEL staining, frozen sections are fixed with 4% paraformaldehyde, incubated with the reaction solution according to the kit instructions, and apoptotic cells are counted under a fluorescence microscope; for flow cytometry detection, Annexin V-FITC / PI double staining is used. Cells are washed with PBS, resuspended in Binding Buffer, incubated with the fluorescent probe, and detection is completed within 1 hour. The proportion of early apoptotic (Q3) and late apoptotic (Q2) cells is analyzed using FlowJo software. In this invention, the TUNEL assay specifically labels DNA break ends, reflecting the late apoptosis / necrosis process, while the Annexin V-FITC / PI double staining method distinguishes between phosphatidylserine eversion (Q3, early apoptosis) and loss of membrane integrity (Q2, late apoptosis / necrosis), achieving a staged analysis of the apoptosis process. The two methods show a high degree of consistency in the SAH model (TUNEL positivity rate of 32.7%±3.2% in the SAH+ZsGreen group, and Q2+Q3 proportion of 34.1%±2.9% in flow cytometry), forming a closed loop of evidence across scales. After Fyn interference, this proportion decreased synchronously (TUNEL positivity rate decreased to 14.3%±1.8%, and Q2+Q3 proportion decreased to 15.6%±1.5%), indicating that Fyn intervention can effectively block the initiation and progression of apoptosis.

[0024] As a specific implementation method, this invention provides a functional study method for Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The specific criteria for brain injury and functional assessment in step 7) include: Neurological function scoring uses a modified Garcia scoring scale, with scores ranging from 0 to 3 points across six dimensions including spontaneous activity, forelimb abduction, and climbing ability, for a total score of 0–18 points; in brain water content determination, brain tissue is weighed after drying in a 70℃ oven for 48 hours and calculated using the formula [(wet weight − dry weight) / wet weight] × 100%; in behavioral experiments, the Y-maze records the time it takes for mice to enter the new arm, the open field test analyzes the dwell time and movement distance in the central region, and the Morris water maze records the time to reach the platform and the dwell time in the target quadrant. In this invention, the modified Garcia score encompasses multidimensional neurological functions such as motor, sensory, and reflex functions, sensitively reflecting the severity of early brain injury (SAH+ZsGreen group score 8.2±0.9, significantly lower than sham-operated group score 16.4±0.7); the brain water content formula directly quantifies vasogenic and cytotoxic edema (SAH+ZsGreen group brain water content 79.6%±0.8%, significantly higher than sham-operated group 77.1%±0.5%); the entry time of the new arm in the Y-maze (SAH+ZsGreen group 12.3±1.1 s vs sham-operated group 28.7±1.5 s), the dwell time in the open field center (SAH+ZsGreen group 24.5±2.3 s vs sham-operated group 87.6±3.1 s), and the dwell time in the target quadrant of the Morris water maze (SAH+ZsGreen group 38.2%±2.7% vs sham-operated group 87.6±3.1 s) were also measured. The sham surgery group (62.4% ± 3.0%) showed complementary coverage of working memory, anxiety state, and spatial learning ability, confirming that Fyn intervention not only reduces pathological damage but also restores higher brain functions. All indicators have internationally recognized validity, supporting the translational medicine value.

[0025] As a specific implementation method, this invention provides a method for functional study of Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. The specific methods for exploring the molecular mechanism in step 8) include: in proteomics analysis, LC-MS / MS technology is used to identify proteins, and GSEA and KEGG analysis is used to enrich related pathways; in the qPCR experiment, β-actin is used as an internal reference gene, and the relative gene expression level is calculated using the 2−ΔΔCt method; in the COIP experiment, after cell lysis, Flag or Myc-specific antibodies are added and incubated overnight, agarose beads are added to capture immune complexes, and Western blot is used to detect interacting proteins. In this invention, unbiased LC-MS / MS screening combined with GSEA / KEGG enrichment analysis identifies Sirt1 as the core downstream target of Fyn from differentially expressed proteins between the OxyHb stimulation group and the siFyn group (log2FC=−2.37, p=1.2×10⁻). 8Furthermore, its pathway was significantly enriched in anti-inflammatory and anti-apoptotic modes (KEGG pathway ID: mmu04151, NES=−2.93); qPCR results showed no significant difference in Sirt1 mRNA levels between the two groups (p=0.62), ruling out transcriptional regulation; COIP experiments confirmed direct interactions between Fyn and C-cbl, and between C-cbl and Sirt1 (WB detected Myc-C-cbl and HA-Sirt1 after Flag-Fyn IP, and WB detected HA-Sirt1 after Myc-C-cbl IP), indicating that Fyn mediates Sirt1 ubiquitination by recruiting C-cbl to form a ternary complex. This synergistic effect is the key molecular basis for driving Sirt1 protein degradation and relieving its negative regulation of NLRP3.

[0026] As a specific implementation method, this invention provides a method for functional research on Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage. It also includes a xanthotoxicol (Xol) intervention experiment, the specific steps of which are: screening for Fyn-targeting Xol through molecular docking; verifying the binding stability of the two using cell thermal shift analysis (CETSA) and drug affinity target stability assay (DARTS); setting up different concentrations of Xol treatment groups (5 mg / kg, 10 mg / kg, 15 mg / kg in vivo dose, 2.5 μM, 7.5 μM, 15 μM in vitro dose) in in vivo and in vitro models, detecting the effects of Xol on Fyn expression, NLRP3 inflammasome activation, apoptosis, and animal brain function, thus clarifying the mechanism of action of Xol targeting Fyn. In this invention, molecular docking showed that Xol has a high affinity (binding energy −8.2 kcal / mol) for the ATP-binding pocket of the Fyn kinase domain (PDB ID: 2DQ7), and key residues Lys295, Glu298, and Asp404 form a hydrogen bond network; CETSA experiments confirmed that Xol treatment increased the thermal denaturation temperature (Tm) of Fyn protein by 4.3 °C, and DARTS experiments showed that Xol protects Fyn from proteinase K degradation (IC50). 50=3.1 μM), jointly verifying its direct targeted binding; in vivo gradient dose experiments showed that 10 mg / kg Xol significantly inhibited Fyn protein expression (reduced by 58.4%±3.7% compared to the SAH+Vehicle group), and dose-dependently improved NLRP3 activation (Caspase-1 p20 band intensity decreased by 42.1%±2.9%), neuronal apoptosis (TUNEL positivity rate decreased to 17.8%±1.6%), and neurological function score (increased to 12.9±0.8 points); in vitro treatment of the BV-2 / HT22 co-culture system with 15 μM Xol showed that its NLRP3 inhibition and apoptosis reduction effects were highly consistent with those of the siFyn group (correlation coefficient r=0.96), indicating that Xol replicated the neuroprotective effect of genetic intervention by targeting and inhibiting Fyn kinase activity, providing a solid basis for the clinical translation of natural products.

[0027] Unless otherwise specified, all materials, reagents and instruments used in the embodiments of this invention can be obtained through commercial channels.

[0028] Materials and reagents: Fyn antibody (Wanlei, WL04271; Solarbio, K112610P, K109962P); GAPDH antibody (Proteintech, 15310-1-AP); β-actin antibody (Proteintech, 66009-1-Ig); Iba1 antibody (abcam, ab178846); NeuN antibody (abcam, ab104224); GFAP antibody (Proteintech, 16825-1-AP); Caspase-1p20 antibody (Adipogen) AG-20B-0042-C100); K48 ubiquitination antibody (abclonal, A3606); NLRP3 antibody (abclonal, A24294); ASC antibody (Proteintech, 10500-1-AP); HRP-goat anti-mouse secondary antibody (BOSTER, BA1050); HRP-goat anti-rabbit secondary antibody (BOSTER, BM2006); FITC-goat anti-rabbit secondary antibody (Earthox, E031220); CY3-goat anti-mouse secondary antibody (Earthox, E031610); APC / Cyanine 7 anti-mouse CD45 (Biolegend, 103115); 0.22 μm PVDF membrane (Merck Millipore, SAMP2GVNK); 0.45 μm PVDF membrane (Merck Millipore, SAMP2HVNK); BCA protein concentration assay kit (Biosharp, BL521A); RIPA lysis buffer (Solarbio, R0010); PBS powder (Biosharp, BL601A); glycine (Biosharp, 56-40-6); sodium chloride (Biosharp, 7647-14-5); skim milk powder (Biosharp, 56-40-6); tris(hydroxymethyl)aminomethane (Tris) (Biosharp, 77-86-1); Tween-20 (Biosharp, 9005-6); high glucose DMEM medium (KeyGEN) BioTECH, KGL1201-500; fetal bovine serum (VivaCell, C2910-0500); pancreatic enzyme cell digestion solution (0.25% EDTA-free (VICMED, VC2005); B27 (Gibco, 17504-044); L-glutamine (Gibco, 25030-081); Hemoglobin (Sigma-Aldrich, H7379); GM-CSF (Thermo Fisher Scientific, 315-03-20UG); IL-1β-ELISA kit (Thermo Fisher Scientific, 88-7013A-88); IL-6-ELISA kit (Thermo Fisher Scientific, BMS603-2); TNF-α-ELISA reagent Kit (Thermo Fisher Scientific, BMS607-3); Nissl staining kit (Beyotime, C0117); Immunohistochemical staining kit (Boster Biological, SA1020); TUNEL kit (Vazyme, A113-02); RNA transfection kit (Beyotime, C0526); HE kit (Beyotime, C0105M); AAV2 / 9-CX3CR1-ZsGreen (Hanheng Biotechnology); pAAV-CX3CR1-siFyn-ZsGreen (Hanheng Biotechnology).

[0029] Instruments and equipment: Stereotype instrument for mouse brain (Stoelting, 51730); confocal laser scanning microscope (Zeiss, LSM 900); Western blot electrophoresis and transfer system (Bio-Rad, Mini-PROTEAN Tetra & Trans-Blot Turbo); microplate reader (BioTek, Synergy H1); flow cytometer (BD, FACSCanto II); real-time quantitative PCR instrument (Bio-Rad, CFX96 Touch); ultra-high performance liquid chromatography-tandem mass spectrometry (Waters, Xevo G2-XS QTof); behavioral video tracking system (Shanghai Xinruan, XR-XJ108).

[0030] Characterization and testing methods: Western blot: Protein samples were transferred to PVDF membranes after SDS-PAGE electrophoresis (10% separating gel), blocked with 5% skim milk powder for 1 h, incubated with primary antibody at 4°C overnight, incubated with HRP-labeled secondary antibody at room temperature for 1 h, developed by ECL, and analyzed by Image Lab software. Immunohistochemistry: After dewaxing and hydration of paraffin sections, antigen retrieval was performed, endogenous peroxidase was inactivated with 3% H2O2, primary antibody was incubated overnight at 4°C, DAB staining was performed, hematoxylin was counterstained, and ≥3 random fields of view images were collected under an optical microscope. Immunofluorescence: Frozen sections were fixed with acetone, permeabilized with 0.3% Triton X-100, blocked with 5% BSA, incubated with primary antibody at 4°C overnight, incubated with FITC / CY3-labeled secondary antibody in the dark for 1 h, stained with DAPI, Z-stack images were acquired by confocal microscopy, and Pearson colocalization coefficients were calculated using ZEN software. ELISA: Follow the kit instructions strictly. The standard curve was fitted using a four-parameter logistic model, and the concentration was calculated based on the average value of three replicates. Flow cytometry: Annexin V-FITC / PI double staining was performed within 1 h. FlowJo v10.8 software was used to analyze the proportion of Q2 (PI⁺ / Annexin V⁺, late apoptosis / necrosis) and Q3 (PI⁻ / Annexin V⁺, early apoptosis). Behavioral analysis: The Y-maze, open field, and Morris water maze experiments were all conducted in a quiet, dark environment. The video tracking system automatically recorded the trajectory and parameters. Each group had n≥10 participants, and the experimenters were blinded during the analysis.

[0031] Example 1: This embodiment aims to construct a complete Fyn-Sirt1-NLRP3 axis function research system to verify the core role of Fyn in mediating neuroinflammation and neuronal apoptosis in early brain injury after SAH by regulating the activation of microglia NLRP3 inflammasomes.

[0032] Male SD rats (6–8 weeks old, 220–250 g) were anesthetized by intraperitoneal injection of 100 mg / kg ketamine. A midline incision was made in the neck to separate the bifurcation region of the internal carotid artery. The external carotid artery was double-ligated, and blood flow between the internal carotid artery and the left common carotid artery was temporarily blocked. A small incision was made 3 mm below the ligation point at the distal end of the external carotid artery, and a microcatheter was inserted retrogradely to the origin of the middle cerebral artery. After penetrating the intima of the blood vessel, the catheter was withdrawn to simulate bleeding. The sham-operated group underwent the same surgical procedure but without puncturing the blood vessel. SAH rats were grouped at 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h, with n=12 in each group; the 24 h group was used for subsequent mechanism studies. Simultaneously, cortical tissue from newborn Balb / c mice was harvested, washed with pre-cooled PBS, digested with 0.125% EDTA-free trypsin at 37°C for 8 min, filtered through a 70 μm cell sieve to obtain a single-cell suspension, and centrifuged. The suspension was then seeded into primary neurons cultured in DMEM containing 2% B27, 1% L-glutamine, and 1% penicillin antibiotics, and primary microglia cultured in DMEM containing 10% FBS and 1% penicillin antibiotics at high glucose levels. After 7 days of culture, NeuN immunofluorescence staining showed a neuronal purity of 96.2% ± 1.3%, and Iba1 staining showed a microglia purity of 92.7% ± 0.9%. Primary microglia and HT22 neurons were co-cultured at a 1:5 ratio for 48 h, followed by stimulation with 50 μM OxyHb for 24 h to construct an in vitro SAH model. Western blot analysis showed that Fyn protein in the hippocampus of SAH rats peaked at 24 h (3.21 ± 0.24 times higher than the sham-operated group, p < 0.001). Immunofluorescence colocalization analysis showed that the colocalization coefficient of Fyn and Iba1 was 0.82 ± 0.05, significantly higher than that of Fyn / NeuN (0.31 ± 0.04, p < 0.001). pAAV-CX3CR1-siFyn-ZsGreen virus was constructed and injected into the hippocampus of SAH mice at a dose of 1 μL (1 × 10¹³ vg / mL) using a stereotaxic instrument (AP −2.0 mm, ML ± 1.5 mm, DV −1.8 mm). The AAV-ZsGreen group served as a control. Brain tissue was harvested 3 days later, and Western blot analysis confirmed that the knockdown efficiency of Fyn protein was 73.2% ± 4.1%.NLRP3 inflammasome detection showed that the band intensities of NLRP3 protein (118 kDa) and Caspase-1 p20 (20 kDa) in the siFyn group were reduced by 52.4%±3.7% and 61.8%±4.2% respectively compared with the ZsGreen group (p<0.001); ASC immunofluorescence showed that the number of ASC oligomerization spots in the siFyn group decreased by 68.3%±5.1%; ELISA detection showed that the concentrations of IL-1β, IL-6, and TNF-α decreased by 57.2%±4.3%, 53.6%±3.9%, and 49.8%±3.5% respectively (p<0.001). TUNEL staining showed that the number of apoptotic cells in the CA1 region of the hippocampus in the siFyn group was 14.3±1.8 cells / field, which was significantly lower than that in the ZsGreen group (32.7±3.2 cells / field) (p<0.001); flow cytometry analysis showed that the proportion of HT22 cells Q2+Q3 in the co-culture system was 15.6%±1.5%, which was significantly lower than that in the OxyHb+ZsGreen group (34.1%±2.9%) (p<0.001). Neurological function scores showed that the siFyn group scored 12.9±0.8 points, significantly higher than the ZsGreen group's 8.2±0.9 points (p<0.001); brain water content was 77.8%±0.6%, significantly lower than the ZsGreen group's 79.6%±0.8% (p<0.001); the entry time into the new arm of the Y maze was 24.5±1.5 s (vs ZsGreen group 12.3±1.1 s), the dwell time in the open field center area was 62.4±3.1 s (vs ZsGreen group 24.5±2.3 s), and the dwell time in the target quadrant of the Morris water maze was 56.3%±2.8% (vs ZsGreen group 38.2%±2.7%). All indicators showed p<0.001. Proteomics LC-MS / MS analysis identified Sirt1 as a key downstream target of Fyn (log2FC=−2.37). qPCR confirmed no change in its mRNA level (p=0.62). COIP verified the formation of the Fyn-C-cbl-Sirt1 ternary complex. K48 ubiquitination antibody detection showed that Sirt1 ubiquitination level decreased by 64.2%±5.3% in the siFyn group (p<0.001).

[0033] The results show that this embodiment successfully constructed a complete chain of evidence for Fyn's regulation of microglial NLRP3 inflammasome activation in early brain injury after SAH. It confirms that Fyn drives Sirt1 to undergo K48-linked ubiquitination and degradation through phosphorylation of C-cbl, thereby relieving its negative regulation of NLRP3 and inducing neuroinflammation and neuronal apoptosis. This system has high reproducibility and verifiability, laying the foundation for further in-depth research on mechanisms and drug intervention.

[0034] Example 2

[0035] Under the same preparation conditions as in Example 1, only the SAH model construction time point specified in this application was adjusted from 24 h to 3 h to obtain the SAH rat model. The results showed that the model could still detect upregulated Fyn protein expression (1.42±0.18 times higher than the sham-operated group, p<0.01), and the band intensities of NLRP3 protein and Caspase-1 p20 increased by 28.3%±2.6% and 31.7%±3.2% respectively (p<0.05), and the number of TUNEL-positive cells was 8.7±1.2 per field of view (p<0.05). This demonstrates that within the time range of 3–72 h, the technical solution of this invention has good time adaptability and stability for detecting dynamic Fyn expression and NLRP3 activation.

[0036] Example 3: Under the same preparation conditions as in Example 1, only the trypsin digestion time specified in this application was adjusted from 8 min to 7 min to obtain primary microglia. The results showed that the purity of the microglia obtained under these conditions was 90.4% ± 1.1% (Iba1 staining), which was not significantly different from that of Example 1 (92.7% ± 0.9%) (p = 0.12). Moreover, the activation degree of NLRP3 after OxyHb stimulation (Caspase-1 p20 band intensity) was 96.3% ± 2.8% of that in Example 1, demonstrating that the technical solution of this invention has good process tolerance for the activity and functional response of primary cells within the digestion time range of 7–10 min.

[0037] Example 4: Under the same preparation conditions as in Example 1, only the siRNA transfection dose specified in this application was adjusted from 100 pmol to 50 pmol to prepare Fyn-interfering BV-2 cells. The results showed that at this dose, the Fyn protein knockdown efficiency was 48.6% ± 3.5% (p < 0.01), and the band intensities of NLRP3 protein and Caspase-1 p20 were reduced by 32.4% ± 2.9% and 38.7% ± 3.1% respectively compared to the control group (p < 0.01). This demonstrates that within the range of 50–100 pmol, the technical solution of this invention has a dose-dependent response to Fyn functional intervention, and the lower limit still achieves a significant biological effect.

[0038] Example 5: With all other preparation conditions the same as in Example 1, only the Western blot molecular weight identification standard defined in this application was adjusted from 118 kDa to 120 kDa (NLRP3 precursor), and protein detection results were obtained. The results showed that the intensity of the 120 kDa band was not significantly different between the SAH group and the siFyn group (p=0.76), while the 118 kDa band showed the expected downward trend (p<0.001), proving that the identification standard of 118 kDa for mature NLRP3 monomers in this invention has molecular specificity, eliminates interference from precursor proteins, and ensures the accuracy of the detection results.

[0039] Example 6: With all other preparation conditions the same as in Example 1, only the drying temperature for brain water content specified in this application was adjusted from 70°C to 65°C to obtain brain tissue dry weight data. The results showed that the difference between the dry weight obtained after drying at 65°C for 48 h and the dry weight under the condition of 70°C was 0.8% ± 0.2% (p = 0.08), and the deviation of the calculated brain water content was 0.3% ± 0.1%, which did not affect the statistical difference between groups (p < 0.001), proving that the present invention's limitation of 70°C as the standard drying temperature has sufficient process robustness.

[0040] Example 7: With all other preparation conditions the same as in Example 1, only the COIP antibody incubation time specified in this application was adjusted from overnight (12 h) to 6 h to obtain the immunoprecipitation product. The results showed that after 6 h of incubation, the Fyn-C-cbl interaction signal intensity was 89.4% ± 3.7% of the overnight group, and the Sirt1 co-precipitation amount was 85.2% ± 4.1% of the overnight group. The ternary complex could still be clearly detected (p < 0.01), proving that overnight incubation is the preferred condition specified in this invention, but 6 h can also meet the basic experimental requirements, demonstrating the practicality of the method.

[0041] Example 8: Under the same preparation conditions as in Example 1, only the in vivo dose of Xol specified in this application was adjusted from 10 mg / kg to 5 mg / kg to prepare Xol-treated SAH mice. The results showed that 5 mg / kg Xol significantly inhibited Fyn protein expression (reduced by 39.7% ± 3.2% compared to the Vehicle group, p < 0.01), NLRP3 activation (Caspase-1 p20 band intensity decreased by 28.4% ± 2.7%, p < 0.05), TUNEL positivity rate decreased to 24.1% ± 2.0% (p < 0.05), and neurological function score increased to 10.8 ± 0.7 points (p < 0.05), demonstrating that the Xol intervention regimen of this invention has a clear dose-response relationship within the dose range of 5–15 mg / kg, and the lower limit still has an observable therapeutic effect.

[0042] Example 9: (Core support for creativity) Table 1 Results of the effect test Sample number Test Project Test results (mean ± SD) Remark SAH+ZsGreen Fyn protein (relative expression) 3.21±0.24 The sham surgery group was set at 1.00. SAH+siFyn Fyn protein (relative expression) 0.87±0.09 It decreased by 73.2% compared to the ZsGreen group. SAH+ZsGreen Caspase-1 p20 (relative strength) 2.83±0.31 The sham surgery group was set at 1.00. SAH+siFyn Caspase-1 p20 (relative strength) 1.08±0.14 61.8% lower than the ZsGreen group SAH+ZsGreen IL-1β (pg / mg prot) 184.2±12.7 SAH+siFyn IL-1β (pg / mg prot) 79.3±6.2 57.2% lower than the ZsGreen group SAH+ZsGreen Number of TUNEL-positive cells ( / field of view) 32.7±3.2 SAH+siFyn Number of TUNEL-positive cells ( / field of view) 14.3±1.8 56.3% lower than the ZsGreen group SAH+ZsGreen Neurological function score (points) 8.2±0.9 Total score 0–18 SAH+siFyn Neurological function score (points) 12.9±0.8 It was 57.3% higher than the ZsGreen group. SAH+ZsGreen Brain water content (%) 79.6±0.8 SAH+siFyn Brain water content (%) 77.8±0.6 2.3% lower than the ZsGreen group Table 2 Characterization Results Sample number Test Project Test results (mean ± SD) Remark SAH+ZsGreen Fyn / Iba1 colocation coefficients 0.82±0.05 Pearson coefficient SAH+siFyn Fyn / Iba1 colocation coefficients 0.35±0.04 It decreased by 57.3% compared to the ZsGreen group. SAH+ZsGreen ASC oligomer spot count ( / field of view) 42.6±4.1 SAH+siFyn ASC oligomer spot count ( / field of view) 13.6±1.7 68.1% lower than the ZsGreen group SAH+ZsGreen Sirt1 K48 ubiquitination (relative strength) 2.94±0.27 The sham surgery group was set at 1.00. SAH+siFyn Sirt1 K48 ubiquitination (relative strength) 1.06±0.12 64.2% lower than the ZsGreen group The comparative proportions were set as follows: ① Blank control group (sham surgery + PBS treatment); ② Samples outside the parameter range of this invention (SAH + 100 pmol scramble siRNA); ③ The closest prior art group (SAH + MCC950, NLRP3 specific inhibitor, 10 mg / kg); ④ Groups lacking function (SAH + AAV-siFyn but without injection, i.e., craniotomy without drug administration). The tests covered physicochemical properties (Fyn protein expression, NLRP3 activation, Sirt1 ubiquitination) and biological properties (neuronal apoptosis, neurological function score, brain water content). The results are shown in Tables 1 and 2: Compared with the sham-operated group, the SAH+ZsGreen group showed significant deterioration in Fyn protein, NLRP3 activation, inflammatory factors, apoptosis rate, cerebral edema, and neurological dysfunction (p<0.001); the scramble siRNA group showed no difference in any of the indicators compared with the ZsGreen group (p>0.05), excluding the non-specific effect of siRNA sequence; although the MCC950 group could inhibit NLRP3 activation (Caspase-1 p20 decreased by 58.3%±3.4%) and apoptosis (TUNEL positive rate 15.2±1.6%), Fyn protein expression was not inhibited (3.18±0.22 times), and Sirt1 ubiquitination level remained unchanged (2.89±0.25 times), proving that its target site is located downstream of NLRP3 and cannot interfere with the upstream Fyn-Sirt1 axis; the Fyn protein and NLRP3 activation levels in the loss-of-function group were consistent with those in the ZsGreen group, confirming that the AAV injection operation itself does not produce an intervention effect. The technical effects of this invention are significant (p<0.001), reproducible (n≥10), and unpredictable: For the first time, it is revealed that Fyn regulates NLRP3 through K48 ubiquitination and selective degradation of Sirt1. This mechanism is different from the known TLR4 / NF-κB or ROS / MAPK pathways, and the Xol intervention effect is highly consistent with siFyn (r=0.96), indicating that targeting Fyn can achieve neuroprotection equivalent to genetic intervention, providing a new target and strategy for SAH treatment.

[0043] Example 10: In this embodiment, the test samples included SAH model animal brain tissue and in vitro co-cultured cells prepared according to each of the embodiments in Examples 1 to 8, and the test results are shown in Tables 1 and 2. The experimental results show that the Fyn functional intervention system (including AAV-siFyn, siRNA and Xol) prepared in this invention can significantly inhibit Fyn expression, reduce NLRP3 inflammasome activation, reduce neuronal apoptosis, and improve cerebral edema and neurological function under their respective parameter conditions (24 h time point, 7–10 min digestion, 50–100 pmol siRNA, 118 kDa recognition, 70℃ drying, overnight COIP incubation, 5–15 mg / kg Xol). Therefore, it can be used to prepare drugs for the prevention and / or treatment of early brain injury caused by subarachnoid hemorrhage.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention / invention, and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention / invention.

Claims

1. A method for functionally investigating Fyn-mediated microglial inflammation in early brain injury induced by subarachnoid hemorrhage, characterized in that, Includes the following steps: 1) Constructing an in vivo model of subarachnoid hemorrhage (SAH): A rat and mouse model of SAH was constructed using vascular intervention. A sham-operated group and an SAH group were set up. The SAH group was divided into groups at different time points. The sham-operated group underwent the same surgical procedure but without vascular puncture. 2) In vitro model construction: Primary microglia and primary neurons were extracted, and primary microglia were treated with hemoglobin (OxyHb) to construct a co-culture system of BV-2 microglia and HT22 neuronal cell lines to simulate the in vitro SAH model. 3) Fyn expression and cell localization detection: The expression level of Fyn protein at different time points in the SAH model was detected by Western blot. Immunohistochemistry was used to detect the expression of Fyn in the hippocampus and cortex. Immunofluorescence staining was used to detect the co-localization of Fyn with microglia (Iba1), neurons (NeuN) and astrocytes (GFAP). 4) Fyn functional intervention experiment: Adeno-associated virus (AAV-siFyn) carrying Fyn interference sequence was constructed and injected into the hippocampus of mice using stereotaxic brain technology. An AAV-ZsGreen control group was set up. In in vivo and in vitro models, siRNA was used to interfere with or overexpress Fyn. 5) Detection of inflammation-related markers: The expression of NLRP3 inflammasome-related proteins such as NLRP3 and Caspase-1p20 was detected by Western blotting, ASC oligomerization was detected by immunofluorescence, and the levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). 6) Apoptosis detection: TUNEL staining was used to detect neuronal apoptosis in vivo, and flow cytometry was used to detect the apoptosis rate of HT22 cells or primary neurons in the co-culture system. 7) Brain injury and functional assessment: The degree of brain injury is assessed by neurological function scores and brain water content measurement. Behavioral experiments such as Y maze, elevated maze, open field test, and Morris water maze are used to assess the animal’s cognitive, learning and exploration functions. 8) Molecular mechanism exploration: downstream regulatory genes of Fyn were screened through proteomics analysis, gene transcription levels were detected by real-time quantitative PCR (QPCR), protein-protein interactions were verified by co-immunoprecipitation (COIP), and ubiquitination modification and degradation pathway of Sirt1 protein were detected. 9) Target reverse screening verification: Xol, a small molecule compound that targets and inhibits Fyn, was screened by molecular docking. The binding ability of Fyn and Xol was detected by cell thermal displacement analysis (CETSA) and drug affinity target stability assay (DARTS). The protective effect of Xol against SAH was evaluated by in vivo and in vitro models. AAV-siFyn reverse genetics was used to evaluate whether Xol depends on Fyn to exert a protective effect against SAH.

2. The research method according to claim 1, characterized in that, The specific procedures for constructing the SAH model in step 1) include: after anesthetizing the experimental animals with an intraperitoneal injection of 100 mg / kg ketamine, a midline incision is made in the neck to separate the bifurcation area of ​​the internal carotid artery, the external carotid artery is double-ligated and the blood flow between the internal carotid artery and the left common carotid artery is temporarily blocked, a puncture is made 3 mm below the ligation point at the distal end of the external carotid artery and a microcatheter is inserted retrogradely to the origin of the middle cerebral artery, the catheter is withdrawn after penetrating the vascular intima to simulate bleeding; the time groups of the SAH rat model include 3h, 6h, 12h, 24h, 48h, and 72h, and 24h is selected as the key time point for subsequent experiments.

3. The research method according to claim 1, characterized in that, Step 2) The specific operations for primary cell extraction and culture include: taking cortical tissue from newborn mice, washing it with pre-cooled PBS, digesting it with 0.125% EDTA-free trypsin at 37°C for 7-10 minutes, and filtering it through a cell sieve to obtain a single-cell suspension; primary neurons are cultured in DMEM medium containing 2% B27, 1% L-glutamine and 1% penicillin antibiotics, and primary microglia are cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin antibiotics; the morphology and purity of primary neurons are identified by NeuN, and the morphology and purity of primary microglia are identified by Iba1 staining.

4. The research method according to claim 1, characterized in that, The specific parameters for Fyn detection in step 3) include: in the Western blot experiment, Fyn target antibody and GAPDH internal reference protein are used to calculate the relative expression level of Fyn; in the immunohistochemical experiment, Fyn target antibody is used, and Fyn-positive cells are observed under a microscope by DAB staining; in the immunofluorescence staining, Fyn target antibody and Iba1 / Neun / GFAP neural markers are used, and Fyn is co-localized with different nerve cells by staining with FITC-labeled goat anti-rabbit secondary antibody and CY3-labeled goat anti-mouse secondary antibody, as well as DAPI staining of the nucleus.

5. The research method according to claim 1, characterized in that, Step 4) includes the following operational parameters for virus injection and cell transfection: AAV injection is performed using a mouse brain stereotaxic instrument to accurately locate the mouse hippocampus, with an injection volume of 0.5 μL per mouse. Interference efficiency is detected after 4 weeks. For in vitro cell transfection, 100 pmol siRNA per well is mixed with 5 μL of liposome transfection reagent. After incubation in serum-free DMEM medium for 5 minutes to form a complex, the complex is added to the cell culture system. After 4 hours of transfection, the medium is replaced with complete medium containing 10% FBS. Interference or overexpression efficiency is detected after 3-5 days of culture.

6. The research method according to claim 1, characterized in that, The specific conditions for detecting inflammatory markers in step 5) include: Western blot detection of a 118kDa NLRP3 target band, Caspase-1p20 detection of a 20kDa target band; immunofluorescence detection of punctate aggregated fluorescence signals as ASC oligomerization signals; and ELISA detection of OD values ​​of IL-1β, IL-6, and TNF-α at an OD wavelength of 450nm.

7. The research method according to claim 1, characterized in that, The specific procedure for apoptosis detection in step 6) includes: during TUNEL staining, frozen sections are fixed with 4% paraformaldehyde and incubated with the reaction solution according to the kit instructions, and apoptotic cells are counted under a fluorescence microscope; during flow cytometry detection, Annexin V-FITC / PI double staining is used, cells are washed with PBS, resuspended in Binding Buffer, and incubated with the fluorescent probe for 1 hour before detection is completed, and the proportion of early apoptosis (Q3) and late apoptosis (Q2) cells is analyzed using FlowJo software.

8. The research method according to claim 1, characterized in that, The specific criteria for brain injury and functional assessment in step 7) include: the neurological function score uses the modified Garcia score, scoring on a scale of 0-3 from 6 dimensions including spontaneous activity, forelimb abduction, and climbing ability, with a total score of 0-18; in the brain water content determination, the brain tissue is weighed after being dried in a 70℃ oven for 48 hours, and the weight is calculated using the formula [(wet weight - dry weight) / wet weight] × 100%; in the behavioral experiments, the Y maze is used to record the time for mice to enter the new arm, the open field test is used to analyze the time spent in the central region and the distance traveled, and the Morris water maze is used to record the time to reach the platform and the time spent in the target quadrant.

9. The research method according to claim 1, characterized in that, The specific methods for exploring the molecular mechanisms in step 8) include: in proteomics analysis, LC-MS / MS technology was used to identify changes in oxyhemoglobin and proteins in microglia treated with siFyn; GSEA and KEGG analyses were used to enrich related pathways; and β-actin was used as an internal reference gene in the qPCR experiment. -ΔΔCt The method calculates the relative expression levels of genes; in the COIP experiment, after cell lysis, Flag or Myc specific antibodies are added and incubated overnight, then agarose beads are added to capture immune complexes, and Western blot is used to detect interacting proteins.

10. The research method according to claim 1, characterized in that, The study also included an Xol intervention experiment, the specific steps of which were: screening Xol targeting Fyn through molecular docking, verifying the binding stability of the two using cell thermal displacement analysis (CETSA) and drug affinity target stability assay (DARTS); setting up different concentrations of Xol treatment groups (5 mg / kg, 10 mg / kg, 15 mg / kg in vivo dose, 2.5 μM, 7.5 μM, 15 μM in vitro dose) in in vivo and in vitro models, detecting the effects of Xol on Fyn expression, NLRP3 inflammasome activation, apoptosis and animal brain function, and clarifying the mechanism of action of Xol targeting Fyn.