Application of ULK1 possibly serving as target spot for treating ischemic stroke

Abstract

The invention belongs to the technical field of ischemic stroke treatment, and discloses application of ULK1 possibly serving as a target spot for treating ischemic stroke. According to the invention, a photothrombotic stroke model is established, a ULK1 inhibitor SBI-0206965 (SBI), LYN1604 hydrochloride (LYN) and a ULK1 agonist are administered, and the ULK1 agonist is used for regulating the activity of ULK1 in vivo. Examples assess the outcome of sensory motor deficits, neuronal apoptosis, and microglia / macrophage activated neurological function. Immunofluorescence detection results show that ULK1 is mainly located in microglial cells in a post-ischemic infarction area of China. Upregulated ULK1 is treated by LYN, so that the infarct volume is remarkably reduced, the motor function is improved, and the increase of inflammatory microglial cells is promoted. In conclusion, the ULK1 promotes the repair of neurons and promotes the formation of 13 paths of anti-inflammatory microglial cell paths after ischemic injury.

Description

Technical Field

[0001] This invention belongs to the field of treatment of ischemic stroke, and particularly relates to the application of ULK1 as a potential target for the treatment of ischemic stroke. Background Technology

[0002] Ischemic stroke is a prevalent brain disease, accounting for over 70% of all cerebrovascular diseases and associated with significant morbidity and mortality. Therefore, research into the cellular and molecular mechanisms of ischemic stroke is crucial. Despite some advances in understanding the pathology, effective clinical treatments for ischemic stroke remain lacking. There is an urgent need to explore and develop key mechanisms for novel therapeutic targets. Ischemic stroke induces a series of complex pathophysiological processes, including neuroinflammation, oxidative stress, excitotoxicity, and hypoxia. Neuroinflammation plays a key role in the development of secondary brain injury following ischemic stroke, primarily through microglia activation. Microglia respond to ischemic brain injury within minutes of an ischemic event. Activated microglia subsequently undergo morphological changes and secrete various substances and cytokines that promote the progression of neuroinflammation. Following cerebral ischemia, microglia release a series of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interleukin-1β (IL-1β), triggering an inflammatory cascade that disrupts the blood-brain barrier (BBB), leading to tissue edema and neuronal death. Conversely, microglia also release anti-inflammatory cytokines, such as growth factor-β (TGF-β) and interleukin-10, to mitigate neuronal damage caused by ischemic stroke. Therefore, microglia exhibit beneficial effects by shifting from a pro-inflammatory to an anti-inflammatory phenotype, making them a potential therapeutic target for ischemic stroke. Autophagy is an important catabolic process crucial for cellular metabolic homeostasis. During stress conditions such as hypoxia, nutrient deficiency, and infection, cells engulf damaged cytoplasmic proteins or organelles, encapsulating them and fusing with lysosomes to form autolysosomes. These autolysosomes degrade their contents, providing nutrients and energy. Recent research has shown that autophagy plays a role in neuronal survival after ischemic brain injury. Unc-51, similar to autophagy-activated kinase 1 (ULK1), a serine / threonine kinase, acts as a central promoter, regulating downstream autophagy flux by recruiting signals from upstream sensors. LC3, p62, and Beclin1 are key downstream proteins of ULK1 in the autophagy process. Induction of autophagy leads to the binding of the diffusely distributed autophagosome marker protein LC3 (LC3-I) to phosphatidylethanolamine, resulting in lipidized LC3 (LC3-II). This autophagy cargo protein p62 is degraded, while another classic autophagy protein, Beclin1, a biomarker, is upregulated during autophagy. Inhibition of ULK1 by knockdown in multiple cells suppresses autophagy. Previous studies have reported that certain drugs can enhance neuronal recovery from ischemic stroke by upregulating ULK1. (Han et al.) It was demonstrated that pharmacological inhibition or suppression of ULK1 expression impairs autophagy activity and eliminates the neuroprotective effect of PPARγcoactivator1α after acute ischemic stroke.However, the role of ULK1 in neuroinflammation and microglial activation after ischemic stroke remains unclear.

[0003] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0004] The neuroprotective and microglial activation effects of ULK1 after ischemic stroke are still unclear. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an application of ULK1 as a potential target for the treatment of ischemic stroke.

[0006] This invention is implemented as follows: an application of ULK1 as a potential therapeutic target for ischemic stroke includes:

[0007] Step 1: Photothrombotic stroke; group and pharmacological treatment; neurobehavioral assessment;

[0008] Step 2, histological tissue processing; Niels staining; TTC staining (for example, TTC staining); TUNEL staining; immunofluorescence staining;

[0009] Step 3: Western blotting; microglia morphology; real-time quantitative PCR; statistical analysis.

[0010] Furthermore, the aforementioned photothrombotic stroke (PT):

[0011] The method for inducing focal cortical photothrombotic ischemic stroke in mice has been described in previous studies; simply put, mice were intraperitoneally injected with 10 mg / ml Rose Bengal (200 μl, Sigma; dissolved in saline); 15 min later, mice were anesthetized and intraperitoneally injected with sodium pentobarbital (80 mg / kg); under anesthesia, the skull was exposed to the cortical region of the forelimb covering the skull; photothrombotic processes in the cortical region of the forelimb were activated (anteroposterior: +0.25 mm; medial and lateral: + relative to +2.5 mm), and a 2 mm diameter cold laser beam (50 mW, 473 nm; technique) was applied to the skull for 15 min; throughout the procedure, the mice's body temperature was maintained at 37°C using a rectal probe; the same procedure was performed on animals undergoing pseudo-ischemic surgery, except for the injection of Rose Bengal.

[0012] Furthermore, the aforementioned group and drug treatment:

[0013] The mice were randomly divided into three groups: the sham group received no cortical PT induction; the vehicle treatment group received ischemic surgery and saline treatment respectively; and the drug treatment group was further divided into: (1) the LYN treatment group treated with LYN1604 hydrochloride; (2) the SBI treatment group treated with SBI-0206965; (3) the 3-MA treatment group treated with 3-methyladenine (3-MA) ​​after PT surgery; and (4) the RAPA treatment group treated with rapamycin (RAPA) after PT surgery. Mice in the vehicle, SBI, LYN, 3-MA and RAPA groups were sacrificed 3 days after PT surgery. The animal experiment diagram and protocol are as follows. Figure 2 As shown in Figure A; 20SBI and LYN were dissolved in normal saline and administered via tail vein at doses of 0.5 mg / kg and 0.1 mg / kg, respectively, 30 min after ischemic stroke; 3-MA (10 mg / kg) was dissolved in DMSO, then diluted 1:10 with normal saline and injected intravenously 30 min after ischemic stroke; RAPA (3 mg / kg) was dissolved in 60% (v / v) diluted DMSO and injected intravenously 30 min after ischemia; SBI, LYN, 3-MA and RAPA were injected 30 min after ischemic stroke.

[0014] 2.4.5-Bromo-2′deoxyuridine (BrdU) administration: To label mitotic cells in the cortical infarct area, labeling was performed in mice in the sham-operated, vector, SBI, and LYN groups. For example, mice were intraperitoneally injected with 50 mg / kg BrdU (Sigma, St. Louis, Missouri, USA) 30 min after ischemia, once daily for 4 consecutive days.

[0015] Furthermore, the neurobehavioral assessment:

[0016] Cylinder and grid walking tests were performed on day 1 prior to ischemic surgery and on days 1 and 3 post-surgery. In the cylinder test, as previously described, the mouse was placed in a glass cylinder (13.6 cm in diameter, 19.9 cm in height) and allowed free walking for 63 min during the test. Two mirrors were positioned at 90° angles behind the cylinder for clear and detailed observation of all behaviors. The number of paw-to-cylinder contact points was recorded for 3 mins during the vertical exploration. Notably, only contact with the load-bearing wall was scored by one or both forelimbs against the wall. Due to significant contralateral muscle weakness caused by damage to the left primary motor cortex, the mouse frequently used the undamaged (left) forelimb for support during spontaneous vertical exploration. The forelimb asymmetry index was calculated as undamaged forelimb contact relative to total forelimb contact. The percentage of left paw contact count / (left paw contact count + right paw contact count + both paw contact count); Simultaneously, a grid walking test was performed to assess motor function; simply put, mice were placed in a 1 cm line grid (27.5 cm in diameter, 22 cm in height) square grid fixed 50 cm above a laboratory table for 1 minute; a camera was placed below the grid to film all the mice's movements; due to left primary motor dysfunction leading to right forelimb muscle weakness and cortical damage, the right forelimb often could not provide support to guide the right paw through a grid hole (called a foot fault); the damaged right forelimb (non-foot fault steps and foot fault steps) was calculated in frame-by-frame video analysis; the foot fault score means the ratio between the number of right foot faults to the total number of right foot faults: right foot faults / (right foot faults + right non-faults).

[0017] Furthermore, the histological tissue processing:

[0018] Following standard procedures, mice in each group were anesthetized with sodium pentobarbital (80 mg / kg, i;p) and perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) via cardiac perfusion, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PB, pH 7.4). The brains of 29 mice were removed, fixed in the same fixative for 12 h, and then transferred to 30% sucrose until they sank. Subsequently, the brains were embedded with OCT and coagulated at -20°C. Thus, each brain was coronally sectioned to a thickness of 30 μm in a cryostat (Leica, Wezlar, Germany).

[0019] Furthermore, the Niels staining method:

[0020] Nissl staining was performed according to published methods; simply put, brain sections were stained in 0.1% cresol violet solution (Sigma-Aldrich, USA) and left at room temperature for 10 min; afterwards, the sections were rinsed with distilled water and dehydrated in 95% and 100% ethanol; finally, the brain sections were cleaned with xylene and coated with Canada balsam (Sigma-Aldrich, USA); morphological measurements of the infarct area of ​​the cresol violet-stained sections were performed using images with Adobe Photoshop (San Jose, USA) and ImageJ software (NIH, USA); the infarct volume was calculated by subtracting the volume of the ipsilateral hemisphere stained with Nissl stain from the contralateral hemisphere [Infarct volume = contralateral hemisphere volume - healthy volume of ipsilateral hemisphere].

[0021] Furthermore, the TTC (2,3,5-triphenyltetrachloride) staining example was performed to observe the lesion size on the 3rd day after ischemic stroke. Simply put, the tissue was directly frozen at -20°C for 20 minutes, and then directly placed in 1% TTC solution and incubated in a 37°C oven away from light for 10-20 minutes, with the tissue gently shaken every 1-2 minutes to ensure uniform staining. After staining, the tissue was fixed with 4% PFA for 5 minutes, and then the fixed tissue photograph was stored in the dark and taken by a camera.

[0022] Furthermore, the TUNEL staining:

[0023] Dual fluorescence staining was used to detect neuronal apoptosis 3 days after ischemic injury using NeuN (a neuronal marker) and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in mouse cortex after PT. The TUNEL kit (Roche) was used according to the manufacturer's instructions for the detection day. For NeuN immunofluorescence staining, as previously described, tissues were treated with mouse anti-Neun antibody (1:800, ab104224, Abcam, USA) and incubated with donkey anti-mouse Alexa Fluor 594 (1:1000, 715-585150, Jackson Center for Immunology, USA). 29 images were observed using a Nikon microscope (Li2). Data were calculated as TUNEL-numbers, with 1 positive neuron per millimeter in the selected field.

[0024] Furthermore, the immunofluorescence staining:

[0025] Immunofluorescence staining was performed according to previous studies; in short, each tissue section was fixed for 30 min, then retrieval with sodium citrate antigen at 96°C for 10 min using extract (C1032, Solarbio). The tissue sections were treated with primary antibodies, including mouse anti-NEUAN antibody (1:800, ab104224, Abcam, USA), mouse anti-GFAP (1:500, 8#3670, Cell Signaling Technologies, MA, USA), goat anti-IBA1 (1:500, ab5076, Abcam, USA), rabbit anti-IBA1 (1:500, ab178847, Abcam, USA), and rabbit anti-ULK1. (1:200, #8054, Cell Signaling Technologies, MA, USA), rabbit anti-brdu (1:200, ab6326, Abcam, USA), mouse anti-iNOS11 (1:1000, ab49999, USA), goat anti-cd206 (1:500, AF2535, R&D Systems, USA), rabbit anti-LC3A / B (1:500, #4108, Cell Signaling Technologies, USA), mouse anti-p62 (1:200, ab56416, Abcam, USA) were incubated overnight at 4°C; then the tissues were immersed in secondary antibodies including donkey anti-rabbit Alexa Fluor 488 (1:1000, #8054, Cell Signaling Technologies, MA, USA). 711-545152, Jackson Institute for Immunology, USA), Donkey anti-mouse AlexaFluor488 (1:1000, 715-545150, Jackson Institute for Immunology, USA), Donkey anti-rabbit AlexaFluor594 (1:1000, 711-585152, Jackson Institute for Immunology, USA), Donkey anti-mouse AlexaFluor594 (1:1000, 715-585150, Jackson Institute for Immunology, USA), Donkey anti-goat AlexaFluor59418 (1:1000, 705-585-003, Jackson Institute for Immunology, USA), in the laboratory The samples were placed at room temperature for 2 hours; images were obtained using a confocal microscope (TCSSP8, Lecia) or a microscope (Li2, Nikon) and analyzed using Photoshop (Adobe); all measurements were performed under blinded conditions by two observers for each experiment, with 22 samples under the same conditions; in addition, the mean cell count was calculated from at least 3 microscopic fields of view in the region of interest (ROI), 3 parts of each brain, and at least 3 animals per group of 24 people; data are expressed as mean cell count per square millimeter; 25 cases of the peri-infarcted region of the cerebral cortex were selected as ROIs to assess neuronal damage after ischemic stroke.

[0026] Furthermore, the Western blot method:

[0027] Western blotting was performed according to the published paper; mice were anesthetized with sodium pentobarbital (80 mg / kg, i; 3 days after ischemic injury); after deep anesthesia, the entire brain of the mice was removed as soon as possible; the peri-infarct area was immediately excised with a scalpel under magnification; the tissue was homogenized and centrifuged, and the supernatant was used for immunoblotting analysis; the following immunoblots were incubated with primary antibodies, including rabbit anti-ulk1 (1:1000, #8054, Cell Signaling Technologies, USA), rabbit... Anti-iba1 (1:500, ab178847, Abcam, USA), rabbit anti-LC3A / B (1:1000, #4108, Cell Signaling Technologies, MC., MA, USA), mouse anti-p62 (1:1000, ab56416, Abcam, USA), rabbit anti-il1β (1:1000, ab2105, Abcam, USA), rabbit anti-TGFβ (1:1000, ab92486, Abcam, USA), rabbit anti-TNF -α (1:1000, ab6671, Abcam, USA), rabbit anti-il10 (1:1000, 9#12163, Cell Signaling Technologies, MA, USA) and mouse anti-β-actin (1:3000, ab8226, Abcam, USA) were incubated overnight at 4°C; the next day, the blots were reacted with secondary antibodies, including goat anti-antibody, to rabbit IgG (1:5000, ab6721, Abcam, USA) and goat anti-mouse IgG (1:5000, ab6671, Abcam, USA). (ab6789, Abcam, USA) was incubated at room temperature for 2 hours for 12 hours; finally, it was detected using the Omni-ECL assay kit (EpiZyme, China); Western blot analysis was performed using the published method; in short, scanning was performed using a chemical microscope touch (Clinx, China), and density analysis was performed using Image software (NIH, USA) to quantify the bands; the ratio of the bars was calibrated to %, and the sham-operated group was specified as 100%; β-actin was used as an internal control;

[0028] The microglia morphology described:

[0029] Microglial morphology was used as a basis for previous studies, with minor modifications. In a brief overview, 30 μm tissue sections were stained with Iba1 and images were prepared using a confocal microscope with Z-pillar imaging. In each group, there were 56–84 Iba1+ microglia (at least 3 cells per section, 3 cells per mouse per section, 7 mice per group), which were analyzed by researchers without considering grouping. The researchers determined the longest distance across the cell body, setting the first radius to 1 μm to begin the Sholl analysis; subsequently, the researchers defined the radius as intervals of 5 μm, and the results were calculated using ImageJ with the Sholl analysis plugin. The calculation is as follows: The convex hull contains the entire process of each microglia, and a circle defines the convex hull further; the hull and circle plugins are defined by ImageJ, with hull and circle plugins; the demonstration results include: the cell area is the total number of pixels of iba1 positive cells, the robustness is the ratio of the cell area to the total area of ​​the convex area (the convex area is the total number of pixels in the convex hull of the image, which includes a polygon connecting the end processes), the roundness value to 1 means a perfect circle, and the intersections represent the intersections of microglia processes with concentric circles; the above three data points were collected using a confocal microscope (Olympus, Japan);

[0030] The real-time quantitative PCR (RT-qPCR):

[0031] Three days after PT, total RNA was extracted from brain tissue using a tissue RNA extraction kit 7 (Qiagen, Hilden, Germany). RNA was reverse extracted and transcribed into cDNA using a HiScript III qRT SuperMix (Vazyme, China). Real-time quantitative polymerase chain reaction (qPCR) was performed using a ChamQ universal SYBR qPCR MasterMix 10 (Vazyme). Data collection was performed on an RT-PCR system (Applied Biosystems, USA). Sample 11 was independently amplified at least three times. Relative gene expression levels were compared to GAPDH using the 2-12ΔΔCt method. Primers are listed in the supplementary file: Table S1.

[0032] The statistical analysis is as follows:

[0033] Data are expressed as mean ± standard deviation; Student's t-test was used for comparisons between two groups of 16 independent samples with normal distributions; for multiple comparisons, one-way ANOVA was performed followed by a post-hoc Bonferroni test; for comparing changes from different levels of multiple categorical variables, a two-way ANOVA was performed using a post-hoc Bonferroni test; statistical analysis was performed using GraphPad Prilm (USA); statistical significance was considered at P < 0.05 (attached file: Figures S1C-E); an animal model of ischemic stroke was established using photochemical embolization (attached file: Figure S2A); the lesion area was located in the S1FL region, and mice developed sensorimotor dysfunction opposite the upper limb cortical infarction after PT (attached file: Figure S2B); 4. Western blot was used to detect the expression level of ULK1 in the five areas surrounding the cortical infarction and the infarct core at different stages after ischemic stroke; it was found that ULK1 expression increased significantly 6 times 1 day after ischemic injury, and reached its peak 3 to 5 days after 7 ischemic injuries (attached file: Figure S2B); Figure 1 A, B), whose expression trend was similar to that of the infarct area and the infarct area after ischemia, and behavioral and functional degeneration (attached file: Figure S2C-I); in order to further investigate the changes in ULK1 localization in the lesion area after ischemic injury, double 10 immunofluorescence staining was performed to mark the cellular expression of ULK1; 11 increased ULK1 expression was detected, mainly located in Iba1+ microglia in the peri-infarct area 123 days after ischemic injury ( Figure 1 CE); The above results indicate that ULK113 is significantly increased in the early stage of ischemic stroke, mainly located in microglia around the infarct, and may be involved in the regulation of neuronal death. Figure 1 ; Expression of ULK1 in the cerebral cortex after ischemic stroke; A and B represent the quantitative analysis of ULK1 protein levels at different time points after ischemic injury using blot strips; n3 = 4. *p < 0.05, p < 0.01 vs; sham group; C Schematic diagram of the infarct area induced by PT; D ULK1 (red) with neurons (NeuN, green), astrocytes (GFAP), and microglia in the peri-infarct area 3 days after ischemic injury (Iba1, green); nuclei are stained with DAPI (blue); arrows: cells with common labels; scale bar = 100 μm, 50 μm; E Quantitative analysis of ULK1 in cells of + / NeuN+, ULK1+ / GFAP+, and ULK1+ / Iba1+; n = 5. *p < 0.01 vs; ULK1 count in + / NeuN+ cells &&p < 0.01 vs; ULK1 count in + / GFAP+ cells.

[0034] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0035] First, this invention established a photothrombotic stroke model and administered the ULK1 inhibitor SBI-0206965 (SBI) and LYN1604 hydrochloride (LYN), a ULK1 agonist, to regulate ULK1 activity in vivo. The results were used to evaluate sensorimotor deficits, neuronal apoptosis, and microglia / macrophage activation of neuronal function. Immunofluorescence assays showed that ULK1 is primarily localized in microglia in the infarcted region after ischemic stroke. LYN treatment upregulated ULK1, significantly reducing infarct volume, improving motor function, and promoting the increase of anti-inflammatory microglia. In summary, ULK1 promotes neuronal repair and facilitates the formation of 13 pathways related to anti-inflammatory microglia after ischemic injury.

[0036] Second, this invention established a photothrombotic stroke model and administered the ULK1 inhibitor SBI-0206965 (SBI) and LYN1604 hydrochloride (LYN), a ULK1 agonist, to regulate ULK1 activity in vivo. The results were used to assess sensorimotor deficits, neuronal apoptosis, and microglia / macrophage activation of neuronal function. Immunofluorescence assays showed that ULK1 is primarily localized in microglia in the infarcted region after ischemic stroke. LYN treatment upregulated ULK1, significantly reducing infarct volume, improving motor function, and promoting the increase of anti-inflammatory microglia. In summary, ULK1 promotes neuronal repair and facilitates the formation of 13 pathways related to anti-inflammatory microglia after ischemic injury.

[0037] Third, based on the information you provided, the following is an analysis of the application of ULK1 as a therapeutic target for ischemic stroke and its significant technological advancements:

[0038] Application steps overview:

[0039] 1) Thrombotic stroke and related management:

[0040] Focal cortical photothrombotic ischemic stroke was induced in mice using a photothrombotic stroke (PT) model.

[0041] Mice were injected with Rose Bengal, then anesthetized and their skulls were exposed to activate photothrombosis processes in the cortical region of the forelimb.

[0042] Lasers are applied to specific areas of the skull to induce thrombosis.

[0043] The mice's body temperature was kept constant during the surgery.

[0044] 2) Histological treatment and staining:

[0045] Processing tissue samples may include steps such as fixation and sectioning.

[0046] Methods such as Nissl staining, TTC staining, TUNEL staining, and immunofluorescence staining were used to observe and evaluate tissue damage, cell death, and the expression of specific proteins.

[0047] 3) Protein analysis and gene expression assay:

[0048] Western blotting was used to detect the expression levels of specific proteins, such as ULK1.

[0049] Assessing morphological changes in microglia may be associated with the inflammatory response in ischemic stroke.

[0050] We used real-time quantitative PCR to determine gene expression levels and understand the regulatory role of ULK1 and related genes in ischemic stroke.

[0051] 4) Statistical analysis:

[0052] Statistical analysis was performed on the collected data to verify the reliability and significance of the experimental results.

[0053] Significant technological advancements:

[0054] 1) Model establishment: A photothrombotic stroke model was successfully established. This model can simulate the pathological process of human ischemic stroke, providing a solid foundation for the study of ULK1 as a therapeutic target.

[0055] 2) Multimodal staining technique: Combining multiple histological staining methods, it can comprehensively assess pathological changes in brain tissue, such as brain tissue damage, apoptosis, and inflammatory response.

[0056] 3) Molecular level analysis: The mechanism of action of ULK1 in ischemic stroke was explored in depth at the molecular level using techniques such as Western blotting and real-time quantitative PCR.

[0057] 4) Refined surgical procedures: The body temperature of the mice was precisely controlled during the operation, which reduced the impact of the operation on the experimental results and improved the accuracy and reproducibility of the experiment.

[0058] 5) Comprehensive assessment system: A comprehensive assessment system including neurobehavioral assessment, histological staining, protein analysis and gene expression assay has been established, which provides strong support for comprehensively evaluating the potential of ULK1 as a therapeutic target for ischemic stroke. Attached Figure Description

[0059] Figure 1 This is a flowchart illustrating the potential application of ULK1 as a target for treating ischemic stroke, as provided in this embodiment of the invention.

[0060] Figure 2 This is an embodiment of the invention showing the expression of ULK1 in the cerebral cortex after ischemic stroke. A and B represent the quantitative analysis of ULK1 protein levels at different time points after ischemic injury using Western blot strips. n3 = 4. *p < 0.05, p < 0.01 and sham group. C is a schematic diagram of the infarct area induced by PT. D ULK1 (red) is associated with neurons (NeuN, green), astrocytes (GFAP), and microglia in the peri-infarct region (Iba1, green) 3 days after ischemic injury. The nucleus is stained with DAPI (blue). Arrows: cells with common markers. Scale bar = 100 μm, 50 μm. E Quantitative analysis of ULK1 in cells of + / NeuN+, ULK1+ / GFAP+, and ULK1+ / Iba1+. n = 5. *p < 0.01 vs. ULK1 count in + / NeuN+ cells and *p < 0.01 vs. ULK1 count in + / GFAP+ cells.

[0061] Figure 3 This invention provides the neuroprotective effect of ULK1 after ischemic stroke. The experimental protocol is illustrated in the animal diagram. Induced photothrombotic stroke was performed, and mice were sacrificed on day 3 after the stroke. Representative images of BNissl staining and TTC staining show one infarct area per group. Scale bar = 500 μm, 1 mm. C. Quantitative analysis of the ischemic lesion volume in each group of patients was performed. n = 4-6. *p < 0.05, p < 0.01. Representative image of DTUNEL co-localization (green). Neurons (NeuN, red) are present in the peri-infarct area after ischemic injury; arrows point to neurons with combined TUNEL / NeuN. Scale bar = 50 μm. Quantitative analysis of DTUNEL + / NeuN+ cells in 5 individuals per group. n = 9. p < 0.01. Quantitative analysis of the behavior of mice in groups F and G on day 1: before ischemic injury, 1 day after injury, and 3 days after injury, cylinder test (F), grid walking test (G). n = 3. * p < 70.001, p < 0.0001 vs. . Drug-loaded group 1 day after ischemia; & p < 0.05, &&&&p < 0.0001 vs. . Vehicle group 3 days after ischemia.

[0062] Figure 4This invention provides the effect of ULK1 on the proliferation and morphology of microglia / macrophages after ischemia. a) Representative images showing the co-localization of proliferating cells (BrdU, green) and microglia (Iba1, red), and schematic diagrams of different microglia morphologies after different treatments. Arrows point to cells where BrdU / Iba1 are combined. B) Quantitative analysis of BrdU+ / Iba1+ cells and cells (1 mm²) in each region of Iba1+. n = 8. *p < 0.05, *p < 0.001, p < 0.0001. D) Patterns for shell, hull, and circular analysis. E) Cell morphological characteristics analysis results: cell area / perimeter, relative robustness, roundness, and mean.

[0063] Figure 5 This invention illustrates the role of ULK1 in ischemic injury-induced microglial activation in an inflammatory phenotype. A, 3B: Representative images of microglia (Iba1, green) co-localized with pro-inflammatory markers (iNOS, red; arrows pointing to iNOS / iba1 combined cells) and anti-inflammatory markers (CD206, red; two arrows pointing to CD206 / iba1 combined cells). Scale bar = 100 μm, 50 μm. C, Quantitative analysis of Iba1: Cells (1 mm²) in each region of Iba1+ / iNOS+ cells and Iba1+ / CD206+ cells. n = 8. E-G4 pro-inflammatory mRNA levels, including CD86 (E), CD32 (F), and iNOS5 (G), were assessed. HJ anti-inflammatory mRNA levels, including CD206 (H), YM1 / 2 (I), and Arg1 (J), were assessed. n = 6. *p < 0.05, p < 0.01, *p < 0.001, p < 0.0001; ns, not significant. Intersection point. n = 7. I and J represent the protein blot bands for quantitative analysis. n = 5. *p < 0.05, p < 0.01; ns, not significant.

[0064] Figure 6 This invention relates to the effect of ULK1 on the secretion of inflammatory factors induced by ischemic injury. Representative blot bands from AC strips were used to quantitatively analyze the IL1β and TNF-α protein levels in four groups. Representative blot bands from DF strips were analyzed at five levels for quantitative analysis of TGFβ and IL10 proteins. n=5. The H mRNA levels of G, IL1β (G), and TNF-α (H) were detected. The mRNA levels of I and J were detected in 6 cases of TGFβ (I) and IL10 (J) in each group. n=4-6. *p<0.05, p<0.01, *p<0.001, 7p<0.0001; ns, not significant.

[0065] Figure 7This invention relates to the effect of ULK1 on autophagy after ischemic injury. Representative immunoblot bands 21 were used to quantitatively analyze the protein levels of LC3, p62, and Beclin1 in each group. n = 5-7. E. Representative images of co-localization of autophagosomes (LC3, green) and microglia (Iba1, red) in each group (3 images per group). Arrows point to cells where LC3 / Iba1 are combined. Scale bar = 50 μm. F. Representative images of co-localization of autophagy (p62, green) and microglia (Iba1, red) in each group, and arrows at 5 points where p62 / Iba1 are combined. Scale bar = 50 μm. G. Quantitative analysis of HLC3+ / Iba1+ cells (6 cells) and P62+ / Iba1+ cells (cells in each region, 1 mm²). n = 8. *p<0.05, p<0.01, *p<0.001, 7p<0.0001 vs. . Vehicle group. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0067] Based on the application of ULK1 as a therapeutic target for ischemic stroke, the following are two specific implementation examples and their solutions:

[0068] Example 1: Study on the role of ULK1 inhibitors in the treatment of ischemic stroke

[0069] 1) Establishment of a photothrombotic stroke model:

[0070] Focal cortical ischemic stroke was induced in mice using the above-described photothrombotic stroke method.

[0071] 2) Drug treatment:

[0072] One group of mice received ULK1 inhibitor treatment immediately after ischemic stroke; the other group of mice served as a control group and received an equal amount of saline.

[0073] 3) Neurobehavioral assessment:

[0074] Neurobehavioral assessments were performed on mice at different time points after treatment to evaluate the recovery of neurological function.

[0075] 4) Histological analysis and Western blotting:

[0076] Niels staining, TTC staining, TUNEL staining, and immunofluorescence staining were performed to assess brain tissue damage and apoptosis.

[0077] The expression of ULK1-related proteins was detected using Western blotting.

[0078] Example 2: The role of ULK1 in microglial activation after ischemic stroke

[0079] 1) Establishment of a photothrombotic stroke model:

[0080] Similar to the above embodiments, focal cortical ischemic stroke was induced in mice.

[0081] 2) Observation of microglia:

[0082] Brain tissue samples were collected at different time points, and immunofluorescence staining was used to observe the activation and morphological changes of microglia.

[0083] 3) Molecular biological analysis:

[0084] We used real-time quantitative PCR to detect gene expression associated with microglial cell activation.

[0085] Statistical analysis was performed to evaluate the role of ULK1 in microglia activation.

[0086] These two examples will contribute to a better understanding of the potential role of ULK1 in the treatment of ischemic stroke, and its impact on microglial activation and neuroinflammatory processes after stroke. These findings may be significant for developing new treatments for ischemic stroke, improving treatment outcomes, reducing neurological impairment, and providing new targets for clinical treatment of ischemic stroke. These examples also allow for a more comprehensive evaluation of the efficacy and safety of ULK1 inhibitors, laying the foundation for future clinical applications.

[0087] To enable those skilled in the art to fully understand how the present invention is specifically implemented, this section provides an explanatory description of the embodiments that expand upon the technical solutions of the claims.

[0088] Materials and methods:

[0089] Twenty-one C57BL / 6 mice (8 weeks old, 25-30g) were provided and cared for at the Laboratory Animal Center of Wenzhou Medical University. All experiments were conducted according to arrival guidelines. The mice were housed in a constant temperature (20-24℃), humidity (approximately 50-70%), and 24-hour light (lights on from 07:00 to 19:00). The experimental protocol was approved by the Animal Care and Use Committee of Wenzhou Medical University, China.

[0090] like Figure 1 As shown, the present invention provides an application of ULK1 as a potential target for treating ischemic stroke, comprising the following steps:

[0091] S101, photothrombotic stroke; group and pharmacological treatment; neurobehavioral assessment;

[0092] S102, Histological tissue processing; Niels staining; TTC staining; TUNEL staining; Immunofluorescence staining;

[0093] S103, Western blotting; microglia morphology; real-time quantitative PCR; statistical analysis.

[0094] The photothrombotic stroke (PT) provided by this invention:

[0095] The method for inducing focal cortical photothrombotic ischemic stroke in mice has been described in previous studies; simply put, mice were intraperitoneally injected with 10 mg / ml Rose Bengal (200 μl, Sigma; dissolved in saline); 15 min later, mice were anesthetized and intraperitoneally injected with sodium pentobarbital (80 mg / kg); under anesthesia, the skull was exposed to the cortical region of the forelimb covering the skull; photothrombotic processes in the cortical region of the forelimb were activated (anteroposterior: +0.25 mm; medial and lateral: + relative to +2.5 mm), and a 2 mm diameter cold laser beam (50 mW, 473 nm; technique) was applied to the skull for 15 min; throughout the procedure, the mice's body temperature was maintained at 37°C using a rectal probe; the same procedure was performed on animals undergoing pseudo-ischemic surgery, except for the injection of Rose Bengal.

[0096] The population and drug treatments provided by this invention:

[0097] The mice were randomly divided into three groups: the sham group received no cortical PT induction; the vehicle treatment group received ischemic surgery and saline treatment respectively; and the drug treatment group was further divided into: (1) the LYN treatment group treated with LYN1604 hydrochloride; (2) the SBI treatment group treated with SBI-0206965; (3) the 3-MA treatment group treated with 3-methyladenine (3-MA) ​​after PT surgery; and (4) the RAPA treatment group treated with rapamycin (RAPA) after PT surgery. Mice in the vehicle, SBI, LYN, 3-MA and RAPA groups were sacrificed 3 days after PT surgery. The animal experiment diagram and protocol are as follows. Figure 2As shown in Figure A; 20SBI and LYN were dissolved in normal saline and administered via tail vein at doses of 0.5 mg / kg and 0.1 mg / kg, respectively, 30 min after ischemic stroke; 3-MA (10 mg / kg) was dissolved in DMSO, then diluted 1:10 with normal saline and injected intravenously 30 min after ischemic stroke; RAPA (3 mg / kg) was dissolved in 60% (v / v) diluted DMSO and injected intravenously 30 min after ischemia; SBI, LYN, 3-MA and RAPA were injected 30 min after ischemic stroke.

[0098] 2.4.5-Bromo-2′deoxyuridine (BrdU) administration: To label mitotic cells in the cortical infarct area, labeling was performed in mice in the sham-operated, vector, SBI, and LYN groups. For example, mice were intraperitoneally injected with 50 mg / kg BrdU (Sigma, St. Louis, Missouri, USA) 30 min after ischemia, once daily for 4 consecutive days.

[0099] The neurobehavioral assessment provided by this invention:

[0100] Cylinder and grid walking tests were performed on day 1 prior to ischemic surgery and on days 1 and 3 post-surgery. In the cylinder test, as previously described, the mouse was placed in a glass cylinder (13.6 cm in diameter, 19.9 cm in height) and allowed free walking for 63 min during the test. Two mirrors were positioned at the 90-degree angles behind the cylinder for clear and detailed observation of all behaviors. The number of paw-to-cylinder contact points was recorded for 3 mins during the vertical exploration. Notably, only contact with the load-bearing wall was scored by one or both forelimbs against the wall. Due to significant contralateral muscle weakness caused by damage to the left primary motor cortex, the mouse frequently used the undamaged (left) forelimb for support during spontaneous vertical exploration. The forelimb asymmetry index was calculated as the ratio of undamaged forelimb contact to total forelimb contact. Percentage of left paw contact number / (left paw contact number + right paw contact number + both paw contact number); Simultaneously, a grid walking test was performed to assess motor function. Simply put, mice were placed in a 1 cm line grid (27.5 cm in diameter, 22 cm in height) with the square grid fixed 50 cm above the lab bench for 1 min; a camera was placed under the grid to record all the mice's movements; due to left primary motor damage leading to right forelimb muscle weakness and cortical injury, the right forelimb often could not provide support to guide the right paw through a grid hole (called foot fault); the damaged right forelimb (non-foot fault steps and foot fault steps) was calculated in the frame-by-frame analysis of the video; the foot fault score means the ratio between the number of right foot faults and the total number of right foot faults 23 [right foot faults / (right foot faults + right non-faults].

[0101] The histological tissue processing provided by this invention:

[0102] Following standard procedures, mice in each group were anesthetized with sodium pentobarbital (80 mg / kg, i;p) and perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) via cardiac perfusion, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PB, pH 7.4). The brains of 29 mice were removed, fixed in the same fixative for 12 h, and then transferred to 30% sucrose until they sank. Subsequently, the brains were embedded with OCT and coagulated at -20°C. Thus, each brain was coronally sectioned to a thickness of 30 μm in a cryostat (Leica, Wezlar, Germany).

[0103] The Nissell staining method provided by this invention:

[0104] Nissl staining was performed according to published methods; simply put, brain sections were stained in 0.1% cresol violet solution (Sigma-Aldrich, USA) and left at room temperature for 10 min; afterwards, the sections were rinsed with distilled water and dehydrated in 95% and 100% ethanol; finally, the brain sections were cleaned with xylene and coated with Canada balsam (Sigma-Aldrich, USA); morphological measurements of the infarct area of ​​the cresol violet-stained sections were performed using images with Adobe Photoshop (San Jose, USA) and ImageJ software (NIH, USA); the infarct volume was calculated by subtracting the volume of the ipsilateral hemisphere stained with Nissl stain from the contralateral hemisphere [Infarct volume = contralateral hemisphere volume - healthy volume for ipsilateral hemisphere].

[0105] The TTC (2,3,5-triphenyltetrachloride) staining example provided by this invention was used to observe the size of lesions on the 3rd day after ischemic stroke. Simply put, the tissue was directly frozen at -20°C for 20 minutes, and then directly placed in a 1% TTC solution and incubated in a 37°C oven away from light for 10-20 minutes, with the tissue gently shaken every 1-2 minutes to ensure uniform staining. After staining, the tissue was fixed with 4% PFA for 5 minutes, and then the fixed tissue photograph was stored in the dark and taken by a camera.

[0106] The TUNEL staining provided by this invention:

[0107] Dual fluorescence staining was used to detect neuronal apoptosis 3 days after ischemic injury using NeuN (a neuronal marker) and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) in mouse cortex after PT. The TUNEL kit (Roche) was used according to the manufacturer's instructions for the detection day. For NeuN immunofluorescence staining, as previously described, tissues were treated with mouse anti-Neun antibody (1:800, ab104224, Abcam, USA) and incubated with donkey anti-mouse Alexa Fluor 594 (1:1000, 715-585150, Jackson Center for Immunology, USA). 29 images were observed using a Nikon microscope (Li2). Data were calculated as TUNEL-numbers, with 1 positive neuron per millimeter in the selected field.

[0108] The immunofluorescence staining provided by this invention:

[0109] Immunofluorescence staining was performed according to previous studies; in short, each tissue section was fixed for 30 min, then retrieval with sodium citrate antigen at 96°C for 10 min using extract (C1032, Solarbio). The tissue sections were treated with primary antibodies, including mouse anti-NEUAN antibody (1:800, ab104224, Abcam, USA), mouse anti-GFAP (1:500, 8#3670, Cell Signaling Technologies, MA, USA), goat anti-IBA1 (1:500, ab5076, Abcam, USA), rabbit anti-IBA1 (1:500, ab178847, Abcam, USA), and rabbit anti-ULK1. (1:200, #8054, Cell Signaling Technologies, MA, USA), rabbit anti-brdu (1:200, ab6326, Abcam, USA), mouse anti-iNOS11 (1:1000, ab49999, USA), goat anti-cd206 (1:500, AF2535, R&D Systems, USA), rabbit anti-LC3A / B (1:500, #4108, Cell Signaling Technologies, USA), mouse anti-p62 (1:200, ab56416, Abcam, USA) were incubated overnight at 4°C; then the tissues were immersed in secondary antibodies including donkey anti-rabbit Alexa Fluor 488 (1:1000, #8054, Cell Signaling Technologies, MA, USA). 711-545152, Jackson Institute for Immunology, USA), Donkey anti-mouse AlexaFluor488 (1:1000, 715-545150, Jackson Institute for Immunology, USA), Donkey anti-rabbit AlexaFluor594 (1:1000, 711-585152, Jackson Institute for Immunology, USA), Donkey anti-mouse AlexaFluor594 (1:1000, 715-585150, Jackson Institute for Immunology, USA), Donkey anti-goat AlexaFluor59418 (1:1000, 705-585-003, Jackson Institute for Immunology, USA), in the laboratory The samples were placed at room temperature for 2 hours; images were obtained using a confocal microscope (TCSSP8, Lecia) or a microscope (Li2, Nikon) and analyzed using Photoshop (Adobe); all measurements were performed under blinded conditions by two observers for each experiment, with 22 samples under the same conditions; in addition, the mean cell count was calculated from at least 3 microscopic fields of view in the region of interest (ROI), 3 parts of each brain, and at least 3 animals per group of 24 people; data are expressed as mean cell count per square millimeter; 25 cases of the peri-infarcted region of the cerebral cortex were selected as ROIs to assess neuronal damage after ischemic stroke.

[0110] The protein blotting method provided by this invention:

[0111] Western blotting was performed according to the published paper; mice were anesthetized with sodium pentobarbital (80 mg / kg, i; 3 days after ischemic injury); after deep anesthesia, the entire brain of the mice was removed as soon as possible; the peri-infarct area was immediately excised with a scalpel under magnification; the tissue was homogenized and centrifuged, and the supernatant was used for immunoblotting analysis; the following immunoblots were incubated with primary antibodies, including rabbit anti-ulk1 (1:1000, #8054, Cell Signaling Technologies, USA), rabbit... Anti-iba1 (1:500, ab178847, Abcam, USA), rabbit anti-LC3A / B (1:1000, #4108, Cell Signaling Technologies, MC., MA, USA), mouse anti-p62 (1:1000, ab56416, Abcam, USA), rabbit anti-il1β (1:1000, ab2105, Abcam, USA), rabbit anti-TGFβ (1:1000, ab92486, Abcam, USA), rabbit anti-TNF -α (1:1000, ab6671, Abcam, USA), rabbit anti-il10 (1:1000, 9#12163, Cell Signaling Technologies, MA, USA) and mouse anti-β-actin (1:3000, ab8226, Abcam, USA) were incubated overnight at 4°C; the next day, the blots were reacted with secondary antibodies, including goat anti-antibody, to rabbit IgG (1:5000, ab6721, Abcam, USA) and goat anti-mouse IgG (1:5000, ab6671, Abcam, USA). (ab6789, Abcam, USA) was incubated at room temperature for 2 hours for 12 hours; finally, it was detected using the Omni-ECL assay kit (EpiZyme, China); Western blot analysis was performed using the published method; in short, scanning was performed using a chemical microscope touch (Clinx, China), and density analysis was performed using Image software (NIH, USA) to quantify the bands; the ratio of the bars was calibrated to %, and the sham-operated group was specified as 100%; β-actin was used as an internal control;

[0112] The microglia morphology described:

[0113] Microglial morphology was used as a basis for previous studies, with minor modifications. In a brief overview, 30 μm tissue sections were stained with Iba1 and images were prepared using a confocal microscope with Z-pillar imaging. In each group, there were 56–84 Iba1+ microglia (at least 3 cells per section, 3 cells per mouse per section, 7 mice per group), which were analyzed by researchers without considering grouping. The researchers determined the longest distance across the cell body, setting the first radius to 1 μm to begin the Sholl analysis; subsequently, the researchers defined the radius as intervals of 5 μm, and the results were calculated using ImageJ with the Sholl analysis plugin. The calculation is as follows: The convex hull contains the entire process of each microglia, and a circle defines the convex hull further; the hull and circle plugins are defined by ImageJ, with hull and circle plugins; the demonstration results include: the cell area is the total number of pixels of iba1 positive cells, the robustness is the ratio of the cell area to the total area of ​​the convex area (the convex area is the total number of pixels in the convex hull of the image, which includes a polygon connecting the end processes), the roundness value to 1 means a perfect circle, and the intersections represent the intersections of microglia processes with concentric circles; the above three data points were collected using a confocal microscope (Olympus, Japan);

[0114] The real-time quantitative PCR (RT-qPCR):

[0115] Three days after PT, total RNA was extracted from brain tissue using a tissue RNA extraction kit 7 (Qiagen, Hilden, Germany). RNA was reverse extracted and transcribed into cDNA using a HiScript III qRT SuperMix (Vazyme, China). Real-time quantitative polymerase chain reaction (qPCR) was performed using a ChamQ universal SYBR qPCR MasterMix 10 (Vazyme). Data collection was performed on an RT-PCR system (Applied Biosystems, USA). Sample 11 was independently amplified at least three times. Relative gene expression levels were compared to GAPDH using the 2-12ΔΔCt method. Primers are listed in the supplementary file: Table S1.

[0116] The statistical analysis is as follows:

[0117] Data are expressed as mean ± standard deviation; Student's t-test was used for comparisons between two groups of 16 independent samples with normal distributions; for multiple comparisons, one-way ANOVA was performed followed by a post-hoc Bonferroni test; for comparing changes from different levels of multiple categorical variables, a two-way ANOVA was performed using a post-hoc Bonferroni test; statistical analysis was performed using GraphPad Prilm (USA); statistical significance was considered at P < 0.05 (attached file: Figures S1C-E); an animal model of ischemic stroke was established using photochemical embolization (attached file: Figure S2A); the lesion area was located in the S1FL region, and mice developed sensorimotor dysfunction opposite the upper limb cortical infarction after PT (attached file: Figure S2B); 4. Western blot was used to detect the expression level of ULK1 in the five areas surrounding the cortical infarction and the infarct core at different stages after ischemic stroke; it was found that ULK1 expression increased significantly 6 times 1 day after ischemic injury, and reached its peak 3 to 5 days after 7 ischemic injuries (attached file: Figure S2B); Figure 2 A, B), whose expression trend was similar to that of the infarct area and the infarct area after ischemia, and behavioral and functional degeneration (attached file: Figure S2C-I); in order to further investigate the changes in ULK1 localization in the lesion area after ischemic injury, double 10 immunofluorescence staining was performed to mark the cellular expression of ULK1; 11 increased ULK1 expression was detected, mainly located in Iba1+ microglia in the peri-infarct area 123 days after ischemic injury ( Figure 2 CE); The above results indicate that ULK113 is significantly increased in the early stage of ischemic stroke, mainly located in microglia around the infarct, and may be involved in the regulation of neuronal death. Figure 2 ; Expression of ULK1 in the cerebral cortex after ischemic stroke; A and B represent the quantitative analysis of ULK1 protein levels at different time points after ischemic injury using blot strips; n3 = 4. *p < 0.05, p < 0.01 vs; sham group; C Schematic diagram of infarct area induced by PT; D ULK1 (red) and neurons (NeuN, green), astrocytes (GFAP), microglia in the peri-infarct area 3 days after ischemic injury (Iba1, green); nuclei are stained with DAPI (blue); arrows: cells with common labels; scale bar = 100 μm, 50 μm; E Quantitative analysis of ULK1 in cells of + / NeuN+, ULK1+ / GFAP+ and ULK1+ / Iba1+; n = 5. *p < 0.01 vs; ULK1 count of + / NeuN+ cells &&p < 0.01 vs; ULK1 count of + / GFAP+ cells.

[0118] This invention established a photothrombotic stroke model and administered the ULK1 inhibitor SBI-0206965 (SBI) and LYN1604 hydrochloride (LYN), a ULK1 agonist, to regulate ULK1 activity in vivo. The results were used to assess sensorimotor deficits, neuronal apoptosis, and microglia / macrophage activation of neuronal function. Immunofluorescence assays showed that ULK1 is primarily localized in microglia in the infarcted region following ischemic stroke. LYN treatment upregulated ULK1, significantly reducing infarct volume, improving motor function, and promoting the increase of anti-inflammatory microglia. In summary, ULK1 promotes neuronal repair and facilitates the formation of 13 pathways related to anti-inflammatory microglia after ischemic injury.

[0119] It should be noted that embodiments of the present invention can be implemented in hardware, software, or a combination of both. The hardware portion can be implemented using dedicated logic; the software portion can be stored in memory and executed by a suitable instruction execution system, such as a microprocessor or dedicated-design hardware. Those skilled in the art will understand that the above-described devices and methods can be implemented using computer-executable instructions and / or included in processor control code, for example, such code provided on a carrier medium such as a disk, CD, or DVD-ROM, a programmable memory such as read-only memory (firmware), or a data carrier such as an optical or electronic signal carrier. The devices and modules of the present invention can be implemented by hardware circuitry such as very large-scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, or programmable hardware devices such as field-programmable gate arrays, programmable logic devices, etc., or by software executed by various types of processors, or by a combination of the above-described hardware circuitry and software, such as firmware.

[0120] The embodiments of the present invention have achieved some positive results during the research and development or use process, and have indeed great advantages compared with the prior art. The following content describes them in conjunction with the data, charts and other information of the experimental process.

[0121] ULK1 expression is upregulated in microglia in the infarct area after ischemic injury. Until now, ULK1 expression in the brain has remained unclear. To address this challenge, the expression levels of ULK1 protein in other brain regions, including the cerebral cortex and hippocampus, were examined using Western blotting in the midbrain, cerebellum, and olfactory bulb. ULK1 was found in the cortex, although lower than in other brain regions (Additional files: Figures S1A, B). In wild-type (WT) mice, ULK1 was expressed in neurons, astrocytes, and microglia in the cerebral cortex (Additional files: Figures S1C-E). An animal model of ischemic stroke was established using photochemoembolization (Additional file: Figure S2A). The lesion area was located in the S1FL region, and mice developed sensorimotor dysfunction opposite the upper limb cortical infarction after PT (Additional file: Figure S2B). 4. Western blot analysis was performed on the expression levels of ULK1 in the peri-infarct area and infarct core at different stages of ischemic stroke. ULK1 expression was found to increase significantly 6 times 1 day after ischemic injury, and reached its peak 3 to 5 days after 7 ischemic injuries. Figure 2 A, B), whose expression trend was similar to that of the infarct area and the infarct area after ischemia, and behavioral and functional degeneration (attached file: Figure S2C-I). To further investigate the changes in ULK1 localization in the lesion area after ischemic injury, double 10 immunofluorescence staining was performed to label ULK1 cellular expression. Increased ULK1 expression was detected in 11 cells, mainly located in microglia in the peri-infarct area 123 days after Iba1+ ischemic injury. Figure 1 The above results indicate that ULK113 is significantly increased in the early stages of ischemic stroke, mainly located in microglia around the infarct, and may be involved in the regulation of neuronal death.

[0122] To investigate the role of ULK1 in ischemic stroke, the inhibitor SBI and the ULK1 agonist LYN were injected in vivo to modulate ULK1 activity. The experimental protocol is outlined below. Figure 3 a. Nissl staining and TTC staining showed that ischemia-induced infarcts were prone to occur 3 days after PT, with 14 cases observed in the cortex of control mice. An increase in lesion volume was observed after administration of ULK1 inhibitors, while a significant decrease was observed after administration of ULK1 agonists (…). Figure 3 B, C). Then, TUNEL / NeuN immunofluorescence showed an increase in the number of apoptotic neurons in the damaged area of ​​SBI-treated mice compared with drug-treated mice, while the content of 18 neurons was significantly reduced in lyn-treated mice ( Figure 3D, E). Next, behavioral tests such as the cylinder test and grid walking test were used to examine ischemic injury to motor function. In the cylinder test, mice treated with SBI had more left forelimb contact than mice in the vehicle group. Conversely, in the vehicle group, mice treated with Lyn had less left forelimb contact than mice in this group ( Figure 3 F). The results of the grid walking experiment are similar to those of the cylinder experiment 23. Figure 2 G). These findings suggest that ULK1 plays a neuroprotective role after ischemic stroke, reducing infarct volume and improving motor deficits in mice.

[0123] Three days after ischemic injury, ULK1 expression was upregulated, primarily in microglia. ULK1 activity significantly participated in neuronal regeneration in mice, suggesting that ULK1 plays a crucial role in microglia / macrophage activation in the pathophysiology of ischemic stroke. Microglia / macrophage proliferation was detected using BrdU / Iba1 dual immunofluorescence staining. Firstly, compared to the vehicle group, the number of BrdU+ / Iba1+ cells and the number of Iba1+ cells in the lesion were significantly increased in 17 regions in the SBI group, while LYN inhibited the increase induced by drug loading treatment. Figure 4 B, C). Resting microglia exhibit typical morphology, characterized by small and elongated somatic cells. Upon activation, microglia somatic cells significantly enlarge, but this enlargement process is short-lived or disappears entirely, leading to a decrease in cell perimeter and an increase in cell length, resulting in an increased area / perimeter ratio. In this study, LYN treatment significantly altered the characteristics of activated microglia in the peri-infarct region, exhibiting various morphological features. Cell area / perimeter ratio ( Figure 4 E), LYN's 24 relative solidities ( Figure 4 F), Roundness ( Figure 4 G) and average intersection point ( Figure 4 The H group showed significant recovery compared to the vehicle group. However, the morphology of Iba1- was similar in the SBI group, with 26 positive microglia, similar to the drug-loaded group. Microglial protein levels were significantly increased in the SBI group and significantly decreased in the LYN group compared to the vehicle group. Figure 4 (I, J) This result is consistent with immunofluorescence staining. The above results indicate that ULK1 inhibits cell proliferation and microglial activation after ischemic injury.

[0124] ULK1 promotes the activation of anti-inflammatory microglia / macrophage phenotypes and the secretion of anti-inflammatory factors after ischemic injury. After ischemic stroke, microglia exhibit pro-inflammatory and anti-inflammatory phenotypes in response to brain injury.

[0125] Therefore, after assessing the phenotypic switching of microglia / macrophages, 10 cases of ischemic injury were observed using immunofluorescence staining. iNOS can serve as a pro-inflammatory marker for 11 microglia / macrophages, while CD206 indicates anti-inflammatory microglia / macrophages. Results showed that the number of iNOS+ / Iba1+ pro-inflammatory microglia / macrophages was increased in the periinfarct area in the SBI group compared to the control group, but decreased in the LYN group (14 groups). Figure 5 A, B). Meanwhile, compared to the vehicle group, the 15CD206+ / Iba1+SBI group showed a decrease in microglia / macrophages, while conversely, the LYN group showed a significant increase in microglia / macrophages on day 3 after ischemic injury (16 cases). Figure 5 C, D). Phenotypic characteristics of microglia 18 days post-ischemic stroke were further confirmed by RT-qPCR evaluation of three loci. These microglia included CD86, CD32, and 19 iNOS molecules, which were upregulated in the SBI group and downregulated in the LYN group. Figure 5 EG). Conversely, the mRNA levels of anti-inflammatory factors CD206, Ym1 / 2, and Arg1 were all significantly downregulated in the SBI group, but significantly upregulated in the LYN group. Figure 5 (HJ). This data indicates that ULK1 promotes microglial activation, leading to a shift towards an anti-inflammatory phenotype.

[0126] Previous studies have shown that pro-inflammatory microglia are involved in mediating neurotoxicity, while anti-inflammatory microglia have a neuroprotective effect in stroke. Therefore, this study examined the secretion of inflammatory factors in the cortical infarct area on postoperative day 3 using Western blotting in cases of ischemic stroke. Figure 6 A, B). Protein levels of pro-inflammatory factors, including IL1, β, and TNF-α, were elevated in the SBI group, while decreased in the control group and the LYN group (…). Figure 6 AC). Furthermore, anti-inflammatory cytokinesis, such as IL10 and TGFβ, was significantly lower in SBI-treated mice than in vector-treated mice; however, in mice treated with lyn alone ( Figure 6 Similar to the protein level results, IL1β mRNA levels and TNF-α decreased, but IL10 and TGFβ were upregulated after LYN treatment (in cases of ischemic injury). Figure 6 GJ). SBI treatment upregulated IL1β mRNA levels in 18 patients treated with vehicle-borne drugs following ischemic injury (GJ). Figure 6 G). Detection of TNF-α, IL10, and 19 TGFβ showed no significant changes. Figure 6 These additional data suggest that ULK1 may stimulate the activation of anti-inflammatory microglia in the acute phase of ischemic stroke.

[0127] Previous studies have reported that AMPK and mTOR regulate autophagy by modulating ULK1. To examine the effect of ULK1 on autophagy, several autophagy indicator proteins, including p62, LC3, and Beclin1, were examined. These are 13 key downstream proteins in the autophagy process regulated by ULK1, using Western blotting. Three days after stroke injury, immunoblotting analysis showed downregulation of LC3-II protein levels, upregulation of p62 protein levels, and downregulation of Beclin1 levels in the SBI and 3-MA groups. Conversely, the LYN and RAPA groups showed differential upregulation of LC3-II and Beclin1 protein levels, while p62 protein levels were downregulated. Figure 7 AD). The number of LC3+ / Iba1+ cells increased after LYN injection, with similar results observed in the RAPA group (2). Figure 7 E, G). Furthermore, the number of p62 cells was also increased; 3 cells were found in both the SBI and 3-MA groups (+ / Iba1+). Figure 7 (F, H). These findings suggest that 4ULK1 upregulates the initiation of autophagy following ischemic injury.

[0128] Autophagy is activated in various cell types, including neurons, glial cells, and microvascular cells, such as after ischemic brain injury. Currently, over 35 autophagy-associated genes (ATGs) and proteins have been identified, including 13. Among them, the first identified autophagy protein, Atg1, is expressed in yeast and plays a crucial role in promoting autophagosome formation. ULK1, a mammalian homolog of Atg1, promotes autophagosome formation and participates in cellular functional autophagy in response to injury and various stimuli. Results showed that ULK1 reduced the degree of damage to cortical neurons and the number of apoptotic neurons in the cerebral cortex caused by ischemic brain injury in mice. Furthermore, it alleviated motor impairments, improving motor function in 19 functions through the cylinder test and the grip walking test. These results indicate that ULK1 reduces the degree of cortical neuronal damage, improves behavioral function deficits, and plays a neuroprotective role after ischemic stroke. Studies have shown that in mammals, the ULK1 complex is primarily found on phagocytes. In the current study, ULK1 levels were positive / Iba1 positive post-surgery, and the number of cells in the periinfarct region was significantly increased in ischemic injuries, suggesting that ULK1 is primarily expressed in microglia. Microglia play a crucial role in the physiology and pathology of the central nervous system (CNS), including ischemic stroke. Following microglia injury, rapid transcriptional regulation and morphological changes occur, varying according to time (e.g., acute, subacute, or chronic), injury location (e.g., cortex and hippocampus), and age. Pro-inflammatory microglia induce the production of various mediators, such as nitric oxide, reactive oxygen species, and pro-inflammatory cytokines, exacerbating ischemic brain injury; however, anti-inflammatory microglia release mediators that contribute to angiogenesis, debris clearance, and brain tissue repair. Therefore, the induction of intracellular metastasis to induce a shift in microglia activation from a pro-inflammatory to an anti-inflammatory phenotype after brain injury warrants consideration. Thus, it is important to focus on the shift in treatment approaches to microglia activation from a pro-inflammatory to an anti-inflammatory phenotype after brain injury. In this study, the results showed that LYN activation of ULK1 reduced the number of Iba1+ microglia and BrdU+ / Iba1+- proliferating microglia, and promoted the restoration of microglia morphology, namely the typical long processes and small bodies. Therefore, the neuroprotective effect of ULK1 in ischemic stroke may be due to the inhibition of microglia proliferation. SBI inhibition of ULK110 increased the number of iNOS+ / Iba1+ cells but decreased the number of CD206+ / Iba1+ cells in the ischemic cortex of mice. Furthermore, LYN also reduced the levels of both pro-inflammatory mediators (12 mRNA and protein levels) and increased the levels of anti-inflammatory mediators. In contrast, SBI13 produced the opposite effect. These findings suggest that ULK1 inhibits the activation of the inflammatory phenotype of microglia, promotes their transition to the anti-inflammatory phenotype, and secretes 15 anti-inflammatory factors that contribute to the repair of neural tissue.Autophagy is a form of cellular breakdown involving the transfer of substances from the cytoplasm to lysosomes. ULK1 acts as a central initiator, modulating autophagy flux from upstream sensors and downstream sensors. In this study, treatment with the autophagy inducer RAPA enhanced ischemic autophagy induced by the infarct region, increasing LC3-II and Beclin1 protein levels while decreasing p62 protein levels. Upon activation by LYN, ULK1 also exhibited a similar effect to RAPA, while SBI and the autophagy inhibitor 3-MA inhibited autophagy activation. Furthermore, immunofluorescence assays showed that LYN significantly enhanced the autophagic capacity of microglia. These results indicate that ULK1 also affects microglia autophagy by influencing Beclin1, LC3-II, and p62. Current research aims to investigate the role and potential mechanisms of ULK1 by administering agonists and inhibitors to experimental mice. Intravenous injection of LYN after cerebral ischemia-reperfusion injury enhances ULK1 expression, primarily occurring in the peri-infarct region of microglia located in the cortex. Therefore, these activated cells secrete anti-inflammatory factors and nutrients, thereby promoting neuronal nutrition, recovery, and ultimately contributing to the repair of ischemic damage. However, some limitations remain in the research. Recently, Beccari et al. reported that RAPA improved phagocytosis after tMCAO injury and proposed that the functional impairment of microglia in stroke patients was caused by autophagy induction. The results confirmed that ULK1 is involved in mediating microglia / macrophage polarization. However, it is important to note that ULK1 may also have an effect on microglia phagocytosis. Therefore, additional research is needed to obtain more substantial evidence on the effect of ULK1 on phagocytosis in the future. In addition, the molecular mechanisms regulating microglia activation are also very complex. He et al. showed that lipopolysaccharide inhibits ULK1 activity in BV2 microglia by directly phosphorylating p38, leading to decreased autophagy levels and ultimately inducing inflammatory activity. Recently, Han et al. reported the protective effect of PPARγcoactivator1α in ischemic brain injury by regulating ulk1-related autophagy and mitophagy and inhibiting neuroinflammation. Evidence for this mechanism is insufficient, therefore further investigation is needed to confirm the ULK1 signaling pathway in ischemic stroke-induced microglial activation. In summary, it was demonstrated that after ischemic stroke, SBI's inhibition of ULK1 leads to microglial activation into a pro-inflammatory state, resulting in nerve damage and motor dysfunction. Conversely, LYN's activation of ULK1 promotes the activation of microglia into an anti-inflammatory state, thereby reducing nerve damage and inflammation and promoting the recovery of motor function. In short, the findings highlight the involvement of autophagy kinase ULK1 in the neuroinflammatory response, underscoring its potential as a therapeutic target for clinical treatment of ischemic stroke.

[0129] This invention established a photothrombotic stroke model and administered the ULK1 inhibitor SBI-0206965 (SBI) and LYN1604 hydrochloride (LYN), a ULK1 agonist, to regulate ULK1 activity in vivo. The results were used to assess sensorimotor deficits, neuronal apoptosis, and microglia / macrophage activation of neuronal function. Immunofluorescence assays showed that ULK1 is primarily localized in microglia in the infarcted region following ischemic stroke. LYN treatment upregulated ULK1, significantly reducing infarct volume, improving motor function, and promoting the increase of anti-inflammatory microglia. In summary, ULK1 promotes neuronal repair and facilitates the formation of 13 pathways related to anti-inflammatory microglia after ischemic injury.

[0130] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of ULK1 as a potential therapeutic target for ischemic stroke, characterized in that, Includes the following steps: Step 1: Photothrombotic stroke; group and pharmacological treatment; neurobehavioral assessment; Step two, histological tissue processing; Niels' staining method; TTC staining was performed on TTC-stained samples; TUNEL staining; Immunofluorescence staining; Step 3, Western blotting; microglia morphology; Real-time quantitative PCR; statistical analysis.

2. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The aforementioned photothrombotic stroke: The method for inducing focal cortical photothrombotic ischemic stroke in mice has been described in previous studies. Mice were intraperitoneally injected with 10 mg / ml Rose Bengal; 15 min later, mice were anesthetized and intraperitoneally injected with 80 mg / kg sodium pentobarbital; under anesthesia, the skull was exposed to the cortical region of the forelimb covering the skull; the photothrombotic process in the cortical region of the forelimb was activated by applying a 2 mm diameter cold laser to the skull for 15 min; throughout the procedure, the mice's body temperature was maintained at 37°C using a rectal probe; the same procedure was performed on animals undergoing pseudo-ischemic surgery, except for the Rose Bengal injection.

3. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The aforementioned group and drug treatment: Mice were randomly divided into 3 groups: The sham ischemic surgery group did not receive or undergo cortical PT induction; The vehicle handling team performed ischemic surgery and treated with saline solution respectively; The drug treatment group was further divided into: (1) LYN treatment group treated with LYN1604 hydrochloride; (2) SBI-0206965 treatment group. (3) The 3-MA treatment group was treated with 3-methyladenine after PT surgery; (4) The RAPA treatment group was treated with rapamycin after PT surgery; In the vehicle-, SBI-, LYN, 3-MA and RAPA groups, mice were sacrificed 3 days after PT surgery; Animal experiment schematic diagram and protocol; 20 SBI and LYN were dissolved in physiological saline and administered via tail vein at doses of 0.5 mg / kg and 0; 1 were 21 mg / kg, 30 min after ischemic stroke; 3-MA was dissolved in DMSO and then diluted with physiological saline 1:10 and injected i; p. 30 min after ischemic stroke; rapamycin 3 mg / kg was dissolved in 60% v / v diluted DMSO and injected i; p. 30 min after ischemia; 30 min after ischemic stroke, SBI, LYN, 3-MA and RAPA were injected respectively; 2.4.5-Bromo-2′deoxyuridine administration: To label mitotic cells in the cortical infarct area, mice in the sham-operated, vector, SBI, and LYN groups were labeled by intraperitoneal injection of 50 mg / kg BrdU, once daily for 4 consecutive days, 30 min after ischemia.

4. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The neurobehavioral assessment: Cylinder and grid walking tests were performed on day 1 before ischemic surgery and on days 1 and 3 after ischemic surgery. In the cylinder test, the mouse was placed in a glass cylinder and allowed to walk freely for 63 minutes during the test. To observe all behaviors clearly and in detail, two mirrors were arranged at the 90-degree angles behind the cylinder. The number of paw-to-cylinder contactes was recorded for 3 minutes during the vertical exploration. Notably, only contact with the load-bearing wall was scored by one or two forelimbs against the wall. Due to the significant contralateral muscle weakness caused by damage to the left primary motor cortex, the mouse frequently used the undamaged forelimb for body support during spontaneous vertical exploration. The forelimb asymmetry index was calculated as the undamaged forelimb contact relative to the total forelimb contact. Percentage of contact: Number of left contacts / (Number of left paw contacts + Number of right paw contacts + Number of both paw contacts); Simultaneously, a grid walking test was performed to assess motor function, as previously described; Simply put, mice were placed in a 1 cm square grid fixed 50 cm above a laboratory table for 1 min; A camera was placed under the grid to film all the mice's movements; Due to left primary motor impairment resulting in right forelimb muscle weakness and cortical damage, the right forelimb often could not provide support to guide the right paw through the grid holes; The damaged right forelimb was calculated in the frame-by-frame video analysis; Foot failure score means the ratio between the number of right foot failures and the total number of right foot failures: Number of right foot failures / (Number of right foot failures + Number of right foot non-failures).

5. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The histological organization process: Following standard procedures, mice in each group were anesthetized with sodium and pentobarbital, and then perfused with 0.1 M phosphate-buffered saline via cardiac perfusion, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline. The brains of 29 mice were removed, fixed in the same fixative for 12 h, and then transferred to 30% sucrose until they sank. Subsequently, the brain was embedded with OCT and coagulated at -20°C; thus, each brain was coronally sliced ​​to a thickness of 30 μm in a cryostat.

6. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The Nissell staining method: Nissl staining was performed according to published methods; simply put, brain sections were stained in 0.1% cresol violet solution and left at room temperature for 10 min; afterwards, the sections were rinsed with distilled water and dehydrated in 95% and 100% ethanol; finally, the brain sections were cleaned with xylene and coated with Canada balsam; morphological measurements of the infarct area of ​​the cresol violet-stained sections were performed using images with Adobe Photoshop and ImageJ software; the infarct volume was calculated by subtracting the volume of the ipsilateral hemisphere stained with Nissl staining from the volume of the contralateral hemisphere infarct = contralateral hemisphere volume - healthy volume of the ipsilateral hemisphere.

7. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The TTC staining example was performed to observe the lesion size on the 3rd day after ischemic stroke. Simply put, the tissue was directly frozen at -20°C for 20 minutes, and then directly placed in 1% TTC solution and incubated in a 37°C oven away from light for 10-20 minutes, with the tissue gently shaken every 1-2 minutes to ensure uniform staining. After staining, the tissue was fixed with 4% PFA for 5 minutes, and then the fixed tissue photograph was stored in the dark and taken by a camera.

8. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The TUNEL staining: Dual fluorescence staining was used to detect neuronal apoptosis 3 days after ischemic injury using NeuN and terminal deoxynucleotidyl transferase (dUTP) nick-terminal PT markers in mouse cortex for 25 days; the TUNEL kit was used according to the manufacturer's instructions for the detection day; for NeuN immunofluorescence staining, tissues were treated with mouse anti-Neun antibody and incubated with donkey anti-mouse Alexa Fluor 594; 29 images were observed using a Nikon microscope; data were calculated as TUNEL-numbers with 1 positive neuron per millimeter in the selected field.

9. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The immunofluorescence staining: Immunofluorescence staining was performed according to previous studies; in short, tissue sections were fixed for 30 min, then retrieval of the extract with sodium citrate antigen at 96°C for 10 min. The tissue sections were treated with primary antibodies including mouse anti-neun antibody, mouse anti-GFAP, goat anti-iba1, rabbit anti-iba1, rabbit anti-ulk1, rabbit anti-brdu, mouse anti-iNOS11, goat anti-cd206, rabbit anti-LC3A / B, and mouse anti-p62 overnight at 4°C. The tissues were then immersed in secondary antibodies including donkey anti-rabbit AlexaFluor488, donkey anti-mouse AlexaFluor488, donkey anti-rabbit AlexaFluor594, donkey anti-mouse AlexaFluor594, and donkey anti-goat AlexaFluor59418, and incubated at room temperature for 2 hours. Images were obtained using confocal microscopy or microscopy and analyzed using images J and Photoshop. All measurements were performed under blinded conditions by two observers for each experiment, with 22 samples under the same experimental conditions. In addition, the average cell count was obtained from at least three microscopic fields of view in the region of interest, three parts of each brain, and at least three animals per group of 24 people; the data are expressed as the average cell count per square millimeter; 25 cases of the peri-infarct region of the cerebral cortex were selected as the ROI to assess neuronal damage after ischemic stroke.

10. The application of ULK1 as a therapeutic target for ischemic stroke as described in claim 1, characterized in that, The Western blot method involved anesthetizing mice with sodium pentobarbital; after deep anesthesia, the entire brain of the mouse was removed as quickly as possible; the infarct core was immediately excised with a scalpel under a magnifying glass in the area surrounding the infarct. Tissue was homogenized and centrifuged, and the supernatant was used for Western blot analysis. The following immunoblots were incubated with primary antibodies including rabbit anti-ulk1, rabbit anti-iba1, rabbit anti-LC3A / B, mouse anti-p62, rabbit anti-il1β, rabbit anti-TGFβ, rabbit anti-TNF-α, rabbit anti-il10, and mouse anti-β-actin overnight at 4°C. The next day, the blots reacted with secondary antibodies, including goat anti-antibody rabbit IgG and goat anti-mouse IgG, at room temperature for 12 hours for 2 hours. Finally, the results were detected using the Omni-ECL assay kit. Protein blot analysis was performed using published methods; in short, scanning was performed using a chemical microscope touch, and density analysis was performed to quantify the bands using Image software. The ratio of the bars was calibrated to %, with the sham-operated group designated as 100%. β-actin was used as an internal control. Microglia morphology: Microglia morphology was used as a basis for previous studies with minor modifications; in a brief, 30 μm tissue section was stained with Iba1 and images were prepared using a confocal microscope with z-pillar imaging; in each group, there were 56-84 Iba1+ microglia, with at least 3 cells per section, 3 cells per section of the mouse, 7 mice per group, analyzed by the researchers without considering grouping; the experimenters found the longest distance across the cell body, and the first radius was set to a distance of 1 μm to start the Sholl analysis; subsequently, the experimenters defined the radius at intervals of 5 μm, and the results were calculated by ImageJ with the Sholl analysis plugin; simultaneously, the convex hull contained the entire process of each microglia and a circle defined the convex hull for further definition; the hull and circle plugins were defined by ImageJ with hull and circle plugins; The demonstration results include: cell area is the total number of pixels of iba1 positive cells, robustness is the ratio of cell area to cell area convex area, roundness value up to 1 means a perfect circle, and intersection points represent the intersection of microglia processes with concentric circles; the above three data points were collected using a confocal microscope (Olympus, Japan). The real-time quantitative PCR was performed as follows: 3 days after PT, total RNA was extracted from brain tissue using a tissue RNA extraction kit 7, and RNA was reverse extracted and transcribed into cDNA using HiScript III qRT SuperMix (Vazyme, China); real-time quantitative polymerase chain reaction was performed using ChamQ universal SYBR qPCR MasterMix 10; data collection was performed on an RT-PCR system; sample 11 was independently amplified at least 3 times; relative gene expression levels were calculated using the 2-12ΔΔCt method for GAPDH; primers listed in the supplementary file: Table S1; The statistical analysis is as follows: data are expressed as mean ± standard deviation; comparisons between variables were performed using a Student's t-test on 16 normally distributed independent samples between two groups; for multiple comparisons, a one-way ANOVA was performed followed by a post-hoc Bonferroni test; for comparing changes from different levels of multiple categorical variables, a two-way ANOVA was performed using a post-hoc Bonferroni test; statistical analysis was conducted using GraphPad Prilm; statistical significance was considered to be p < 0.05; an animal model of ischemic stroke was established using photochemical embolization; disease... The affected area was located in the S1FL region. Mice developed sensory and motor dysfunction opposite the upper limb cortical infarction after PT.

4. Western blot analysis was performed on the expression level of ULK1 in the cortical infarct area and infarct core at different stages after ischemic stroke. It was found that ULK1 expression increased significantly 6 times 1 day after ischemic injury, and reached its peak 3 to 5 days after 7 ischemic injuries. Its expression trend was similar to that of the infarct area and the infarct area after ischemia.

9. To further explore the changes in ULK1 localization in the lesion area after ischemic injury, a double 10-fold CT scan was performed. Immunofluorescence staining was used to label cellular expression of ULK1; increased expression of ULK1 was detected in 11 areas, mainly located in microglia in the periinfarct region 12-3 days after ischemic injury. These results indicate a significant increase in ULK1 expression in the early stage of ischemic stroke, primarily in microglia around the infarct, which may be involved in regulating neuronal death. ULK1 expression in the cerebral cortex after ischemic stroke was also analyzed. A and B represent the quantitative analysis of ULK1 protein levels at different time points after ischemic injury using Western blot analysis; n3 = 4. * p < 0.05, p < 0.01 and... ; Pseudogroup; C Schematic diagram of PT-induced infarct area; DULK1 and neurons, astrocytes, microglia in the peri-infarct area 3 days after ischemic injury; Nucleus stained with DAPI; Arrow: Commonly labeled cells; Scale bar = 100μm, 50μm; E Quantitative analysis of ULK1 in cells of + / NeuN+, ULK1+ / GFAP+ and ULK1+ / Iba1+; n = 5. p < 0.01 vs; ULK1 count in + / NeuN+ cells && p < 0.01 vs; ULK1 count in + / GFAP+ cells.

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