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CN-121978331-A - ULK1 may be used as target for treating ischemic stroke

CN121978331ACN 121978331 ACN121978331 ACN 121978331ACN-121978331-A

Abstract

The invention belongs to the technical field of ischemic stroke treatment, and discloses an application of ULK1 possibly serving as a target for ischemic stroke treatment. The present invention establishes a photothrombotic stroke pattern and administers the ULK1 inhibitor SBI-0206965 (SBI), and LYN1604 hydrochloride (LYN), an agonist of ULK1, which modulates ULK1 activity in vivo. Examples the results of sensory motor deficits, neuronal apoptosis and microglial/macrophage activation nerve function were evaluated. Immunofluorescence assay results showed that ULK1 was mainly localized to microglial cells in the post-ischemic infarct area of china. Upregulation of ULK1 by LYN treatment significantly reduces infarct volume, improves motor function, promotes anti-inflammatory microglia increase. In conclusion, ULK1 promotes neuronal repair and promotes its formation of 13 pathways that are anti-inflammatory microglial pathways following ischemic injury.

Inventors

  • XIONG YE
  • WANG MINMIN
  • Tan Xianxi
  • CHEN BAIHUI
  • XIAO BOHUAI
  • Jin Liqi

Assignees

  • 温州医科大学附属第一医院

Dates

Publication Date
20260505
Application Date
20240118

Claims (10)

  1. 1. Use of ULK1 as a target for the treatment of ischemic stroke comprising the steps of: Step one, photo-thrombotic stroke, group and drug treatment, neural behavior assessment; Step two, histological tissue treatment, a nisel staining method, TTC staining of TTC staining examples, TUNEL staining and immunofluorescence staining; step three, western blotting, microglial cell morphology, real-time fluorescence quantitative PCR and statistical analysis.
  2. 2. Use of ULK1 according to claim 1 as a target for the treatment of ischemic stroke, wherein the photothrombotic stroke: The induction of focal cortical light thrombosis ischemic stroke in mice by this method was described in the previous study, 10mg/mlRose Mongolian was injected into the abdominal cavity of mice, 80mg/kg sodium pentobarbital was injected into the abdominal cavity after 15min, the skull was exposed to the outer covering anterior limb cortical region under anesthesia, the light thrombosis process cortex of the cell anterior limb region was activated, a 2mm diameter cold laser was applied to the skull for 15min, the body temperature of mice was maintained at 37 ℃ temperature probe by rectal surgery throughout the surgery, the same procedure was performed on the animals of the pseudoischemic surgery, except for the Benglazino rose injection.
  3. 3. Use of ULK1 as claimed in claim 1 as a target for the treatment of ischemic stroke, wherein the population and the drug treatment: mice were randomly divided into 3 groups: The sham-ischemic surgical group was not administered and did not receive cortical PT induction; the vehicle treatment group respectively performs ischemia operation and physiological saline treatment; The medicine treatment group is further divided into (1) LYN treatment group treated by LYN1604 hydrochloride and (2) SBI-case treatment group which is SBI-0206965 group; (3) The 3-MA treatment group is treated by 3-methyladenine after PT operation, the RAPA treatment group is treated by rapamycin after PT operation, the mice in the vehicle-, SBI-, LYN,3-MAANDRAPA groups are respectively killed at 3d after PT operation, animal experiments show a protocol, 20SBI and LYN are dissolved in physiological saline and are respectively administered through tail veins with dosages of 0.5mg/kg and 0;1 respectively, 30min after ischemic cerebral apoplexy, 3-MA is dissolved in DMSO and then diluted by physiological saline 1:10, i is injected after p. ischemic cerebral apoplexy, 3mg/kg is dissolved in DMSO diluted by 60% v/v, i is injected, p. 30min stroke after ischemia, 30min after ischemic apoplexy, SBI, LYN,3-MA and RAPA are respectively injected; 2.4.5-bromo-2' deoxyuridine administration to label mitotic cells in the cortical infarct zone, labeling examples were injected intraperitoneally 50mg/kgBrdU, once daily for 30min after ischemia, for 4 consecutive days, in sham-operated, vehicle, SBI, LYN group mice.
  4. 4. Use of ULK1 according to claim 1 as a target for the treatment of ischemic stroke, wherein the neurobehavioral assessment: The cylinder test and the grid walking test are carried out on the 1 st day before the ischemia operation, and the 1 st and the 3 rd days after the ischemia operation; in the cylinder test, the mice were placed in a glass cylinder and allowed to walk freely for 63min during the test, two mirrors were arranged behind the cylinder for clarity and detail to observe all the behaviors, the number of paw and column wall contacts was recorded for 3min during the vertical exploration, notably, only the bearing wall contact was scored by one or both forelimbs on the counter wall, because of the apparent ipsilateral muscle weakness caused by left primary motor cortex injury, the mice frequently used the body support of the intact forelimb during the spontaneous vertical exploration, the calculated forelimb asymmetry index was the percentage of intact forelimb contact relative to total forelimb contact: left contact number/(left paw contact number + right paw contact number + double paw contact number contact person), while the grid walk test was performed to evaluate the motor behavior function, as before, the mice were placed in a1 cm square grid, were usually fixed 50 cm above the laboratory bench, 1min, one below the camera had a visual support line, the left and right eyes were not used to provide a failure score for the total number of intact forelimb, the failure was measured by the visual support of the left and right foot, the failure rate was measured by the left and right eye loss of the left and right foot support line.
  5. 5. Use of ULK1 according to claim 1 as a target for the treatment of ischemic stroke, wherein the histological tissue processing: According to the general procedure, each group of mice was anesthetized with sodium pentobarbital, perfused with 0;1M phosphate buffered saline followed by 4% paraformaldehyde in 0;1M phosphate buffer, brain removed 29 the mice, fixed in the same fixative for 12h, transferred to 30% sucrose until they were submerged, then the brains were embedded with OCT and coagulated at-20℃and therefore, the brains were coronally sectioned in a cryostat to a thickness of 30. Mu.m.
  6. 6. Use of ULK1 according to claim 1 as a target for the treatment of ischemic stroke, wherein the nisel staining method: The Nile staining was performed according to published methods, briefly, sections of brain were stained in 0;1% cresyl violet solution, left at room temperature for 10min, then sections were rinsed with distilled water and dehydrated in 95% and 100% ethanol, finally sections of brain were cleared with xylene and balsam Canadian, morphological measurements of infarct area of cresyl violet stained sections were performed with image and Adobephotoshop and image J software, infarct volume was calculated by subtracting the ipsilateral hemispheric infarct volume = contralateral hemispheric volume-healthy volume as ipsilateral hemispheres.
  7. 7. Use of ULK1 according to claim 1 as a possible target for the treatment of ischemic stroke, wherein TTC staining is performed on TTC staining examples, the lesion size is observed on day 3 after ischemic stroke, briefly, the tissue is frozen directly at-20 ℃ for 20min, then directly in a 1% TTC solution, incubated in a 37 ℃ oven for 10-20min away from the light, the tissue is stained evenly by gently shaking every 1-2min, after staining the tissue, the tissue is fixed with 4% pfa for 5min, and then a fixed photograph of the tissue is stored in the dark and taken by a camera.
  8. 8. The use of ULK1 according to claim 1 as a target for the treatment of ischemic stroke, wherein the TUNEL staining: The neuronal apoptosis NeuN and terminal deoxynucleotidyl transferase dUTP notch terminal mark PT 3 days after ischemic injury were detected by double fluorescent staining, 25% was used in the mouse cortex, TUNEL kit was used to carry out the daily instructions for detection according to the manufacturer's requirements, neuN immunofluorescent staining, tissue as exemplified by treatment with mouse anti-NeuN antibody and incubation with donkey anti-mouse AlexaFluor594, 29 images were observed by digital use of Nikon microscope, and 1 positive neurons 2 per millimeter were calculated in the data as TUNEL-number in the selected field.
  9. 9. Use of ULK1 according to claim 1 as a target for the treatment of ischemic stroke, wherein the immunofluorescent staining: Immunofluorescence staining was performed according to the previous study, briefly, after 30min fixation of each tissue section, 10min extracts were repaired with sodium citrate antigen at 96 ℃, tissue sections were treated with primary antibodies including mouse anti-neun antibody, mouse anti-GFAP, goat anti-iba 1, rabbit anti-iba 1, rabbit anti-ulk 1, rabbit anti-brdu, mouse anti-iNOS 11, goat anti-cd 206, rabbit anti-LC 3A/B, mouse anti-p 62 at 4 ℃ overnight, then tissues were immersed in secondary antibodies including donkey anti-rabbit AlexaFluor488, donkey anti-mouse AlexaFluor488, donkey anti-rabbit AlexaFluor594, donkey anti-mouse AlexaFluor59418, and left at room temperature for 2 hours, images were obtained using confocal microscopy or microscopy and analyzing images J and Photoshop, all measurements were performed under conditions of at least two areas of the same area, and at least two areas of interest were measured in order to ensure that the brain were not less than 3mm, and the average number of brain injury was at least 3 per animal cell area was measured, and the average number of brain injury was at least 3mm was measured per animal area per unit, and the average number of brain injury was measured.
  10. 10. The use of ULK1 as a target for the treatment of ischemic stroke according to claim 1, wherein the western blotting method comprises the steps of anesthetizing mice with sodium pentobarbital, removing the whole brain of the mice as soon as possible after deep anesthesia, immediately removing the infarct core with a surgical blade in the area around the infarct, performing immunoblotting analysis on the supernatant by tissue homogenization centrifugation, performing immunoblotting with a first antibody comprising rabbit anti ULK1, rabbit anti iba1, rabbit anti LC3A/B, mouse anti p62, rabbit anti il1β, rabbit anti tgfβ, rabbit TNF- α, rabbit anti il10 and mouse anti- β -actin, performing a second day of blotting to react only rabbit IgG and anti-mouse IgG with a second antibody comprising goat anti-antibody at 12 goat room temperature for 2 hours, and finally performing scanning by a method used for Omni-ECL detection kit, performing a short chemical blotting and performing a quantitative analysis on the samples using a round bar density, performing a quantitative ratio of the samples as a control group of Image of 100% by contrast; The microglial morphology was used as a prior study and was modified slightly, 30 μm tissue sections were stained with Iba1 in brief, image confocal microscopy was prepared with z-stacks, in each group 56-84 iba1+ microglial cells, at least 3 cells per section, 3 mice per section, 7 mice per group, were analyzed by researchers without considering the grouping, experimenters found the longest distance across the cell body, the first radius was set to be starting at Sholl μm from 1 μm, then experimenters defined the radius as 5 μm interval, the result was calculated by ImageJ and Sholl analysis inserts, while the convex hull was further defined by ImageJ including the entire course of each microglial cell and one circle, hull and round inserts with hull and round inserts; the demonstration results include that the total number of pixels of cells with areas iba as positive cells, the firmness as the ratio of the area of the cells to the area of the cells is convex, the roundness value of 1 means a perfect circle, and each intersection point represents the intersection of a microglial protuberance with a concentric circle; The real-time fluorescent quantitative PCR comprises extracting total RNA from brain tissue 3 days after PT, using tissue RNA extraction kit 7, reversely extracting RNA and transcribing the RNA into cDNA by HiScriptIIIqRTSuperMix (Vazyme, china), carrying out real-time quantitative polymerase chain reaction by adopting ChamQ general SYBRqPCRMasterMix10, carrying out data collection on an RT-PCR system, independently amplifying sample 11 at least 3 times, using a 2-12 delta Ct method on GAPDH relative to gene expression quantity, and carrying out the primers listed in the additional file, wherein the primers are shown in Table S1; The statistical analysis is that each data is expressed as mean ± standard deviation; the comparison with the variables adopts the student t test to test 16 normal distributions of independent samples between two groups; for multiple comparisons, one-way analysis of variance was followed by a post Bonferroni test; comparing multiple classification variables from different levels, performing bidirectional variance analysis by post Bonferroni test, performing analysis by GRAPHPADPRILM company in statistics, establishing an animal model of ischemic cerebral apoplexy by photochemical embolism method in consideration of whether the statistical significance is P <0.05, positioning a lesion region in S1FL region, detecting opposite sensory and motor dysfunction of upper limb cortical infarction by mice after PT, 4. Performing westernblot detection on ULK1 expression level in 5 regions around cortical infarction and infarct cores at different stages after ischemic cerebral apoplexy, finding out that ULK1 expression is significantly increased 6 times in 1 day after ischemic injury and reaches peak value in 3 days to 5 days after 7 times of ischemic injury, marking ULK1 cell expression by double 10 immunofluorescent staining in order to further discuss change 9 of lesion region ULK1 positioning after ischemic injury, detecting 11 increase in glial expression of ULK1, and mainly in Ibao+123, taking part in peripheral region after peripheral infarction, and peripheral infarction 1, determining that ULK1 expression is significantly increased in 3 days to 5 days after 7 times of ischemic injury, and measuring ULK1 expression is similar to infarct area, and measuring 5 days after peripheral injury, and measuring 4 = 4, and measuring peripheral nerve cell death of peripheral cerebral infarction, and measuring the peripheral nerve cell injury, and measuring the peripheral nerve injury, and the peripheral nerve injury, the peripheral brain, peripheral nerve injury, peripheral nerve brain, peripheral human, astrocytes, microglia in the periinfarct area 3 days after ischemic injury, nuclei were stained with DAPI, arrows, co-labeled cells, scale bar = 100 μm,50 μm, quantification of the unit cells in each of ulk1 assay+/neun+, ulk1+/gfap+ and ulk1+/iba1+, n = 5.p <0.01vs, number of ulk1+/neun+ cells +.0.01 vs, number of ulk1+/gfap+ cells.

Description

ULK1 may be used as target for treating ischemic stroke Technical Field The invention belongs to the technical field of ischemic stroke treatment, and particularly relates to an application of ULK1 possibly serving as a target for ischemic stroke treatment. Background Ischemic stroke is a ubiquitous brain disease, accounting for over 70% of all cerebrovascular diseases and is associated with significant morbidity and mortality. Thus, the cellular and molecular mechanisms of ischemic stroke are studied. Despite some advances in understanding pathology, effective clinical treatment of ischemic stroke remains lacking. There is an urgent need to explore 7 cases of ischemic stroke, a key mechanism for developing new therapeutic targets. Ischemic stroke induces a complex series of pathophysiological processes including neuroinflammation, oxidative stress, excitotoxicity, and hypoxia. Neuroinflammation is a critical role in the development of brain damage secondary to ischemic stroke, primarily through microglial activation. Microglial cells respond to ischemic events in minutes to cerebral ischemic injury. The activated microglial cells then undergo morphological changes and secrete various species of cytokines, promoting the progression of neuroinflammation. After cerebral ischemia, microglia release a series of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), inducible Nitric Oxide Synthase (iNOS) and interleukin 1 beta (il1β), triggering an inflammatory cascade, disrupting the Blood Brain Barrier (BBB), leading to tissue edema, leading to neuronal death. Microglia, on the other hand, also release anti-inflammatory cytokines such as growth factors (tgfβ) and Interleukin (IL) -10 to alleviate neuronal damage caused by ischemic stroke. Microglia thus exhibit a beneficial effect on inflammatory phenotype by transitioning from a pro-inflammatory phenotype to an anti-inflammatory phenotype, making them potential therapeutic targets for ischemic stroke. Autophagy is an important catabolic process, critical to cellular metabolic homeostasis. Cells phagocytose damaged cytoplasmic proteins or organelles during stress under conditions of hypoxia, nutrient deficiency, and infection, and are encapsulated in fusion with lysosomes to form autophagy lysosomes. These autophagosomes degrade their contents, providing nutrition and energy. Recent studies have shown that autophagy plays a role in neuronal survival following cerebral ischemic injury. Unc-51 resembles autophagy-activated kinase 1 (ULK 1), a serine/threonine kinase, as a central promoter regulates downstream autophagy flux by recruiting signals from upstream sensors. LC3, p62 and Beclin1 are key downstream proteins of ULK1 during autophagy. Induction of autophagy results in a diffuse distribution of binding of the autophagosome marker protein LC3 (LC 3-I) to phosphatidylethanolamine, thereby forming lipidated LC3 (LC 3-II). This autophagy cargo protein p62 is degraded, while the other classical autophagy protein Beclin1 biomarker is up-regulated during autophagy. Inhibiting ULK1 inhibits autophagy by knocking down in multiple cells. Previous studies reported that certain drugs can enhance neurons to restore ischemic stroke by upregulating ULK 1. korean et al. Pharmacological inhibition or inhibition of ULK1 expression has been shown to impair autophagy activity and eliminate neuroprotection by pparγ coactivator1 alpha following acute ischemic stroke. However, the role of ULK1 in neuroinflammation and microglial activation following ischemic stroke remains unclear. Through the above analysis, the problems and defects existing in the prior art are as follows: ULK1 is not clear for neuroprotection and microglial activation after ischemic stroke. Disclosure of Invention Aiming at the problems existing in the prior art, the invention provides an application of ULK1 possibly serving as a target for treating ischemic stroke. The present invention is thus embodied in a ULK1 which may be used as a target for treating ischemic stroke comprising: Step one, photo-thrombotic stroke, group and drug treatment, neural behavior assessment; Step two, histological tissue treatment, a nisel staining method, TTC staining of TTC staining examples, TUNEL staining and immunofluorescence staining; step three, western blotting, microglial cell morphology, real-time fluorescence quantitative PCR and statistical analysis. Further, the photo thrombotic stroke (PT): Induction of focal cortical photoimbogenic ischemic stroke in mice using this method was described in previous studies, briefly, mice were intraperitoneally injected with 10mg/mlRose of Bengali (200 μl, sigma; dissolved in physiological saline), after 15min, mice were intraperitoneally injected with pentobarbital sodium (80 mg/kg) under anesthesia with an anesthetic, the skull was exposed to the outer covering anterior limb cortical region under anesthesia, the photoimbogenic process cortex (anterio