Application of Mbtps1 gene and protein SKI-1 coded by Mbtps1 gene in screening or preparing medicine for treating cerebral arterial thrombosis

Abstract

The invention discloses an application of an Mbtps1 gene and a protein SKI-1 coded by the Mbtps1 gene in screening or preparing a medicine for treating cerebral arterial thrombosis, and particularly discloses an application of the Mbtps1 gene / protein SKI-1 as a medicine target of cerebral arterial thrombosis. A tMCAO mouse model is constructed by specifically knocking out a microglial cell Mbtps1 gene / protein SKI-1 of a tMCAO mouse to verify that the Mbtps1 gene and the protein SKI-1 can be used as drug targets for screening or preparing drugs for treating cerebral arterial thrombosis; the compound PF429242 is screened out and has the functions of repairing brain injury nerves caused by cerebral arterial thrombosis, inhibiting neuroinflammation and repairing fragmentation of a mitochondrial network, namely, the feasibility of the Mbtps1 gene and the protein SKI-1 serving as targets in screening or preparing medicines for preventing or treating cerebral arterial thrombosis is verified.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the use of Mbtps1 gene and the protein SKI-1 encoded by the Mbtps1 gene in screening or preparing a drug for treating ischemic stroke. BACKGROUND

[0002] Ischemic stroke (commonly known as stroke) is a major cerebrovascular disease caused by occlusion of cerebral blood vessels, leading to local tissue ischemia and hypoxia, and further causing nerve cell death. The incidence rate of ischemic stroke accounts for more than 80% of all strokes, and it is one of the main causes of disability and death worldwide. With the continuous aging of the population structure, the disease burden is increasing.

[0003] At present, the standard treatment for ischemic stroke in clinic is vascular recanalization therapy, including intravenous thrombolysis and mechanical thrombectomy. However, these therapies have strict treatment time windows, and are often accompanied by fatal cerebral ischemia-reperfusion injury after vascular recanalization, which seriously affects the prognosis of patients. Existing studies have conducted in-depth research on the molecular pathological mechanism of ischemic stroke. A large number of effective candidate drugs have been found through animal experiments. However, preclinical studies have shown that the above-mentioned candidate drugs and therapeutic targets have failed to provide effective neuroprotection for stroke patients. Therefore, the joint research of drug screening and ischemic stroke treatment targets is more significant for the development of ischemic stroke treatment.

[0004] In the complex pathological network of ischemic stroke, blood-brain barrier disruption is a key link and a key hub of brain injury, which triggers two core events of inflammatory infiltration and mitochondrial damage, which promote each other and synergistically lead to the final neuronal damage. Mbtps1 gene is located on human chromosome 16q23.3-q24.1, which encodes a membrane-bound transcription factor peptidase, namely SKI-1 (Subtilisin Kexin Isozyme-1), also known as Site-1 protease (S1P). SKI-1 is the only core functional protein product of Mbtps1 gene, belongs to the proprotein convertase family, is responsible for the processing of protein precursors such as SREBPs and ATF6 into mature forms, and thus participates in lipid biosynthesis, lysosome biogenesis and unfolded protein response. Further studies have found that SKI-1 is involved in mitochondrial metabolism. Inhibition of SKI-1 can reduce kidney damage by rescuing mitochondria in mouse renal ischemia-reperfusion injury. Inhibition of SKI-1 can effectively block viral replication and protect nerve cells, indicating the potential of targeting SKI-1 in the treatment of neurological diseases. However, the role of SKI-1 in the pathological process of ischemic stroke has not been clearly studied. SUMMARY

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present application provides a use of an Mbtps1 gene and a protein SKI-1 encoded by the Mbtps1 gene in screening or preparing a drug for treating ischemic stroke.

[0006] In order to achieve the above-mentioned purpose, the present application provides a use of an Mbtps1 gene and a protein SKI-1 encoded by the Mbtps1 gene in preparing a drug for treating ischemic stroke.

[0007] As one of the purposes of the application, the present application further provides an application of an Mbtps1 gene / protein SKI-1 as a target point of a drug for treating ischemic stroke.

[0008] As one of the purposes of the application, the present application further provides a method for screening a drug for preventing and treating ischemic brain injury, comprising using an Mbtps1 gene / protein SKI-1 as a target point of the drug for preventing and treating ischemic brain injury.

[0009] As one of the purposes of the application, the present application further provides a drug for treating ischemic stroke, wherein an active component of the drug at least comprises an agent for inhibiting an activity of an Mbtps1 gene / protein SKI-1.

[0010] Preferably, the agent for inhibiting the activity of the Mbtps1 gene / protein SKI-1 can bind to the Mbtps1 gene / protein SKI-1.

[0011] Preferably, the agent for inhibiting the activity of the Mbtps1 gene / protein SKI-1 is PF429242.

[0012] Preferably, the only active component in the drug is PF429242.

[0013] Preferably, in the drug, the dosage of PF429242 is 5-7 mg / kg.

[0014] Further preferably, in the drug, the dosage of PF429242 is 6 mg / kg.

[0015] As one of the purposes of the application, the present application further provides a pharmaceutical composition, which at least comprises the drug as described above; and the drug at least comprises PF429242.

[0016] Exemplarily, in specific embodiments, the present application studies the use of an Mbtps1 gene / protein SKI-1 in screening and preparing a drug for treating ischemic stroke by constructing a tMCAO mouse model.

[0017] Specifically, the Cre-Loxp system is applied to construct a knockout model of the microglial cell Mbtps1 gene / protein SKI-1 of the tMCAO mouse, and the specific deletion of the Mbtps1 gene / protein SKI-1 in the microglial cell effectively protects the structural and functional integrity of the blood-brain barrier.

[0018] Further, the knockout of the Mbtps1 gene / protein SKI-1 can repair nerve injury, inhibit nerve inflammation and repair mitochondrial network fragmentation.

[0019] The application has the following beneficial technical effects: the application constructs a tMCAO mouse model, specifically knocks out the microglial cell Mbtps1 gene / protein SKI-1 of the tMCAO mouse model, and the results obtained through various verification methods show the feasibility and specificity of the Mbtps1 gene and protein SKI-1 as a target, and the compound PF429242 is screened out to have a repairing effect on the nerves of the brain injury caused by ischemic stroke, and also has the functions of inhibiting nerve inflammation and repairing mitochondrial network fragmentation, that is, the Mbtps1 gene and protein SKI-1 as a target are verified to be applied to screening or preparing drugs for preventing or treating ischemic stroke. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figures 1A-1D The immunofluorescence quantitative analysis results of the protein SKI-1 expression in the Sham group and the MCAO / R group in a typical embodiment of the application are compared.

[0021] Figures 2A-2C The primary neuron injury in the Ctrl group, the OGD / R group and the OGD / R+PF429242 group in a typical embodiment of the application.

[0022] Figures 3A-3E The mouse nerve function detection experiment results in the Sham group, the MCAO / R group and the MCAO / R+PF429242 group in a typical embodiment of the application.

[0023] Figures 4A-4C The influence of SKI-1 inhibition on nerve inflammation in the cerebral cortex region of a mouse in a typical embodiment of the application.

[0024] Figures 5A-5F The influence of SKI-1 inhibition on the structure and function of mitochondria in a typical embodiment of the application.

[0025] Figures 6A-6F The influence of specific knockout of Mbtps1 in microglial cells on blood-brain barrier damage after cerebral ischemia-reperfusion injury in a typical embodiment of the application. DETAILED DESCRIPTION

[0026] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application.

[0027] The use of an Mbtps1 gene and a protein SKI-1 in the preparation of a drug for treating ischemic stroke.

[0028] In specific embodiments, based on a tMCAO mouse model,

[0029] (1) Application of a Cre-Loxp system to construct a microglial cell Mbtps1 gene-specific knockout male mouse

[0030] Two loxP sites are inserted on both sides of exon 2 of the Mbtps1 gene of the mouse to construct an Mbtps1 floxed mouse strain-Mbtps1 f / f The Cx3crl gene is used to express a Cre enzyme in microglial cells creERT2 The mouse strain is crossed with the Mbtps1 f / f gene of the offspring Mbtps1 f / f Cx3cr1 creERT2 Mouse is dosed to achieve microglial cell-specific knockout, resulting in a microglial cell knockout (Conditonal knockout, CKO, Mbtps1 f / f Cx3cr1 creERT2 ), which is referred to as CKO mouse in the present application.

[0031] 2-month-old control (WT, Mbtps1 f / f ) and CKO male mice are used to prepare a mouse transient middle cerebral artery occlusion (tMCAO) model using a thread plug method, and neural function scoring and cerebral infarction volume measurement are performed.

[0032] More specifically, 1) Construct a tMCAO mouse model: select C57BL / 6J male mice with a body weight of 20-22 g, and establish using a thread plug method.

[0033] First, the mice were intraperitoneally injected with 3% sodium pentobarbital for anesthesia, fixed in a supine position, and the neck skin was disinfected. A longitudinal incision about 1 cm long was made on the midline of the neck. The fascia was bluntly separated to expose the right vagus nerve, common carotid artery, external carotid artery, and internal carotid artery. A ligature was reserved at the proximal end of the common carotid artery, the external carotid artery was ligated and coagulated at the distal end, the external carotid artery was freed, a ligature was reserved at the proximal end of the external carotid artery, a small vessel clip was used to occlude the proximal common carotid artery and distal internal carotid artery, a small opening was made at the distal end of the external carotid artery with microscissors, and a thread plug was inserted through the common carotid artery into the internal carotid artery. After the operation, the external carotid artery was ligated.

[0034] After 60 minutes of MCAO, the fixation line was loosened, the thread plug was removed, the common carotid artery ligature was loosened to achieve reperfusion, and the wound was sutured routinely. After the operation, the animals were placed in a feeding box with clean bedding and allowed to drink water and eat freely.

[0035] Laser speckle imaging was used to confirm whether the MCAO model was successful.

[0036] After inserting the thread plug, the cerebral blood flow (CBF) in the middle cerebral artery supply area rapidly decreased to less than 20%-30% of the baseline value. During reperfusion, for the tMCAO model, after removing the thread plug, the blood flow should be significantly restored (usually to more than 50%-70% of the baseline value), indicating successful recanalization.

[0037] 2) Neurological function evaluation. The effects of SKI-1 on improving mouse behavior were detected by combining open field test, mouse rotating rod test, mouse pulling test, and Longa score.

[0038] 3) TTC (2,3,5-chlorinated triphenyl tetrazolium) staining was used to evaluate the cerebral infarction volume.

[0039] After 24 hours of reperfusion, the mice were deeply anesthetized and sacrificed. The brain was quickly removed and placed on a slide. The brain tissue was frozen at -80°C. After 2 hours, the olfactory bulb and cerebellum were removed and quickly cut into 6 slices, with 1 slice per well. 3% TTC staining solution was added, and the slices were incubated at 37°C for 15 minutes. During this period, the slices were gently shaken several times to ensure uniform staining. After staining, the slices were gently rinsed with PBS for 1-2 times to terminate the reaction.

[0040] 4% paraformaldehyde solution was added to the brain slices and fixed overnight. Photographs were taken and measurements were made.

[0041] (2) Western blot and immunofluorescence staining were used to detect the expression of mouse protein SKI-1.

[0042] Western blot: Mice in each group were euthanized by decapitation 24 hours after cerebral ischemia-reperfusion surgery. Protein was extracted from the ipsilateral cerebral cortex and homogenized thoroughly at 4°C with RIPA tissue lysis buffer containing protease inhibitors and phosphatase inhibitors. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein content was determined using the BCA method to ensure consistent sample loading. Samples containing equal amounts of protein (20-40 μg) were calculated and prepared. After high-temperature denaturation with loading buffer, the samples were separated by electrophoresis using a 10% SDS-PAGE gel. Proteins were transferred from the gel to a PVDF membrane using a wet transfer method and blocked with TBST buffer containing 3% BSA at room temperature for 1 h to reduce non-specific binding. The membrane was incubated with primary antibody overnight, followed by incubation with the corresponding secondary antibody based on the species of the primary antibody. The membrane was washed three times with 1×TBST, exposed to light, developed, and the expression level of SKI-1 in the brain tissue was analyzed.

[0043] Immunofluorescence staining: Mice were sacrificed 24 hours after reperfusion, and fixed by perfusion with 1×PBS buffer and 4% paraformaldehyde. Brain tissue was harvested, fixed with 4% paraformaldehyde for 24 hours, then dehydrated with 20% sucrose solution until the brain tissue settled. The olfactory bulb and hindbrain were removed. The brain tissue near the olfactory bulb was placed vertically in an embedding cassette lined with OCT gel, and frozen tissue was stored at -80°C. The brain tissue was cut into 10μm sections using a cryostat. The sections were equilibrated at room temperature for 0.5 hours in a humidified chamber, rinsed three times in a glass bath containing pre-chilled PBS, and dried with lint-free paper. The brain tissue was circled with a histochemical pen and incubated with 10% donkey serum at room temperature for 30 minutes, then dried with lint-free paper. The appropriate primary antibody was then added, and the tissue was incubated overnight at 4°C. The sections were rinsed three times with pre-chilled PBS and dried with lint-free paper. The appropriate secondary antibody was added, and the tissue was incubated at room temperature in the dark for 1.5 hours, rinsed three times, and dried with lint-free paper. Mount the slide with a DAPI-containing anti-fluorescence quencher and image using a laser confocal microscope.

[0044] (3) Flow cytometry, Calcein-AM / PI staining and CCK8 assay were used to detect primary neuronal damage.

[0045] Flow cytometry: To quantitatively analyze the effect of SKI-1 on MCAO-induced cell death, flow cytometry was performed using the Annexin V-FITC / PI double staining method. In brief, collected and resuspended cells were first incubated with Annexin V-FITC under light-protected conditions, followed by the addition of PI staining solution before flow cytometry. Detection was performed using a flow cytometer, and analysis was conducted using FlowJo V10 software. Cell populations were gating based on the fluorescence intensity of Annexin V and PI: Annexin V... - / PI - For living cells, Annexin V + / PI -For early apoptotic cells, Annexin V + / PI + These are late-stage apoptotic / necrotic cells. The experiment was repeated three times.

[0046] Cell viability was assessed using a Calein-AM / PI double staining kit. Specifically, cells were incubated with 2 μM Calein-AM and 4.5 μM PI at 37°C for 30 minutes. Subsequently, fluorescence microscopy was used for observation. Live cells produced green fluorescence (Calcein) due to esterase activity, while dead cells produced red nuclear fluorescence due to PI labeling caused by membrane rupture. Multiple fields of view were randomly selected for imaging, and cell viability was calculated using ImageJ software: viability % = number of green fluorescent cells / total cell number × 100%.

[0047] CCK8 assay: Digest and resuspend cells, count cells using a cell counting chamber, adjust cell density to the desired level, seed cells in 96-well plates, and incubate at 37°C with 5% CO2 until cells are fully adherent and enter the logarithmic growth phase (usually 12-24 hours). Replace with fresh medium containing different concentrations of the drug, treat for specific time periods, add CCK8 stock solution, and return to the 37°C incubator for 1-4 hours in the dark, then measure absorbance.

[0048] (4) Evans Blue (EB) staining and FITC-Dextran were used to assess the integrity / permeability of the blood-brain barrier. EB or FITC-Dextran was injected via the tail vein, allowing the dye to circulate in vivo for 2–4 hours. Mice were anesthetized and perfused with physiological saline through the heart until the outflow was colorless to completely remove the dye from the blood vessels. The brain was then harvested, and the blue leakage area was photographed and recorded. Alternatively, OCT-embedded sections were prepared for direct observation under a fluorescence microscope.

[0049] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0050] Example 1

[0051] This embodiment provides the use of the Mbtps1 gene and protein SKI-1 in the preparation of drugs for the prevention / treatment of ischemic stroke, and animal experiments were conducted to verify the pharmacological mechanism based on the MCAO mouse model.

[0052] 1. Pharmacological effects of SKI-1 inhibition on brain injury in MCAO / R mice

[0053] Thirty-two 6-week-old male C57BL / 6 mice were randomly divided into four groups (n=8): the Sham group, the MCAO / R group, and the MCAO / R+PF429242 group. They were housed in a quiet environment with free access to water and food. One week later, the Sham group underwent sham surgery. After anesthesia, the neck skin was incised, and the carotid artery was separated, but no suture was inserted. The Sham group experienced the same anesthesia time, surgical trauma, and exposure as the MCAO / R group. In the MCAO / R+PF429242 group, mice were intraperitoneally injected with 6 mg / kg PF429242 before surgery. The MCAO / R and MCAO / R+PF429242 models of transient middle cerebral artery occlusion (MCAO) in mice were established using the suture occlusion method. The specific method is as follows: Mice were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, fixed in a supine position, and the neck skin was disinfected. A longitudinal incision of approximately 1 cm was made along the midline of the neck. The fascia was bluntly dissected to expose the right vagus nerve, common carotid artery, external carotid artery, and internal carotid artery. A ligation suture was left proximally on the common carotid artery, and the external carotid artery was ligated and coagulated distally. The external carotid artery was freed, and a ligation suture was left proximally on the external carotid artery. A small, non-invasive vascular clamp was used to clamp the proximal common carotid artery and the distal internal carotid artery. A small incision was made at the distal end of the external carotid artery with microsurgical scissors, and a suture occluded was inserted through the common carotid artery into the internal carotid artery. After the procedure, the external carotid artery was ligated. Sixty minutes after MCAO, the fixation sutures were loosened, the suture plug was removed, and the carotid artery ligation suture was released to achieve reperfusion. The wound was then routinely sutured. Postoperatively, the animal was placed in a feeding box with clean bedding and allowed free access to water and food. Laser speckle imaging was used to confirm the success of the MCAO model. After suture plug insertion, cerebral blood flow (CBF) in the middle cerebral artery supply area rapidly decreased to below 20%-30% of baseline. During reperfusion, for the MCAO model, blood flow should significantly recover after suture plug removal (usually recovering to over 50%-70% of baseline), indicating successful vascular recanalization.

[0054] 2. Effects of conditional knockout of Mbtps1 on blood-brain barrier injury in mice

[0055] Male mice with Mbtps1 gene knockout specific in microglia were constructed using the Cre-Loxp system.

[0056] The Mbtps1 floxed mouse strain was constructed by inserting two loxP sites flanking key exon 2 of the Mbtps1 gene in male C57BL / 6 mice. f / f .

[0057] Using microglia to specifically express Cre enzyme Cx3cr1 creERT2 Mouse strains and Mbtps1 f / f Hybridization, in its offspring Mbtps1f / f Cx3cr1 creERT2 Mice were administered the drug to achieve microglia-specific knockout (CKO, Mbtps1) microglia. f / f Cx3cr1 creERT2 In this embodiment, the mice are referred to as CKO mice.

[0058] A 2-month-old control group (WT group, Mbtps1) was used. f / f A transient middle cerebral artery occlusion (tMCAO) model was established in mice using male CKO mice and WT mice via the suture occlusion method. Twenty-four 6-week-old mice were divided into three groups (n=8): WT group, WT+MCAO / R group, and CKO+MCAO / R group, to detect blood-brain barrier damage.

[0059] In this example, fluxed mice carrying homozygous conditional alleles were first used as the control group (WT0 group) and the results were compared with those of the WT group and the experimental group. Previous experimental verification showed that the insertion of the loxP site itself did not cause changes in the relevant phenotypes detected. This result indicates that the experimental group and the control group (WT group) have completely consistent genetic backgrounds. Therefore, any phenotypic differences observed in the experimental group and the WT group can be attributed to the deletion of the Mbtps1 gene.

[0060] Furthermore, the mice in each of the above groups were compared and evaluated separately:

[0061] (1) Neurological function was evaluated using the MCAO mouse model, including open field test, mouse rotarod test, mouse pulling test, and Longa score to detect the effect of SKI-1 inhibition on improving mouse behavior.

[0062] Open field test: A mouse is placed in a square open box, immediately removed, and left to stand still for 10 minutes for video recording. After the video recording, the mouse is removed, the box is cleaned to remove the odor, and then the next mouse is placed in. The spontaneous activity is assessed by analyzing the mouse's movement trajectory.

[0063] Mouse rotundus test: Mice are made to walk on a rotating rotundus, and the time it takes for them to fall off is recorded. This test is mainly used to assess a mouse's motor coordination, balance, and endurance. A shorter fall time generally indicates poorer motor coordination.

[0064] Mouse pulling force test: Have a mouse grasp a T-shaped force-measuring bar with its front paws, then gently pull its tail until its front paws release. Measure the maximum gripping force it can exert before releasing the bar. Repeat the measurement 2-3 times, with short intervals between each measurement. Record the maximum pulling force value and analyze the average of the multiple measurements.

[0065] (2) TTC (2,3,5-triphenyltetrazolium chloride) staining to assess cerebral infarction volume

[0066] Mice were deeply anesthetized and sacrificed 24 hours after reperfusion. Brain tissue was rapidly harvested, placed on glass slides, and frozen at -80°C. After 2 hours, the olfactory bulb and cerebellum were removed, and the tissue was quickly sliced ​​into 6 sections on ice, one section per well. 3% TTC staining solution was added, and the slides were incubated at 37°C for 15 minutes, gently agitating several times during the incubation period to ensure even staining. After staining, the slides were gently rinsed 1-2 times with PBS to terminate the reaction. 4% paraformaldehyde solution was added to the brain slices. The slides were fixed overnight, photographed, and measured.

[0067] (3) Western blot and immunofluorescence staining to detect the expression of mouse protein SKI-1

[0068] Western blot: Mice in each group were euthanized by decapitation 24 hours after cerebral ischemia-reperfusion surgery. Proteins were extracted from the ipsilateral cerebral cortex and homogenized thoroughly at 4°C with RIPA tissue lysis buffer containing protease inhibitors and phosphatase inhibitors. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. Protein content was determined using the BCA method to ensure consistent sample loading. Samples containing equal amounts of protein (20–40 μg) were calculated and prepared. After high-temperature denaturation with loading buffer, the samples were separated by electrophoresis using a 10% SDS-PAGE gel. Proteins were transferred from the gel to a PVDF membrane using a wet transfer method and blocked with TBST buffer containing 3% BSA at room temperature for 1 h to reduce non-specific binding. The membrane was incubated overnight with primary antibody (SKI-1, occluding, ZO-1) and then with mouse secondary antibody. The membrane was washed three times with 1×TBST, exposed to light, developed, and the expression level of SKI-1 in the brain tissue was analyzed.

[0069] Immunofluorescence staining: Mice were sacrificed 24 hours after reperfusion, and fixed by perfusion with 1×PBS buffer and 4% paraformaldehyde. Brain tissue was harvested, fixed with 4% paraformaldehyde for 24 hours, then dehydrated with 20% sucrose solution until the brain tissue settled. The olfactory bulb and hindbrain were removed. The brain tissue near the olfactory bulb was placed vertically in an embedding cassette lined with OCT gel, and frozen tissue was stored at -80°C. The brain tissue was cut into 10μm sections using a cryostat. The sections were equilibrated at room temperature for 0.5 hours in a humidified chamber, rinsed three times in a glass bath containing pre-chilled PBS, and dried with lint-free paper. The brain tissue was circled with a histochemical pen, incubated with 10% donkey serum at room temperature for 30 minutes, and dried with lint-free paper.

[0070] Then, SKI-1 primary antibody was added, and the mixture was incubated overnight at 4°C. After pre-cooling (4°C), the slides were rinsed three times with PBS, dried with lint-free paper, and then mouse secondary antibody was added. The slides were incubated at room temperature in the dark for 1.5 hours, rinsed three times, and dried with lint-free paper. A mounting medium containing DAPI (MCE) was added, and the slides were photographed using a laser confocal microscope.

[0071] (4) Quantitative analysis of the effect of SKI-1 on MCAO-induced cell death

[0072] Flow cytometry

[0073] Flow cytometry analysis was performed using the Annexin V-FITC / PI double staining method. The process included collecting and resuspending cells, incubating them with Annexin V-FITC in the dark, and then adding PI staining solution before flow cytometry. Flow cytometry analysis was then performed using FlowJo V10 software. Cell populations were gating based on the fluorescence intensity of Annexin V and PI: Annexin V... - / PI - For living cells, Annexin V + / PI - For early apoptotic cells, Annexin V + / PI + These are late-stage apoptotic / necrotic cells. The experiment was repeated three times.

[0074] Calcein-AM / PI staining

[0075] Cell viability was assessed using a Calcein-AM / PI double staining kit (Beyotime). Specifically, cells were incubated with 2 μM Calcein-AM and 4.5 μM PI at 37°C for 30 minutes. Subsequently, fluorescence microscopy was used for observation. Live cells produced green fluorescence (Calcein) due to esterase activity, while dead cells produced red nuclear fluorescence due to PI labeling caused by membrane rupture. Multiple images were taken from random angles, and cell viability was calculated using ImageJ software: viability % = (number of green fluorescent cells / total number of cells) × 100%.

[0076] CCK8 Experiment

[0077] Digest and resuspend the cells, count them using a cell counting chamber, adjust the cell density to the desired level, seed the cells in 96-well plates, and incubate at 37°C in a 5% CO2 incubator until the cells are fully adherent and enter the logarithmic growth phase (usually 12-24 hours). Replace the medium with fresh medium containing different concentrations of the drug, treat for specific time periods, add CCK8 stock solution, and return to the 37°C incubator for 1-4 hours in the dark, then measure the absorbance.

[0078] (5) Assess the integrity / permeability of the blood-brain barrier.

[0079] Evans Blue (EB) staining and FITC-Dextran were used to assess the integrity / permeability of the blood-brain barrier. Specifically, EB 5 mg / kg and FITC-Dextran 10 mg / kg were injected via the tail vein, allowing the dye to circulate in vivo for 2-4 hours.

[0080] Mice were anesthetized and perfused with physiological saline through the heart until the outflow was colorless to completely remove the dye from the blood vessels. The brain was then removed, and the blue leakage area was photographed and recorded. Alternatively, the brain was embedded in OCT and frozen sections were prepared for direct observation under a fluorescence microscope.

[0081] See Figure 1A As shown, SKI-1 is basally expressed in normal brain tissue, with its main expression area concentrated in the cerebral cortex; see reference Figure 1B The immunofluorescence quantitative analysis of each experimental group and control group is shown in the figure. Compared with the ischemic contralateral brain region in the Sham group, the positive signal area of ​​SKI-1 in the ischemic ipsilateral brain region was significantly increased.

[0082] See each Figure 1C and Figure 1D The results of Western Blot (WB) and ELISA, as shown, further confirm that SKI-1 expression was significantly upregulated after ischemia-reperfusion, indicating that SKI-1 has a repairing effect on ischemic brain injury.

[0083] Figures 2A-2C The results showed that inhibiting SKI-1 could significantly reduce oxygen-glucose deprivation / reperfusion (OGD / R)-induced primary neuronal damage (S1P in the figure is SKI-1, the same below).

[0084] Figure 2A The study used Calcein AM (labeling live cells) and Propidium Iodide (PI, labeling dead cells) for fluorescent double staining. The results showed that compared with the OGD / R model group, the proportion of live cells in the PF429242 treatment group was significantly increased, while the proportion of dead cells was correspondingly reduced, indicating that inhibiting SKI-1 effectively maintained the integrity of the neuronal membrane and cell viability.

[0085] Figure 2B Quantitative analysis using flow cytometry further confirmed that inhibiting SKI-1 can significantly reduce neuronal mortality induced by OGD / R, indicating that it has a significant neuroprotective effect.

[0086] Figure 2CThe image shows a comparison of CCK-8 proliferation detection results. The results indicate that PF429242 treatment can significantly reverse the decrease in cell activity caused by OGD / R, further confirming from the perspective of metabolic activity that inhibiting SKI-1 has a significant neuroprotective effect on neurons.

[0087] Figure 3A The laser speckle contrast imaging (CBF) image shown illustrates representative blood flow images and quantitative analysis results of the middle cerebral artery supply area in mice at baseline, immediately after occlusion, and after reperfusion. CBF significantly decreased after occlusion and recovered after reperfusion following thrombectomy, confirming the successful establishment of the MCAO / R model.

[0088] Figure 3B The figure shows a comparison of open field tests between mice in the Sham group and the MCAO / R model group. As shown in the figure, compared with the MCAO / R model group, inhibiting SKI-1 in the Sham group significantly increased the total movement distance and central region activity time of mice, indicating that the overall motor function was improved.

[0089] Figure 3C The image shows a comparison of the rotarod test results of mice in each experimental group and the control group. On the accelerated rotarod, the fall latency of the MCAO / RPF429242 group mice was significantly longer than that of the control group, indicating that their motor coordination and balance abilities were improved after ischemic injury.

[0090] Figure 3D The image shows a comparison of grip strength tests between mice in the experimental and control groups. The results indicate that inhibiting SKI-1 significantly prolonged the fall latency and improved grip strength in mice.

[0091] Figure 3E The neurological function of mice in the experimental and control groups was scored using the Longa 5-point scale. The results showed that the neurological function score of mice inhibited by SKI-1 was significantly reduced, indicating that the degree of neurological deficit in the MCAO+RPF429242 group was significantly reduced compared with the MCAO / R model group.

[0092] Figures 3A-3E The results showed that inhibiting SKI-1 could alleviate brain damage and improve neurological function in MCAO / R model mice.

[0093] Figures 4A-4C Proteomics analysis was performed on the Sham group, MCAO / R group, and MCAO / R+RPF429242 group, respectively. The results showed that inhibiting SKI-1 could significantly reduce the neuroinflammatory response after cerebral ischemia-reperfusion (MCAO / R). Figure 4AThe proteomic analysis shown indicates that, compared with the MCAO / R group, the MCAO / R+PF429242 treatment significantly suppressed the inflammatory response. Further validation revealed that the protein expression levels of several key pathway-related inflammatory factors, including S100A8, S100A9, and complement C1qb, were significantly downregulated. Further statistical analysis of the above results showed that... Figure 4B As shown, S100A8 was significantly reduced in the MCAO / R+PF429242 group after treatment with PF429242.

[0094] Figure 4C HE staining results showed that treatment with compound PF429242 reduced inflammatory infiltration in the cerebral cortex of mice, indicating that PF429242 can significantly inhibit inflammatory infiltration after cerebral ischemia-reperfusion (MCAO / R).

[0095] Figure 5A Proteomics analysis showed that inhibiting SKI-1 significantly upregulated differentially expressed proteins associated with the mitochondrial electron transport chain (ETC), especially the core subunits of complex I (NADH dehydrogenase) and complex IV (cytochrome c oxidase), suggesting that it can enhance mitochondrial oxidative phosphorylation function.

[0096] Further statistical analysis of the above results yielded the following results: Figure 5B As shown, the changes in the expression of mitochondrial electron transport chain-related proteins were statistically significant, indicating that SKI-1 inhibition has a clear regulatory effect on ETC function.

[0097] Figure 5C ELISA was used to quantify complex I, and the results further confirmed that inhibiting SKI-1 could significantly increase the protein expression level of the complex, thus verifying its role in restoring mitochondrial function at the biochemical level. Figure 5D To assess changes in mitochondrial membrane potential (ΔΨm), staining was performed using the JC-1 fluorescent probe. As shown in the figure, compared with the OGD / R group, the PF429242 group showed significantly enhanced red fluorescence intensity, weakened green fluorescence, and a significantly increased red / green fluorescence ratio (*P<0.05), indicating that the inhibition of SKI-1 mitochondrial membrane potential was repaired and mitochondrial membrane potential was enhanced. Figure 5E Flow cytometry quantitative analysis further confirmed that inhibiting SKI-1 can significantly reduce the disruption of mitochondrial membrane potential.

[0098] Figure 5F Mito-Tracker staining images for each group confirmed that the fragmentation of the mitochondrial network was repaired after SKI-1 inhibition, indicating that SKI-1 induces cell damage by disrupting the integrity of mitochondrial structure and function after ischemia.

[0099] The aboveFigures 5A-5F The results showed that inhibiting the SKI-1 signaling pathway can significantly improve mitochondrial dysfunction.

[0100] like Figure 6A The results of routine blood tests on mouse serum showed that neutrophils were highly expressed in CKO mice, and the infiltration of inflammatory factors in the brain parenchyma was reduced.

[0101] Figures 6B-6F The protective effect of CKO on the blood-brain barrier (BBB) ​​was demonstrated from different perspectives. Specifically, this included: Figure 6B The results of the EB bleed-out experiment showed that the amount of dye bleed-out in the CKO group was significantly reduced, indicating that the BBB permeability was reduced. Figure 6C These are transmission electron microscope (TEM) images. The results show that the tight junction structure of cerebral vascular endothelial cells was more intact in the CKO group. Figure 6D The in vivo imaging images of small animals further visually demonstrate that the leakage of the circulating fluorescent tracer into the brain parenchyma was significantly reduced in the CKO group; Figure 6E Immunofluorescence techniques revealed a reduction in GLUT-I and FITC co-localization in the CKO group. Figure 6F Western blot analysis confirmed a decrease in occludin and ZO-1 in the CKO group.

[0102] Figures 6A-6F This indicates that specific knockout of SKI-1 in microglia can significantly reduce blood-brain barrier disruption following cerebral ischemia-reperfusion injury.

[0103] The above experimental results indicate that the specific loss of SKI-1 in microglia effectively protects the structural integrity and function of the blood-brain barrier by inhibiting neuroinflammation. The screened compound PF429242 can bind to SKI-1 protein to inhibit inflammatory infiltration after cerebral ischemia-reperfusion injury and reduce mitochondrial damage.

[0104] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by realizing the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. The use of the Mbtps1 gene and its encoded protein SKI-1 in screening or preparing drugs for the treatment of ischemic stroke.

2. Application of Mbtps1 gene / protein SKI-1 as a drug target for ischemic stroke.

3. A method for screening or preparing drugs for ischemic stroke, characterized in that, This includes using the Mbtps1 gene / protein SKI-1 as a target for screening or preparing drugs to prevent and treat ischemic stroke.

4. The method as described in claim 3, characterized in that, This includes conducting animal trials on screened or prepared drugs by constructing tMCAO mouse models.

5. The method as described in claim 3, characterized in that, The method includes knocking out the protein SKI-1 in microglia of a tMCAO mouse model, which protects the structure of the blood-brain barrier from ischemic stroke.

6. A drug for treating ischemic stroke, characterized in that, Its active ingredients include at least a reagent that inhibits the activity of the Mbtps1 gene / protein SKI-1; The reagent that inhibits the activity of the Mbtps1 gene / protein SKI-1 can bind to the Mbtps1 gene / protein SKI-1.

7. The drug as described in claim 6, characterized in that, The reagent used to inhibit the activity of the Mbtps1 gene / protein SKI-1 is PF429242.

8. The drug as described in claim 6, characterized in that, The only active ingredient in the drug is PF429242.

9. The drug as described in claim 6, characterized in that, The dosage of PF429242 is 5–7 mg / kg.

10. A pharmaceutical composition comprising at least the medicament as described in any one of claims 6-9.

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