Application of PEAK1 as cerebral ischemia reperfusion injury diagnosis and treatment target

Abstract

The invention discloses application of PEAK1 as a target spot for diagnosing and treating cerebral ischemia reperfusion injury. The invention provides relevance between PEAK1 and cerebral arterial thrombosis induced blood-brain barrier dysfunction, proves that PEAK1 participates in the pathological process of cerebral ischemia-reperfusion injury, and shows that PEAK1 can be used as a new target for screening drugs for preventing and treating cerebral ischemia-reperfusion injury. Besides, the invention provides a targeted agonist erlotinib hydrochloride of the PEAK1 through database screening and experimental verification, proves that the erlotinib hydrochloride can improve the cerebral ischemia reperfusion injury by improving the expression level of the PEAK1, and provides a new strategy for drugs for treating the cerebral ischemia reperfusion injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to the application of PEAK1 as a diagnostic and therapeutic target for cerebral ischemia-reperfusion injury. Background Art

[0002] Ischemic stroke is a blood circulation disorder caused by cerebral vascular embolism, resulting in adverse effects such as cerebral tissue ischemia and hypoxia. It has characteristics such as high incidence, high recurrence rate, high fatality rate, and high disability rate. It is a common refractory disease that seriously endangers human life health and quality of life. At present, the main treatment methods for ischemic stroke are thrombolytic therapy and mechanical thrombectomy. However, they have characteristics such as a short treatment time window and easy occurrence of reperfusion injury after thrombolysis (i.e., cerebral ischemia-reperfusion (CIRI)). Therefore, it is of great significance to actively search for therapeutic drugs and potential action targets for ischemic stroke.

[0003] Multiple damages will occur after cerebral ischemia-reperfusion. Among them, the destruction of the blood-brain barrier (BBB) is the core initiating link. The BBB is composed of tight junctions and adherens junctions of endothelial cells. Among them, tight junction proteins and the cytoskeleton form a complex protein structure, which determines the integrity of the BBB. In the state of ischemia and hypoxia, rearrangement of the endothelial cell cytoskeleton structure, degradation of tight junction proteins between endothelial cells, and leukocyte transendothelial adhesion and migration will lead to the destruction of the BBB, and then cerebral ischemia will occur. Among them, rearrangement of the endothelial cell cytoskeleton is the main factor for the early destruction of the BBB, which will lead to a change in the planar cell polarity of endothelial cells, and then increase vascular permeability, resulting in the destruction of the BBB. Pseudopodium-enriched atypical kinase 1 (PEAK1) is an atypical tyrosine kinase related to the actin cytoskeleton, located in actin filaments and focal adhesions, and contains binding sites for cytoskeleton effector proteins, which can change the phosphorylation level of cytoskeleton-related proteins. Therefore, it is suggested that PEAK1 may affect the endothelial barrier injury by changing the planar cell polarity of endothelial cells through its cytoskeleton function, and play a role in protecting the BBB. In addition, the research group of the inventor found significant differences in PEAK1 before and after MCAO / R modeling through transcriptomic analysis in the early stage, suggesting that PEAK1 is involved in the pathological process of cerebral ischemia-reperfusion injury. However, whether PEAK1 plays an important role in the BBB injury process after cerebral ischemia-reperfusion has not been reported. Summary of the Invention

[0004] One of the purposes of the present invention is to provide the application of PEAK1 as a diagnostic marker in the preparation of a reagent for diagnosing cerebral ischemia-reperfusion injury.

[0005] The second object of the present invention is to provide the use of a reagent for detecting PEAK1 in the preparation of a diagnostic reagent for cerebral ischemia-reperfusion injury.

[0006] The third object of the present invention is to provide the use of PEAK1 as a therapeutic target in screening drugs for preventing and treating cerebral ischemia-reperfusion injury.

[0007] The fourth object of the present invention is to provide the use of a PEAK1 agonist in the preparation of a drug for preventing and treating cerebral ischemia-reperfusion injury.

[0008] Furthermore, the PEAK1 agonist is erlotinib hydrochloride.

[0009] The present invention provides the correlation between PEAK1 and the disruption of blood-brain barrier function induced by ischemic stroke, confirms that PEAK1 is involved in the pathological process of cerebral ischemia-reperfusion injury, and indicates that PEAK1 can be used as a new target for screening drugs for preventing and treating cerebral ischemia-reperfusion injury. In addition, the present invention provides the PEAK1-targeted agonist erlotinib hydrochloride through database screening and experimental verification, and confirms that erlotinib hydrochloride can improve cerebral ischemia-reperfusion injury by increasing the expression level of PEAK1, providing a new strategy for drugs for treating cerebral ischemia-reperfusion injury.

[0010] Beneficial effects:

[0011] 1. The present invention provides an experimental basis for PEAK1 to be involved in the disruption of blood-brain barrier function induced by ischemic stroke, indicating that PEAK1 can be used as a new target for screening drugs for preventing and treating cerebral ischemia-reperfusion injury.

[0012] 2. The present invention confirms through drug database screening and experimental verification that the marketed drug erlotinib hydrochloride can target PEAK1 and increase the expression level of PEAK1, and provides the PEAK1 agonist erlotinib hydrochloride.

[0013] 3. The present invention first confirms the application of the PEAK1 agonist erlotinib hydrochloride in drugs for treating cerebral ischemia-reperfusion injury, indicating that erlotinib hydrochloride can be used for the new indication of treating ischemic stroke reperfusion injury. The current experimental results provide strong support and basis for the broad application of the new medical use of erlotinib hydrochloride. Description of the Drawings

[0014] Figure 1Expression changes and localization results of PEAK1 in the brain tissues of MCAO / R mice. Panel A shows the protein expression of PEAK1 in the brain tissues of C57BL / 6J mice after 1 h of middle cerebral artery occlusion (MCAO) and reperfusion for 0, 3, 6, 12, and 24 h (n = 6). Panel B shows the immunofluorescence microscopy images of the brain tissues of C57BL / 6J mice after MCAO for 1 h and reperfusion for 3 h. In the figure, green represents PEAK1, red represents CD31, and blue represents the cell nucleus (n = 3). The results are expressed as mean ± SD. # P < 0.05, ## P < 0.01 vs. Sham group).

[0015] Figure 2 Docking results of erlotinib and PEAK1 (AutoDock 4.1 software was used to dock erlotinib and PEAK1, and PyMol software was used for visualization analysis. The binding free energy of erlotinib and PEAK1 was -9.92 kcal / mol).

[0016] Figure 3 Results of MST detection of the interaction between erlotinib and PEAK1 (Erlotinib can bind to PEAK1, and the Kd value is 564.37 ± 91.36 nM).

[0017] Figure 4 Results of erlotinib reducing the cerebral infarction volume and neurological behavior score in MCAO / R model mice (mean ± SD, n = 6, ## P < 0.01 vs. Sham group, ** P < 0.01 vs. MCAO / R group).

[0018] Figure 5 Results of erlotinib improving cerebral blood flow and pathological damage of brain tissues in MCAO / R model mice (mean ± SD, n = 6, ## P < 0.01 vs. Sham group, **P < 0.01 vs. MCAO / R group).

[0019] Figure 6 Results of erlotinib reducing the leakage amount of Evans blue in the brain tissues of mice induced by cerebral ischemia-reperfusion (mean ± SD, n = 6, ## P < 0.01 vs. Sham group, **P < 0.01 vs. MCAO / R group).

[0020] Figure 7Results of erlotinib increasing the expression levels of tight junction proteins ZO-1 and Occludin in the brain tissues of MCAO / R model mice. Panel A shows the detection of Occludin expression in mouse brain tissues using Western blot technique (n = 6). Panel B shows the detection of ZO-1 expression in mouse brain tissues using Western blot technique (n = 6). Panel C shows the determination of ZO-1 expression level in mouse brain tissues using immunofluorescence staining technique (n = 3). The results are expressed as mean ± SD. ## P < 0.01 vs. Sham group, ** P < 0.01 vs. MCAO / R group).

[0021] Figure 8 Results of erlotinib reducing the expression of matrix metalloproteinases MMP-2 / 9 in the brain tissues of MCAO / R model mice (mean ± SD, n = 6, ## P < 0.01 vs. Sham group, **P < 0.01 vs. MCAO / R group).

[0022] Figure 9 Results of the effect of erlotinib on the PEAK1 protein level in the brain tissues of MCAO / R model mice. Panel A shows the detection of the PEAK1 protein level expression in mouse brain tissues using Western blot technique (n = 6). Panel B shows the determination of the PEAK1 protein expression level in the blood-brain barrier of mouse brain tissues using immunofluorescence staining technique (n = 3). The results are expressed as mean ± SD. ## P < 0.01 vs. Sham group, ** P < 0.01 vs. MCAO / R group. Specific implementation manners

[0023] Although great progress has been made globally in the pathological mechanism and diagnosis of ischemic stroke, there is still a lack of effective therapeutic drugs. Currently, the main treatment for acute ischemic stroke is tPA thrombolytic therapy, but it can cause reperfusion injury and further exacerbate brain injury, and relevant therapeutic drugs are still in a very scarce state. The blood-brain barrier is a key pathological initiating link after cerebral ischemia-reperfusion. Therefore, based on the pathological process of early blood-brain barrier permeability changes, searching for drugs to protect the endothelial barrier can provide a new strategy for the prevention and treatment of cerebral ischemia-reperfusion injury, with broad market space. However, the association between the potential drug target PEAK1 and the disorder of the blood-brain barrier in ischemic stroke is not yet clear, and the potential drug based on targeting PEAK1 for the treatment of cerebral ischemia-reperfusion injury, that is, the agonist of PEAK1, has not been reported yet, so it has good development prospects.

[0024] In this invention, Western blot and immunofluorescence techniques were used to confirm that PEAK1 is involved in the pathological process of cerebral ischemia-reperfusion injury. By searching the DrugBank drug database and batch predicting the binding of different drugs to the PEAK1 target, and through molecular docking technology, the marketed drug Erlotinib hydrochloride with the highest score was virtually docked with PEAK1. Finally, the microscale thermophoresis technique was used to verify that there is good binding activity between the marketed drug Erlotinib hydrochloride and PEAK1. Further, a mouse model of middle cerebral artery occlusion and reperfusion was prepared, and methods such as TTC staining, neurological behavior scoring, and cerebral blood flow were used to confirm that Erlotinib can reduce the cerebral infarction volume of model mice, lower the neurological behavior score, increase cerebral blood flow, and improve pathological damage to brain tissue. In addition, methods such as Evans blue leakage and Western blot were used to confirm that Erlotinib has an improving effect on the BBB dysfunction induced by cerebral ischemia-reperfusion injury. Finally, Western blot and immunofluorescence techniques were used to confirm that Erlotinib can significantly increase the expression level of PEAK1 in the brain tissue of model mice. In summary, Erlotinib hydrochloride can improve cerebral ischemia-reperfusion-induced brain injury and blood-brain barrier dysfunction by increasing the protein expression level of PEAK1, providing important reference basis and data support for in-depth exploration of the important pathophysiology and mechanism of action of ischemic stroke, finding new therapeutic targets, and thus improving cerebral ischemia-reperfusion injury.

[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0026] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0027] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0028] Example 1

[0029] Localization of PEAK1 in the brain tissue of MCAO / R mice and the time-course change of protein expression

[0030] I. Experimental methods

[0031] 1. Construction of an animal model of middle cerebral artery occlusion and reperfusion in mice

[0032] A mouse middle cerebral artery occlusion / reperfusion (MCAO / R) model was established based on the reference literature and prepared by the suture method. C57 male mice weighing between 20 - 22 g were selected. The mice were intraperitoneally injected with 3% sodium pentobarbital. After anesthesia, they were fixed in the supine position, and a midline incision was made in the neck. The right common carotid artery, external carotid artery, and internal carotid artery were separated and exposed. A ligature was reserved at the proximal end of the common carotid artery in sequence. The external carotid artery was ligated at the distal end, and the distal end of the external carotid artery was dissected free. A section of the internal carotid artery was dissected deeper along the internal carotid artery, and a ligature was reserved at the proximal end of the external carotid artery. The proximal end of the common carotid artery and the distal end of the internal carotid artery were clamped with a small non-invasive vascular clamp. A small incision was made at the distal end of the external carotid artery with a micro-scissors, and a suture was inserted through the bifurcation of the common carotid artery and into the internal carotid artery. The suture was inserted until slight resistance was encountered. At this time, the tip of the suture was inserted about 1 mm into the anterior cerebral artery. After the suture insertion was completed, the ligature pre-placed on the external carotid artery was tightened. After 60 minutes of MCAO, the external carotid artery was separated and exposed, the fixation suture was loosened, the suture was removed, and the ligature of the common carotid artery was loosened to achieve reperfusion. The neck wound was routinely sutured. During the operation, the rectal temperature of the animals was maintained at 37°C. After the operation, the animals were placed in a breeding box with clean bedding and allowed to drink and eat freely.

[0033] After establishing the MCAO / R model, the animals were divided into a sham operation group (Sham group) and model groups at different reperfusion time periods. Behavioral evaluations were performed at 0, 3, 6, 12, and 24 hours respectively, and the brains were collected for sample collection.

[0034] 2. Detection of the expression level of PEAK1 in the mouse brain tissue by Western blot technique

[0035] Each group of mice was sacrificed at 0, 3, 6, 12, and 24 hours after cerebral ischemia-reperfusion surgery. The heart was perfused with 1×PBS buffer solution, the brain was taken, and the remaining blood streaks were washed away in physiological saline. The ischemic penumbra brain tissue was taken for Western blot to analyze the expression level of PEAK1 in the brain tissue of the two groups of mice.

[0036] Method for extracting histones: After 0, 3, 6, 12, and 24 h of cerebral ischemia-reperfusion surgery in each group of mice, the mice were sacrificed by decapitation. 0.1 g of solid tissue from the ischemic cortex was taken to extract proteins, which were homogenized by adding tissue lysate and lysed at 4°C for 30 min. After centrifugation at 12,000 rpm for 10 min in a 4°C centrifuge, the supernatant was taken to measure the protein content. 5×loading buffer was added to the remaining supernatant. After the proteins were denatured at high temperature, they were respectively loaded onto a 10% SDS-PAGE gel for electrophoresis. The bands after wet transfer were incubated with the corresponding primary antibody overnight, then incubated with the corresponding secondary antibody. Finally, they were developed using an ECL kit, and the bands were exposed using a gel imager. The expression level of the target protein was expressed as the relative value compared with the expression level of the corresponding internal reference protein.

[0037] 3. Detection of the expression level of PEAK1 in the blood-brain barrier of mouse brain tissue by immunofluorescence technique

[0038] (1) Perfusion, fixation, dehydration, embedding, sectioning

[0039] Each group of mice was sacrificed at 0, 3, 6, 12, and 24 h after cerebral ischemia-reperfusion surgery. The hearts were perfused with pre-cooled PBS, the brains were taken out, blotted dry with filter paper, fixed with 4% paraformaldehyde for 48 h, and then replaced with 40% sucrose solution for dehydration until the brain tissue sank to the bottom. The olfactory bulbs and hindbrains of the brain tissue were excised. The part of the brain tissue close to the olfactory bulb was vertically placed in an embedding cassette filled with OCT compound, and the frozen tissue was stored at -80°C. The brain tissue was cut into 5-μm thin sections using a cryostat for standby.

[0040] (2) Staining, photographing

[0041] The brain tissue sections were equilibrated at room temperature in a wet box for 0.5 h, then rinsed 3 times (5 min each time) in a glass trough filled with pre-cooled PBS and dried with lint-free paper. The brain tissue was circled with a histochemical pen, and a rapid blocking solution was added and incubated at room temperature for 30 min, then dried with lint-free paper. Subsequently, the corresponding primary antibodies (PEAK1 diluted at 1:200, CD31 diluted at 1:50) were added and incubated overnight at 4°C. After rinsing 3 times with pre-cooled PBS and drying with lint-free paper, the corresponding secondary antibody was added and incubated in the dark at room temperature for 1.5 h, then rinsed 3 times and dried with lint-free paper. A DAPI-containing anti-fluorescence quenching agent was added for mounting, and photographs were taken using a laser confocal microscope LSM700.

[0042] II. Experimental results

[0043] The Western blot technique was used to investigate the time-course changes in the expression of PEAK1 in mouse brain tissue at 1 h after MCAO and different reperfusion times (0 h, 3 h, 6 h, 12 h, 24 h). Figure 1Figure A shows that the expression of PEAK1 in the mouse brain tissue began to decline at 0 h of reperfusion, significantly decreased at 3 h of reperfusion, and remained at a low level in the brain tissue from 6 to 24 h of reperfusion. Statistically, there was a significant difference compared with the Sham group (P < 0.01). Immunofluorescence technology was used to investigate the localization of PEAK1 in the brain tissue. Figure 1 Figure B shows that PEAK1 overlapped with the endothelial marker protein CD31 and its expression significantly decreased under the condition of the MCAO 1 h / R 3 h model. Statistically, there was a significant difference compared with the Sham group (P < 0.01). The results indicate that PEAK1 is expressed in the blood-brain barrier of the brain tissue. Collectively, it shows that PEAK1 is involved in the pathological process of cerebral ischemia-reperfusion injury in MCAO / R model mice and its expression is significantly reduced in the brain tissue of MCAO / R model mice.

[0044] Example 2

[0045] Selection and preliminary verification of PEAK1 agonists

[0046] 1. Screening of PEAK1 agonists based on the DrugBank drug database

[0047] Agonists related to PEAK1 were mined from the DrugBank drug database. Initially, more than 1,300 potential marketed drugs with the effect of activating PEAK1 were predicted. Excluding drugs that have been reported in the field of stroke and those with relatively large toxic and side effects, the most representative important marketed drugs (the top 50) were classified and listed in descending order of scores. However, drugs containing similar structures were not limited. The screening results are shown in Table 1. The marketed drug with the highest score, erlotinib hydrochloride, was selected, suggesting that it may be an agonist of PEAK1, and in vitro and in vivo experiments were planned to verify it.

[0048] Table 1 Related marketed drugs predicted for PEAK1 agonists using the DrugBank drug database

[0049]

[0050] 2. Binding free energy analysis based on molecular docking technology

[0051] The representative with the highest score, erlotinib, selected from the initially screened marketed drugs, was subjected to molecular docking with the target protein PEAK1 to calculate the free binding energy between molecules and the specific binding sites. The docking results of PEAK1 and erlotinib are as follows Figure 2As shown, the binding free energy is -9.92 kcal / mol. It can be seen that the binding energy of the top-scoring marketed drug erlotinib to PEAK1 < -5 kcal / mol, indicating that the agonist erlotinib screened from the DrugBank database can bind to PEAK1 relatively stably.

[0052] 3. Detection of the interaction between erlotinib and PEAK1 by MST

[0053] First, use the Monolith TM RED-NHS Second Generation Protein Labeling Kit to label the PEAK1 recombinant protein, and use MST technology to investigate the binding ability of erlotinib to PEAK1. The results are as Figure 3 shown. The Kd value is 564.37 ± 91.36 nM, indicating that erlotinib can interact with PEAK1, further confirming that erlotinib can bind to PEAK1.

[0054] Example 3

[0055] Improvement effect of erlotinib on cerebral ischemia-reperfusion-induced brain injury in mice

[0056] I. Experimental methods

[0057] 1. Establishment of an animal model of middle cerebral artery occlusion and reperfusion in mice

[0058] Refer to the mouse middle cerebral artery occlusion and reperfusion (MCAO / R) model established according to the literature. It is prepared by the suture method. Select male C57 mice with a body weight between 20 - 22 g. The mice are intraperitoneally injected with 3% sodium pentobarbital. After anesthesia, they are fixed in the supine position, and a midline incision is made in the neck. The right common carotid artery, external carotid artery, and internal carotid artery are separated and exposed. A ligature is reserved at the proximal end of the common carotid artery in turn. The external carotid artery is ligated at the distal end, and the distal end of the external carotid artery is dissected free. A section of the internal carotid artery is dissected deeper along the internal carotid artery, and a ligature is reserved at the proximal end of the external carotid artery. The proximal end of the common carotid artery and the distal end of the internal carotid artery are clamped with a small non-invasive vascular clamp. A small incision is made at the distal end of the external carotid artery with a micro-scissors, and a suture is inserted through the bifurcation of the common carotid artery and into the internal carotid artery. The suture is inserted until a slight resistance is encountered. At this time, the tip of the suture is inserted about 1 mm into the anterior cerebral artery. After the suture insertion is completed, the ligature pre-placed on the external carotid artery is tightened. After 60 minutes of MCAO, the external carotid artery is separated and exposed, the fixation suture is loosened, the suture is removed, and the ligature of the common carotid artery is loosened to achieve reperfusion. The neck wound is sutured routinely. During the operation, the rectal temperature of the animal is maintained at 37°C. After the operation, the animal is placed in a breeding box with clean bedding and allowed to drink and eat freely.

[0059] After establishing the MCAO / R model, the animals were divided into a sham operation group (Sham), a model group (MCAO / R), a model drug administration group (MCAO / R + erlotinib 50 mg / kg), and a positive control group (MCAO / R + edaravone 3 mg / kg). Except for not inserting the suture, the other treatments of the sham operation group were the same as those of the other groups. During reperfusion, the erlotinib group was given intragastric administration, the edaravone group was given intraperitoneal injection, and the sham operation group and the model group were given intraperitoneal injection of normal saline. Behavioral evaluation was performed 24 hours later, and samples were collected from the brain.

[0060] 2. Administration method of erlotinib

[0061] The method for preparing erlotinib as a drug for treating ischemic stroke: First, grind the erlotinib hydrochloride tablets, dissolve the powder in normal saline to prepare an erlotinib solution of 100 mg / kg, and then dilute it with normal saline to obtain a dosing dose of 50 mg / kg.

[0062] 3. Measurement of cerebral infarction volume by TTC staining

[0063] After 24 hours of cerebral ischemia-reperfusion in mice, the mice were sacrificed and the brains were taken, placed in a -20°C refrigerator until the brain tissue was completely frozen, and then the frozen brain tissue was cut into 5 coronal brain slices about 2 mm thick on an ice box. The cut brain slices were quickly placed into a 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution, and the brain slices were placed in one direction. Subsequently, they were incubated in the dark in an incubator at 37°C for 30 minutes. They were placed on a black cardboard for photographing and transferred to the computer, and the cerebral infarction volume was measured using Image J image analysis software. The calculation formula is: percentage of infarction volume = infarction volume of the right hemisphere / total brain volume × 100%.

[0064] 4. Neurological function score

[0065] Neurological dysfunction is one of the important indicators for evaluating ischemic stroke injury. After 24 hours of ischemia-reperfusion in mice, the neurological dysfunction behavior was evaluated according to the Longa neurological function score. The neurological injury was divided into 5 grades: 0 point: normal, no neurological deficit; 1 point: the right (paralyzed side) front paw cannot be fully extended, but there is no obvious circling during walking, with mild neurological deficit; 2 points: the mouse circles to the right side and walks slowly, with moderate neurological deficit; 3 points: the mouse's body tilts to the right side and slowly turns small circles, with severe neurological deficit; 4 points: unable to walk spontaneously and loss of consciousness.

[0066] 5. H&E staining of brain tissue

[0067] After 24 h of reperfusion, the mice in each group were sacrificed, the brains were removed, fixed with 4% paraformaldehyde for more than 24 h, and then sent to Wuhan Sevier Biotechnology Co., Ltd. for paraffin embedding, sectioning, H&E staining, and then scanned, observed and photographed with a digital pathological section scanner.

[0068] 6. Cerebral blood flow detection

[0069] After 24 h of reperfusion, the mice were anesthetized, placed prone on a thermostatic blanket, the scalp of the mice was disinfected with iodine, the scalp was cut open, and the hair on the skull was removed. The cerebral blood flow of the mice was detected using a Doppler blood flow meter.

[0070] III. Experimental results

[0071] 1. Erlotinib reduces the cerebral infarction volume and neurological function score in MCAO / R model mice

[0072] As Figure 4 shown in A, the mice in the Sham group did not show obvious neurological deficits, and the neurological function score of the mice in the MCAO / R group increased significantly. After ischemia, erlotinib (50 mg / kg) was administered, and the neurobehavioral score of the MCAO / R model mice decreased significantly. As Figure 4 shown in B, the brain tissue of the mice in the Sham group was red and no obvious infarct area was seen. Compared with the Sham group, there were severe white infarcts on the ischemic side of the mice in the MCAO / R group. Compared with the MCAO / R group, the white infarct areas in the right brain of the mice in the MCAO / R + erlotinib (50 mg / kg) group and the MCAO / R + edaravone (Edara) administration group were significantly reduced. In summary, erlotinib can significantly reduce the cerebral infarction volume and neurological dysfunction in MCAO / R model mice.

[0073] 2. Erlotinib improves cerebral blood flow and pathological damage of brain tissue in MCAO / R model mice

[0074] The results measured by the laser Doppler blood flow meter are as Figure 5 shown in A and B. Compared with the Sham group, the cerebral blood flow in the right hemisphere of the brain in the Model group decreased significantly, showing obvious cerebral ischemia. Compared with the Model group, the cerebral blood flow in the right hemisphere of the brain in the Erlotinib group and the Edara group recovered. The results indicate that the erlotinib group (50 mg / kg) can significantly restore the cerebral blood flow in the right hemisphere of MCAO / R mice. As Figure 5 shown in C, compared with the Sham group, the brain tissue of the mice in the MCAO / R group showed vacuolization, nuclear pyknosis, a decrease in the number of nerve cells, and an increase in the perivascular space. After administration, the brain tissue of the mice in the erlotinib group (50 mg / kg) was significantly less damaged compared with the MCAO / R group.

[0075] Example 4

[0076] Erlotinib Improves Blood-Brain Barrier Dysfunction Induced by Cerebral Ischemia-Reperfusion in Mice

[0077] I. Experimental Methods

[0078] 1. Detection of Blood-Brain Barrier Permeability in Mouse Brain Tissue by Evans Blue

[0079] The method of injecting Evans blue via the tail vein was used to investigate the exudation of microvessels. After 22 h of MCAO / R modeling, 2% Evans blue solution was injected into the tail vein of the mice. After 2 h of circulation, the mice were perfused with 1×PBS through the heart until the blood was washed out. The whole brain was removed and photographed. The right hemisphere of the brain (ischemic side of the brain) was taken, the residual blood streaks were washed away in physiological saline, blotted appropriately with filter paper and weighed. Homogenization was performed with pre-cooled formamide and brain tissue at a ratio of 1 mL:0.1 g, transferred to a 1.5 mL EP tube, the centrifuge was pre-cooled to 4 °C, the rotation speed was 12,000 rpm, and centrifuged for 20 min. The supernatant was taken, and the absorbance was measured at a wavelength of 620 nm. Finally, the content was calculated according to the standard curve.

[0080] 2. Detection of the Expression Levels of Tight Junction Proteins and Matrix Metalloproteinases in Mouse Brain Tissue by Western Blot Technique

[0081] Each group of mice was sacrificed 24 h after cerebral ischemia-reperfusion surgery. The heart was perfused with 1×PBS buffer solution, the brain was taken, the residual blood streaks were washed away in physiological saline, and the ischemic penumbra brain tissue was taken for protein immunoblotting to analyze the expression levels of ZO-1, Occludin, MMP-2, and MMP-9 in each group.

[0082] Method for extracting tissue protein: After 24 h of MCAO / R in each group of mice, they were decapitated and sacrificed, 0.1 g of solid tissue from the ischemic cortex was taken to extract protein, tissue lysate was added for homogenization, and lysis was performed at 4 °C for 30 min. Centrifuged at 12,000 rpm in a 4 °C centrifuge for 10 min, the supernatant was taken, and the protein content was measured. The remaining supernatant was added with 5×loading buffer. After protein denaturation at high temperature, it was loaded onto a 10% SDS-PAGE gel for electrophoresis respectively. The bands after wet transfer were incubated with the corresponding primary antibody overnight, then incubated with the corresponding secondary antibody, and finally developed using an ECL kit. The bands were exposed using a gel imager, and the expression level of the target protein was expressed as the relative value to the expression level of the corresponding internal reference protein.

[0083] 3. Detection of the Expression Level of Tight Junction Protein ZO-1 in the Blood-Brain Barrier of Mouse Brain Tissue by Immunofluorescence Technique

[0084] (1) Perfusion, Fixation, Dehydration, Embedding, Sectioning

[0085] Mice in each group were sacrificed 24 h after cerebral ischemia-reperfusion surgery. The heart was perfused with pre-cooled PBS, the brain was removed, blotted dry with filter paper, fixed with 4% paraformaldehyde for 48 h, and then dehydrated with 40% sucrose solution until the brain tissue sank to the bottom. The olfactory bulb and hindbrain of the brain tissue were excised. The part of the brain tissue close to the olfactory bulb was vertically placed into an embedding cassette filled with OCT compound, and the frozen tissue was stored at -80 °C. The brain tissue was cut into 5-μm thin sections with a cryostat for standby.

[0086] (2) Staining and photographing

[0087] The brain tissue sections were equilibrated at room temperature in a wet box for 0.5 h, rinsed 3 times (5 min each time) in a glass trough containing pre-cooled PBS, and dried with lint-free paper. The brain tissue was circled with a histochemical pen, and a rapid blocking solution was added and incubated at room temperature for 30 min, then dried with lint-free paper. Subsequently, the corresponding primary antibodies (dilution ratio of ZO-1: 1:2000, dilution ratio of CD31: 1:50) were added and incubated overnight at 4 °C. The sections were rinsed 3 times with pre-cooled PBS and dried with lint-free paper. The corresponding secondary antibody was added and incubated in the dark at room temperature for 1.5 h, then rinsed 3 times and dried with lint-free paper. A DAPI-containing anti-fluorescence quenching agent was added for mounting, and photographs were taken using a laser confocal microscope LSM700.

[0088] II. Experimental results

[0089] 1. Erlotinib reduces the leakage of Evans blue in the brain tissue of mice induced by cerebral ischemia-reperfusion

[0090] As Figure 6 shown in A and B, compared with the Sham group, the leakage of Evans blue in the brain tissue of model mice in the MCAO / R group was obvious. Compared with the MCAO / R group, the MCAO / R + erlotinib (50 mg / kg) group and the Edara group significantly reduced the leakage of Evans blue in the brain tissue of mice after MCAO / R.

[0091] 2. Erlotinib inhibits the degradation of tight junction proteins in the brain tissue of MCAO / R model mice

[0092] As Figure 7 shown in A and B, compared with the Sham group, the expressions of tight junction proteins ZO-1 and Occludin in the brain tissue of model mice in the MCAO / R group were significantly decreased. After administration of erlotinib (50 mg / kg), the expressions of ZO-1 and Occludin in the brain tissue of model mice were significantly increased. As Figure 7 shown in C, the immunofluorescence results further verified that erlotinib could significantly improve the expression of tight junction protein ZO-1 in the BBB of MCAO / R mice. The results showed that erlotinib could play a protective role on the BBB by inhibiting the degradation of tight junction proteins in the brain tissue of MCAO / R mice.

[0093] 3. Erlotinib reduces the expression of matrix metalloproteinases in the brain tissue of mice in the MCAO / R model

[0094] MMP-2 / 9 is a key molecule that promotes the degradation of tight junction proteins and the cell basement membrane after cerebral ischemia-reperfusion, and is also an important factor involved in the inflammatory response. As Figure 8 shown in A and B below, compared with the Sham group, the expression of MMP-2 and MMP-9 in the brain tissue of model mice in the MCAO / R group was significantly increased. After administration, erlotinib (50 mg / kg) significantly decreased the expression of MMP-2 and MMP-9 in the brain tissue of model mice.

[0095] Example 5

[0096] Effect of the agonist erlotinib on the expression of PEAK1 in the brain tissue of mice induced by MCAO / R

[0097] I. Experimental methods

[0098] 1. Detection of the expression level of PEAK1 in the brain tissue of mice by Western blot technique

[0099] After establishing the MCAO / R model, the animals were divided into a sham operation group (Sham), a model group (MCAO / R), a sham operation + drug administration group (Sham + erlotinib 50 mg / kg), and a model + drug administration group (MCAO / R + erlotinib 50 mg / kg). The sham operation group and the sham operation + drug administration group were treated the same as the other groups except for not inserting the thread embolism. The mice in each group were sacrificed 24 h after cerebral ischemia-reperfusion surgery, perfused through the heart with 1×PBS buffer solution, the brain was taken, the residual blood streaks were washed away in physiological saline, and the ischemic penumbra brain tissue was taken for protein immunoblotting to analyze the expression level of PEAK1 in the brain tissue of each group of mice.

[0100] Method for extracting tissue protein: 24 h after MCAO / R in each group of mice, they were sacrificed by decapitation, 0.1 g of solid tissue from the ischemic cortex was taken to extract protein, tissue lysate was added for homogenization, and lysis was carried out at 4°C for 30 min. Centrifugation was carried out at 12,000 rpm for 10 min in a 4°C centrifuge, the supernatant was taken, and the protein content was measured. The remaining supernatant was added with 5×loading buffer, and after high-temperature protein denaturation, the samples were respectively loaded onto a 10% SDS-PAGE gel for electrophoresis. The bands after wet transfer were incubated with the corresponding primary antibody overnight, then incubated with the corresponding secondary antibody, and finally developed using an ECL kit, and the bands were exposed using a gel imager. The expression level of the target protein was expressed as the relative value of the expression level of the corresponding internal reference protein.

[0101] 2. Detection of the protein expression level of PEAK1 in the blood-brain barrier of the brain tissue of mice by immunofluorescence technique

[0102] (1) Perfusion, fixation, dehydration, embedding, sectioning

[0103] Mice in each group were sacrificed 24 h after cerebral ischemia-reperfusion surgery. The heart was perfused with pre-cooled PBS, the brain was removed, blotted dry with filter paper, fixed with 4% paraformaldehyde for 48 h, and then dehydrated with 40% sucrose solution until the brain tissue sank to the bottom. The olfactory bulb and hindbrain of the brain tissue were excised. The part of the brain tissue close to the olfactory bulb was vertically placed into an embedding cassette filled with OCT compound, and the frozen tissue was stored at -80 °C. The brain tissue was cut into 5-μm thin sections with a cryostat for standby.

[0104] (2) Staining, photographing

[0105] The brain tissue sections were equilibrated at room temperature in a wet box for 0.5 h, rinsed 3 times (5 min each time) in a glass trough containing pre-cooled PBS, and dried with lint-free paper. The brain tissue was circled with a histochemical pen, and a rapid blocking solution was added and incubated at room temperature for 30 min, then dried with lint-free paper. Subsequently, the corresponding primary antibodies (dilution ratio of PEAK1 1:200, dilution ratio of CD31 1:50) were added and incubated overnight at 4 °C. After rinsing 3 times with pre-cooled PBS and drying with lint-free paper, the corresponding secondary antibody was added and incubated in the dark at room temperature for 1.5 h, then rinsed 3 times and dried with lint-free paper. A DAPI-containing anti-fluorescence quenching agent was added for mounting, and photographs were taken using a laser confocal microscope LSM700.

[0106] II. Experimental results

[0107] The effects of erlotinib on the expression of PEAK1 in the brain tissue of mice after cerebral ischemia-reperfusion were investigated by Western blot and immunofluorescence techniques. As shown in A and B below, compared with the Sham group, the expression of PEAK1 in the MCAO / R group was significantly decreased. Figure 9

[0108] Statistically, compared with the Sham group, there was a significant difference (P < 0.01). Administration of erlotinib (50 mg / kg) after ischemia could significantly increase the expression of PEAK1. Statistically, compared with the MCAO / R group, there was a significant difference (P < 0.01). Compared with the Sham group, there was no significant difference in the expression of PEAK1 in the Sham+Erlotinib group. The results showed that erlotinib could significantly increase the expression level of PEAK1 protein in the brain tissue of MCAO / R mice.​

Claims

1. Use of PEAK1 as a diagnostic marker in the preparation of a reagent for diagnosing cerebral ischemia-reperfusion injury.

2. Use of a reagent for detecting PEAK1 in the preparation of a diagnostic reagent for cerebral ischemia-reperfusion injury.

3. The application according to claim 2, characterized in that, The reagent for detecting PEAK1 is selected from: PCR or qPCR primers, antibodies.

4. Use of PEAK1 as a therapeutic target in the screening of drugs for preventing and treating cerebral ischemia-reperfusion injury.

5. Use of a PEAK1 agonist in the preparation of a drug for preventing and treating cerebral ischemia-reperfusion injury.

6. The application according to claim 5, wherein The PEAK1 agonist is a small molecule compound, nucleic acid, protein or a combination thereof.

7. The application according to claim 6, characterized in that, The PEAK1 agonist is erlotinib hydrochloride.

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