Application of polypeptide in preparation of medicine for preventing or treating cerebral apoplexy

Abstract

The invention discloses an application of a polypeptide in preparation of a medicine for preventing or treating cerebral apoplexy. The polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 1. The N15 polypeptide expressed by the sequence can inhibit apoptosis and autophagy of damaged neurons by inhibiting expression of Tom20 and PARP proteins, promote expression of SYN-1 protein to promote a patient with ischemic cerebral apoplexy to recover learning, memory and cognitive functions, can comprehensively protect neuronal damage caused by ischemic cerebral apoplexy, can promote recovery of damaged nerve cells of an OGD model, and has a good application prospect. The polypeptide has an obvious protection effect on neuronal cells, the polypeptide is connected with a cell transmembrane carrier and can penetrate through a blood brain barrier to enter brain tissue to directly act on the neuronal cells, and an ischemic cerebral apoplexy animal model verifies that the polypeptide has an effect of treating cerebral apoplexy. Particularly, the cognitive ability and learning and memory ability of a mouse with cerebral arterial thrombosis can be recovered to a normal level, and a good treatment effect on cerebral arterial thrombosis is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to the use of a polypeptide in preparing a drug for preventing or treating cerebral stroke. Background Art

[0002] Stroke is a series of clinical symptoms caused by sudden rupture and bleeding of cerebral blood vessels, or embolism, infarction, and ischemia under the influence of multiple genetic and environmental factors. It is generally caused by local neuronal necrosis, among which brain tissue damage is the pathological basis for the clinical symptoms of stroke. Due to the high incidence, mortality, disability, recurrence and medical expenses of stroke, it has become a very serious health and social problem. It is the first and second cause of death and the main cause of disability in China and the world respectively. The current treatment goal of stroke is to reduce brain damage and restore the normal function of brain nerves.

[0003] Stroke can be divided into two types: hemorrhagic stroke and ischemic stroke. Among stroke cases, ischemic stroke accounts for 60%-70%, and hemorrhagic stroke accounts for 30%-40%. Ischemic stroke mainly includes cerebral thrombosis and cerebral embolism. The former is caused by arterial stenosis, gradual formation of thrombus in the lumen and eventual blockage of the artery, while the latter is caused by the detachment of thrombus or other emboli entering the bloodstream and blocking the cerebral artery. It is usually manifested as sudden fainting and unconsciousness. Common symptoms include facial paralysis, speech impairment, hemiplegia, etc. Hemorrhagic stroke is divided into cerebral hemorrhage and subarachnoid hemorrhage according to the different bleeding sites. Cerebral hemorrhage, commonly known as "cerebral hemorrhage", is caused by rupture of intracerebral arteries and blood overflowing into brain tissue. Subarachnoid hemorrhage is caused by rupture of blood vessels on the surface or bottom of the brain, and blood directly enters the subarachnoid space and cerebral cisterns containing cerebrospinal fluid. It is mainly manifested as aphasia and hemiplegia. In severe cases, the patient is unconscious. More than half of the patients have headaches and vomiting.

[0004] Currently, the only drug approved by the FDA is tissue plasminogen activator (tPA), which is used to dissolve blood clots in the treatment of ischemic stroke. However, tPA has a narrow treatment time window, limited contraindications, and dangerous complications. According to statistics, only 3% to 5% of patients receive effective treatment. In addition, the rate of vascular recanalization after tPA treatment is low, making the overall effectiveness of this drug low. Existing research on stroke-related drugs is mainly divided into blood volume expanders, thrombus dissolving drugs, anticoagulants, and drugs to prevent platelet aggregation, but these drugs still have some defects.

[0005] Among them, drugs that improve microcirculation and expand blood volume, such as low-molecular-weight dextran, are currently used more frequently, but patients with heart disease should use them with caution, otherwise they may cause heart failure; and drugs that dissolve thrombin, such as urokinase, often require large doses when used systemically intravenously, which may cause the risk of bleeding. Although anticoagulant therapy such as heparin can prevent blood coagulation, it is necessary to check the prothrombin time and activity every day when using it, which cannot be performed in hospitals with poor conditions and anticoagulant therapy may also cause bleeding. Platelet aggregation is often the beginning of cerebral thrombosis. Although drugs that prevent platelet aggregation, such as aspirin, can effectively block platelet aggregation and prevent further formation of cerebral thrombosis, the effect of using such drugs in the acute stage of stroke is not ideal. It can be seen that such drugs are suitable as drugs for preventing cerebral thrombosis. From the existing drugs for preventing or treating stroke, there is no real special drug. Therefore, further research on drugs for preventing or treating stroke is of great significance. Summary of the invention

[0006] In view of the above defects or improvement needs of the prior art, the present invention provides an application of a polypeptide in the preparation of a drug for preventing or treating stroke, the purpose of which is to find that the N15 polypeptide as shown in SEQ ID NO: 1 can restore the function of damaged nerve cells and has a significant effect on protecting neurons. It has an effective therapeutic effect on stroke and has little toxic and side effects. It can be used to prepare drugs for preventing or treating stroke, especially for preparing drugs for preventing or treating ischemic stroke, thereby solving the technical problem that existing drugs for preventing or treating stroke are poorly effective and have obvious side effects.

[0007] To achieve the above object, according to one aspect of the present invention, there is provided a use of a polypeptide in the preparation of a drug for preventing or treating stroke, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1.

[0008] Preferably, the polypeptide is used in the preparation of a drug for preventing or treating cerebral stroke, and the polypeptide is used in the preparation of a drug for preventing or treating ischemic stroke.

[0009] Preferably, the use of the polypeptide in the preparation of a drug for preventing or treating stroke, wherein the polypeptide further comprises an amino acid sequence expressing a cell-penetrating vector;

[0010] The cell-penetrating vector comprises the cell-penetrating polypeptide TAT.

[0011] Preferably, the polypeptide is used in the preparation of a drug for preventing or treating stroke, and the polypeptide is used in the preparation of a drug for repairing brain nerve damage caused by ischemic stroke.

[0012] According to another aspect of the present invention, a drug for preventing or treating stroke is provided, wherein the active ingredient of the drug is a polypeptide, and the polypeptide includes the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence of a cell-penetrating vector for expression.

[0013] Preferably, in the drug for preventing or treating stroke, the amino acid sequence of the cell-penetrating vector expressing the drug is as shown in SEQ ID NO:2.

[0014] Preferably, the drug for preventing or treating stroke, the polypeptide, and its amino acid sequence are as shown in SEQ ID NO:3.

[0015] Preferably, the drug for preventing or treating stroke has a content of the polypeptide in the drug for preventing stroke of ≥10 ug / ml.

[0016] Preferably, in the drug for preventing or treating stroke, the content of the polypeptide in the drug for treating stroke is ≥ 0.01%wt.

[0017] Preferably, the drug for preventing or treating stroke further comprises a pharmaceutically acceptable excipient.

[0018] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0019] The present invention finds that the N15 polypeptide shown in SEQ ID NO: 1 can promote the recovery of damaged nerve cells in the OGD model and has a significant protective effect on the nerve cells. The cell-penetrating vector connected thereto can penetrate the blood-brain barrier into the brain tissue and directly act on the nerve cells. The ischemic stroke animal model verifies that the N15 polypeptide can promote the recovery of nerve function in stroke model mice, enhance the physical fitness, body balance and coordination of the mice, reduce the volume of cerebral infarction, and improve the cognitive ability and learning and memory ability of the mice. The N15 polypeptide can be used to prepare drugs for preventing or treating stroke, especially for preparing drugs for preventing or treating ischemic stroke.

[0020] The drug for preventing or treating stroke provided by the present invention comprises a polypeptide as an active ingredient, and the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1, and an amino acid sequence of a cell-penetrating vector, which can penetrate the blood-brain barrier, enter brain tissue and directly act on neurons. The drug is an innovative drug that is effective in preventing or treating stroke, is safe, has low toxicity and controllable quality, is easy to produce and has low cost, and is helpful in reducing the cost required for treating stroke patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the observation of N15 polypeptide in brain tissue in Example 1;

[0022] Figure 2 is the effect of N15 peptide on the survival rate of SH-SY5Y cells;

[0023] Figure 3 The effect of N15 peptide on the cell viability of SH-SY5Y cells in the oxygen-glucose deprivation / reperfusion injury model;

[0024] Figure 4 is the effect of N15 peptide on the expression of Tom20, SYN-1, and PARP proteins;

[0025] Figure 5 is the neurological deficit score of mice in each group;

[0026] Figure 6 is the time mice stay in the rotarod test (rotarodtest) at 20 rpm in each group of mice;

[0027] Figure 7 is the time mice stay in the rotarod test (rotarodtest) at 40 rpm in each group of mice;

[0028] Figure 8 is the TTC staining image of the mouse brain in each group;

[0029] Fig. 9 It is the time to locate the platform in each group of mice during the 5-day training experiment in the Morris water maze experiment;

[0030] Fig.10 is the movement trajectory of each group of experimental mice in Morris water maze;

[0031] Fig.11 This is the result of statistical analysis of the time it takes each group of experimental mice to locate the platform in the Morris water maze. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] The present invention finds that the N15 polypeptide with an amino acid sequence as shown in SEQ ID NO: 1 can promote the recovery of damaged nerve cells in the OGD model and has a significant protective effect on nerve cells. A cell-penetrating vector is connected to the N15 polypeptide, which can pass through the blood-brain barrier and smoothly enter the brain tissue to directly act on nerve cells. Furthermore, in an ischemic stroke animal model, it is found that injection of the N15 polypeptide connected to the cell-penetrating vector has a significant therapeutic effect on stroke model mice, which can significantly reduce the area of ​​cerebral infarction and improve the balance and coordination of mice, promote the recovery of motor ability and neurological function of stroke mice, and the cognitive and learning and memory abilities of stroke mice show a dose effect with the N15 polypeptide, wherein injection of 1.5 mg of the N15 polypeptide connected to the cell-penetrating vector can restore the cognitive ability and learning and memory abilities of ischemic stroke model mice to normal levels.

[0034] Based on this, the present invention provides an application of a polypeptide in the preparation of a drug for preventing or treating stroke, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO:1.

[0035] The application is preferably used in the preparation of drugs for preventing or treating ischemic stroke.

[0036] The polypeptide also includes an amino acid sequence expressing a cell-penetrating vector;

[0037] In some embodiments, the cell-penetrating vector is a cell-penetrating polypeptide TAT (47-57), whose amino acid sequence is shown in SEQ ID NO: 2: YGRKKRRQRRR;

[0038] Adding more than 10ug / ml of N15 peptide connected to cell-penetrating peptide (TAT-N15 peptide) can significantly promote the recovery of damaged SH-SY5Y neuronal cells, while injecting 0.5mg of TAT-N15 peptide can inhibit brain nerve necrosis caused by cerebral infarction and promote the recovery of brain nerve damaged tissue. It has a significant nerve-protecting effect and can be used to prevent or treat stroke caused by nerve damage.

[0039] In the process of treating ischemic stroke model mice, the N15 polypeptide can use cell-penetrating peptides such as TAT ​​(47-57) to cross the blood-brain barrier into the brain tissue and directly act on neurons. The injection of 0.5 mg of N15 polypeptide connected with cell-penetrating peptide can significantly reduce the volume of cerebral infarction in ischemic model mice, promote the recovery of neurological function in ischemic stroke model mice, improve their balance and coordination, and facilitate the recovery of the mouse's motor ability. The injection of a high dose of the N15 polypeptide (1.5 mg) can significantly promote the recovery of the cognitive and learning and memory abilities of ischemic stroke model mice to normal levels. The N15 polypeptide can be used to prepare drugs for treating ischemic stroke, especially for preparing drugs for repairing brain nerve damage caused by ischemic stroke.

[0040] In addition, the present invention also provides a drug for preventing or treating stroke, wherein the active ingredient of the drug is a polypeptide, and the amino acid sequence of the polypeptide includes MMPYSTELIFYIEMDP as shown in SEQ ID NO: 1, and the amino acid sequence of a cell-penetrating vector for expression;

[0041] In some embodiments, the cell-penetrating vector is a cell-penetrating peptide TAT (47-57), whose amino acid sequence is shown in SEQ ID NO: 2, YGRKKRRQRRR, that is, the polypeptide, whose amino acid sequence is shown in SEQ ID NO: 3, YGRKKRRQRRRMMPYSTELIFYIEMDP;

[0042] The content of the polypeptide in the drug for preventing ischemic stroke is ≥10ug / ml;

[0043] The content of the polypeptide in the drug for treating ischemic stroke is ≥ 0.01%wt;

[0044] The medicine also includes pharmaceutically acceptable excipients.

[0045] The following are examples:

[0046] Example 1

[0047] Due to the blood-brain barrier of brain diseases such as stroke and Alzheimer's disease, it is difficult for therapeutic drugs to reach the brain tissue smoothly. In this embodiment, in order to enable the N15 polypeptide to enter the brain tissue, a transmembrane carrier is connected to the N15 polypeptide, wherein the N15 polypeptide contains 16 amino acids, and its amino acid sequence is shown in SEQ ID NO: 1 MMPYSTELIFYIEMDP.

[0048] The transmembrane carrier connected to the N15 polypeptide is TAT ​​(47-57), which contains 11 amino acids, as shown in SEQ ID NO: 2, YGRKKRRQRRR.

[0049] The above-mentioned N15 polypeptide is linked to TAT (47-57) to form TAT-N15 polypeptide, whose amino acid sequence is shown in SEQ ID NO: 3: YGRKKRRQRRRMMPYSTELIFYIEMDP.

[0050] The following experiments were performed to verify that the TAT-N15 peptide can pass through the blood-brain barrier and enter the brain tissue, as follows:

[0051] Experimental animals: Twelve C57 mice, 10 weeks old, were randomly divided into three groups, namely blank control group, short-term group and long-term group, with 4 mice in each group.

[0052] Experimental materials: N15 polypeptide antibody, chloral hydrate, paraformaldehyde, DAB color developing solution, PBS, anti-tissue proteinase K, paraffin, slicer, microscope, image analysis system ImageJ, 3DHISTECH'sSlide Converter, CaseViewer, etc.

[0053] Experimental steps:

[0054] The mice in the short-term group and the long-term group were injected with 1 mg of TAT-N15 peptide through the tail vein, and the blank control group was injected with an equal volume of saline through the tail vein. Brain tissues of mice in the short-term group were taken 1 hour later, and brain tissues of mice in the long-term group and the blank control group were taken 12 hours later for immunohistochemistry.

[0055] (1) Obtaining brain tissue

[0056] 1. Animal anesthesia: Anesthetize the mice with 350 mg / kg body weight of chloral hydrate. After the animals are anesthetized, fix them on the perfusion operation platform in the supine position;

[0057] 2. Animal perfusion: Starting from the xiphoid process of the sternum, cut the mouse skin along the lower ribs on both sides to the armpits to fully expose the heart. Insert the needle from the left ventricle and directly insert the perfusion needle into the ascending aorta. When perfusion begins, the right atrial appendage can be seen to be filled. Use physiological saline to quickly flush the blood. When no obvious blood flows out, switch to 4% paraformaldehyde for perfusion, about 100-150ml.

[0058] 3. Open the skull and remove the brain: Pierce the skull from the back of the cerebellum to the front to obtain the brain tissue.

[0059] 4. Fix overnight: Immerse the brain tissue in 4% paraformaldehyde and fix it for 12 hours.

[0060] (2) Paraffin embedding of brain tissue

[0061] 1. Tissue gradient dehydration and transparency: Take out the brain tissue fixed overnight and place it in the embedding window, mark it accordingly, rinse it with running water for about 10 minutes to remove the remaining impurities, and perform gradient alcohol dehydration.

[0062] 2. Wax immersion of tissue: Drain excess xylene from the transparent tissue block as much as possible to complete the wax immersion.

[0063] 3. Tissue block embedding: Use small tweezers to pick up the tissue block after burning and place it in the center of the depression of the embedding tank. Gently place the embedding window on the platform of the embedding tank, and try to ensure that the heights on both sides are the same. After standing, separate the wax block from the embedding tank.

[0064] (3) Immunohistochemistry of paraffin sections

[0065] Prepare a 40℃ constant temperature water bath, slicer blades, coated slides, curved tweezers, brushes, pencils, slice boxes, and ice boxes.

[0066] Install the blade and adjust the angle of the wax block to make the slice symmetrical on the left and right. At the same time, pay attention to the position of the target area and slice. Clamp the complete and appropriate slices into the water-soluble pot and flatten them. Stick the slices on the slides, place them on the edge of the water bath to dry and make corresponding marks.

[0067] Select appropriate tissue slices, place them on a slice rack, place them in a 60°C oven, and bake for 2 hours.

[0068] Dewaxing and gradient rehydration of sections: After baking, quickly place the sections in the prepared xylene glass jar for dewaxing and rehydration.

[0069] Microwave antigen retrieval: Prepare citrate buffer and adjust to pH 6.0.

[0070] Place the buffer solution in the tissue box and heat it in the microwave until it boils. Place the rehydrated slices in the boiling buffer solution (, place the tissue box in the microwave again and repair it with intermittent microwave heating. Specific requirements: keep the repair solution temperature above 95 degrees without boiling.

[0071] After the repair is completed, take the tissue box out of the microwave, place it at room temperature, and cool it naturally to the wide temperature.

[0072] (4) Chemical staining observation

[0073] First, place the slices in an incubation box with 3% H 2 O 2 Evenly drip on the slices and incubate in dark for 15 minutes. After incubation, wash with PBS for 5 minutes and repeat 3 times. Use a brush to circle the area where the tissue is located along the perimeter of the tissue slice. Place the slices in an incubation box, evenly drip 5% BSA on the tissue slices, distribute it in the entire circled area, and incubate in dark for 1 hour. After incubation, wash with PBS in the tissue box for 5 minutes and repeat three times.

[0074] Dilute TAT antibody to working concentration with PBS, add primary antibody to the slices, spread evenly, cover and incubate at 4°C overnight. Take out the incubation box and let it warm up at room temperature for 30 minutes, wash with PBS for 5 minutes, and repeat three times. Add secondary antibody and incubate at room temperature for 1 hour. Prepare an appropriate volume of DAB colorimetric solution, rinse with PBS for 5 minutes after the secondary antibody incubation, and repeat three times. After staining, place the slices in hematoxylin staining solution to stain the cell nucleus.

[0075] After staining is completed, the excess differentiation solution and excess ammonia are washed away with tap water, and the cells are dehydrated and made transparent. Imaging observation is performed after overnight.

[0076] Take a photo under a microscope, copy the photo and use the image analysis system ImageJ, 3DHISTECH's SlideConverter, and CaseViewer to observe and analyze the results. Figure 1 shown.

[0077] Depend on Figure 1 It can be seen that the N15 polypeptide was observed in the brain tissue, indicating that the TAT-N15 polypeptide can pass through the blood-brain barrier and smoothly enter the brain tissue. In the present invention, the cell-penetrating polypeptide connected to the N15 polypeptide can also be other polypeptides that can pass through the blood-brain barrier, including but not limited to the cell-penetrating peptide TAT (47-57) in Example 1.

[0078] Example 2 Evaluation of cytotoxicity of N15 polypeptide

[0079] Experimental materials and instruments: human neuroblastoma SH-SY5Y cell line; DMEM culture medium and fetal bovine serum were purchased from HyClone; MTT cell viability and cytotoxicity detection kit, antibodies Tom20, SYN-1 and PARP, β-actin, and tri-gas incubator were purchased from Thermo Scientific; IX71 fluorescence microscope was purchased from Olympus, etc.

[0080] The N15 polypeptide is the N15 polypeptide connected to the transmembrane carrier in Example 1.

[0081] The experimental method is as follows:

[0082] (1) SH-SY5Y cell culture: The cells were placed in DMEM containing 10% fetal bovine serum and incubated at 37°C and 5% CO 2 The cells were cultured in a saturated water vapor incubator, and the culture medium was changed every 48 hours. When the monolayer cells were confluent, they were subcultured.

[0083] (2) Establishment of OGD model and grouping: The well-grown SHSY5Y cells were selected and replaced with sugar-free and serum-free medium for incubation. The cells were placed in a 37°C three-gas incubator (1% O 2, 5% CO 2 and 94% N 2 ) for 12 hours to complete the model establishment. With normal cells as the blank control group (control), the model cells were randomly divided into OGD group (0ug / ml), 2.5ug / ml, 5ug / ml, 10ug / ml, 20ug / ml, 40ug / ml, 80ug / ml, 160ug / ml and 200ug / ml N15 polypeptide solution groups. Each group was set up with 6 parallel wells, and the experiment was repeated 3 times; DMEM complete medium was used to prepare polypeptide solutions of different concentrations.

[0084] (3) Detection of cell survival rate: MTT colorimetric assay, specifically:

[0085] The cells were plated at 5×10 7 / L density was inoculated in a 96-well culture plate, 100 μL per well, and the cells were treated after 24 hours. After the action was completed, 10 μL of MTT (5 mg / mL) was added to each well and placed in an incubator for incubation for 4 hours. Then, 100 μL of Formanzan cell lysis solution was added to each well and incubated in the cell incubator for another 4 hours. The absorbance (absorbance, A) of each well was measured at 570 nm using an enzyme-linked immunosorbent assay to evaluate the cell survival rate, where the SH-SY5Y survival rate = A value of the experimental group / A value of the control group. In this embodiment, the cell survival rate of the control group was set to 1. The evaluation results are shown in the figure. Figure 2 shown.

[0086] Depend on Figure 2 It can be seen that the cell survival rate of the OGD model group was 1, and there was no statistical difference in the cell survival rate between the 2.5-200ug / ml N15 polypeptide solution group and the OGD model group (0ug / ml N15 polypeptide) (p>0.05). This shows that the N15 polypeptide with a concentration of 2.5-200ug / ml has no obvious cytotoxic effect on SH-SY5Y cells, indicating that the N15 polypeptide with a concentration of 200ug / ml has no cytotoxicity.

[0087] (4) Detection of cell variation: Divide the model cells into 6 groups, add N15 polypeptide solution with concentrations of 0ug / ml, 10ug / ml, 20ug / ml, 40ug / ml, 80ug / ml, and 160ug / ml to each group, and culture the cells in an incubator for 24 hours. Add 10μl of 5mg / ml MTT solution to each group of cells, mix gently, and then continue to culture the cells in an incubator for 4 hours. Add 100μL Formanzan cell lysate to each well and continue incubating in the cell incubator for another 4 hours. The absorbance value was measured at 490nm by a spectrophotometer to evaluate the cell viability. The results are as follows: Figure 3 shown.

[0088] Depend on Figure 3 It can be seen that the viability of cells in the OGD model group was significantly lower than that in the blank control group. Compared with the OGD model group, the addition of 10-160ug / ml of N15 polypeptide can improve cell viability, and with the increase of N15 polypeptide concentration, cell viability gradually increased, showing a certain dose effect, especially when the N15 polypeptide concentration was above 20ug / ml, the cell viability of the experimental group was statistically different from that of the model group, indicating that N15 polypeptide can significantly improve the viability of SH-SY5Y oxygen-glucose deprivation / reperfusion injured cells and can be used for the repair of nerve damaged cells.

[0089] Example 3 Protective effect of N15 polypeptide on SH-SY5Y neuronal cells

[0090] In recent years, the oxygen-glucose deprivation (OGD) model of nerve cells has been used to simulate the clinical ischemic stroke process at the cellular level, and mainly to quickly restore oxygen and glucose supply after oxygen and glucose deprivation, thereby simulating cerebral ischemia-reperfusion injury for cell autonomous repair and drug research. Among them, SH-SY5Y cells are a kind of neuron-like cells, originating from human neuroblastoma, with cell morphology, neurotransmitter synthesis and release, synapse formation and function with the basic characteristics of neurons. This example uses SH-SY5Y neuronal cells as the object, simulates the local cerebral infarction process of ischemic stroke by establishing an oxygen-glucose deprivation model, and further studies the protective effect of N15 polypeptide on oxygen-glucose deprivation / reperfusion injury of SH-SY5Y cells, wherein the N15 polypeptide is the N15 polypeptide connected to the transmembrane carrier in Example 1, as follows:

[0091] (1) SH-SY5Y cell culture: same as in Example 1;

[0092] (2) Establishment of OGD model: As in Example 1, normal cells were used as a blank control group (control), and model cells were randomly divided into an OGD group (0 ug / ml), 20 ug / ml, 40 ug / ml, 80 ug / ml, and 160 ug / ml N15 polypeptide solution groups. Six parallel wells were set up in each group, and the experiment was repeated 3 times.

[0093] (3) Detection of protein expression: Harvest cells using Western blot to detect protein expression, specifically:

[0094] Wash once with 4℃ precooled PBS, add appropriate amount of cell lysis buffer, scrape cells with a cell scraper, centrifuge at 12000rpm for 10min, take the supernatant, and discard the precipitate. Determine the protein content of each group by BCA method, load 25μL protein in each well, electrophoresis by 15% SDS-PAGE 80V for 2h, transfer membrane at 300mA constant flow for 40min, block at room temperature with 5% skim milk for 1h, incubate with I antibody at 4℃; block II antibody with 5% skim milk at room temperature, and display the target band and internal reference by ECL development. Use ImageJ software to analyze the absorbance value, which represents the relative level of protein expression. The results are shown in Figure 4 shown.

[0095] Depend on Figure 4 It can be seen that the expression levels of Tom20 and PARP in the OGD model group were higher than those in the blank control group, while the expression levels of Tom20 and PARP in the 20-160ug / ml N15 polypeptide group were significantly lower than those in the OGD model group, especially the N15 polypeptide group with a concentration of more than 40ug / ml.

[0096] Among them, Tom20 is a protein in the outer membrane of mitochondria, which is involved in the biosynthesis, repair and function maintenance of mitochondria. Studies have shown that Tom20 plays an important role in processes such as apoptosis and mitochondrial autophagy. When mitochondria are dysfunctional or damaged, cells will degrade them through autophagosomes. Tom20 protein can act as a receptor protein to transport mitochondria to autophagosomes, thereby achieving autophagic degradation of mitochondria. As the concentration of N15 polypeptide increases, the expression of Tom20 gradually decreases, indicating that N15 polypeptide can inhibit the expression of Tom20, and inhibiting the expression of Tom20 can inhibit the autophagy and apoptosis of damaged neurons, which is beneficial to protecting neurons.

[0097] PARP (poly(ADP-ribose)polymerase) is a poly(ADP-ribosyl)ase that participates in many biological processes, including gene expression, DNA repair, and cell signal transduction. PARP plays an important role in DNA damage repair. In addition, PARP is also involved in biological processes such as apoptosis and autophagy. When cells are stressed, PARP will be activated, consuming intracellular ATP, leading to an increase in intracellular calcium ion concentration, thereby inducing apoptosis. As the concentration of N15 polypeptide increases, the expression of PARP gradually decreases, and there is a certain dose effect, indicating that N15 polypeptide can inhibit the expression of PARP, and reducing the protein expression level of PARP can reduce the apoptosis of damaged neurons, thereby providing comprehensive protection for neurons.

[0098] In addition, SYN-1 protein is a neuron-specific protein, also known as Synapsin I. It is a phosphorylated protein that plays an important role in synaptic plasticity and neuronal sustainability. SYN-1 protein is mainly expressed on the presynaptic membrane. It is involved in the assembly and release of synaptic vesicles and has an important impact on synaptic plasticity and cognitive functions such as learning and memory. In addition, SYN-1 protein is also involved in the regulation of neuronal survival and death.

[0099] Depend on Figure 4 It can be seen that the expression of SYN-1 in different concentrations of N15 peptide groups is higher than that in the blank control group and OGD cell model group, indicating that N15 peptide can promote the expression of SYN-1 protein. Promoting the expression of SYN-1 protein may promote the recovery of learning, memory and cognitive function in patients with ischemic stroke.

[0100] In summary, N15 peptide can inhibit the apoptosis and autophagy of damaged neurons by inhibiting the expression of Tom20 and PARP proteins, comprehensively protect neuronal damage caused by ischemic stroke, and promote the recovery of memory and cognitive ability by regulating the expression of SYN-1 protein. It is a potential drug that can be used for the prevention and treatment of stroke, especially for the prevention of stroke.

[0101] Example 4 The therapeutic effect of N15 polypeptide on ischemic stroke in mice

[0102] In this example, a mouse ischemic stroke model was established, and the mice were randomly divided into groups and given different concentrations of N15 polypeptide drug treatment, wherein the N15 polypeptide is the N15 polypeptide connected to the transmembrane carrier in Example 1, and the neurological deficit score (MNSS score) of the mice, the rotarod test of the mice, the TTC staining of the brain tissue, and the Morris water maze test were used to observe the effect of the N15 polypeptide on the recovery of the neurological function of the mice, and to evaluate its effect on the physical fitness, body balance ability, coordination, and cerebral infarction volume of the mice. The details are as follows:

[0103] (1) Establishment of mouse ischemic stroke model (MCAO model):

[0104] Prepare 0.2 ml of 3.8% chloral hydrate and inject it subcutaneously into C57 mice. After 5 minutes, the mice were anesthetized and placed on an electric blanket. The teeth and limbs were fixed with thread and tape. A longitudinal skin incision of about 1 cm was made vertically on the left side of the neck, and the fascia was cut. The thyroid gland was peeled off, and the "Y"-shaped blood vessels were found under a 10X microscope. Double knots were tied on the outside of the neck: a dead knot at the distal end and a slipknot at the proximal end. The proximal end was as low as possible, and the neck was tied with a dead knot. Ophthalmic scissors made a "V" incision on the outside of the neck, and the thread plug was inserted downward into the knot of the neck, and the slipknot on the outside of the neck was tied. The thread plug was turned down so that the thread plug originally in the neck was turned inside the neck. Continue to push the thrombus upward until there is a sense of resistance, and suture the skin of the mouse. The thread plug was pulled out after 1.5 hours, and behavioral tests were performed 24 hours later. The scores showed that the model was successfully established. Repeat the above method to establish 16 C57 mice.

[0105] (2) N15 peptide drug treatment:

[0106] Experimental groups: 16 normal mice (sham group) were randomly divided into two groups, the normal control group (sham) and the N15 polypeptide drug treatment group (sham+N15 polypeptide), with 8 mice in each group; the above 16 ischemic stroke MCAO model mice were randomly divided into two groups, the model control group (MCAO) and the N15 polypeptide drug treatment group (MCAO+N15 polypeptide), with 8 mice in each group.

[0107] Drug administration: The drug group in normal mice was injected with 0.5 mg N15 polypeptide through the tail vein every day, and the normal control group was injected with an equal volume of normal saline through the tail vein, and each group was treated with the drug for 7 consecutive days; the drug group in model mice was injected with 0.5 mg N15 polypeptide for the first time 1 hour after modeling, and then 0.5 mg N15 polypeptide was injected through the tail vein every day, and the model control group was injected with an equal volume of normal saline through the tail vein at the same time, and each group was treated with the drug for 7 consecutive days.

[0108] ① Mouse neurological deficit score (MNSS score)

[0109] After the mouse ischemic stroke model was successfully established, the neurological severity score (NSS) of the mice was measured at 24 hours and 7 days after drug administration. The results are as follows: Figure 5 The scoring criteria are as follows:

[0110] 0 points: normal neurological function;

[0111] 1 point: mild neurological deficit (flexion of the left forelimb when lifting the tail);

[0112] 2 points: moderate neurological deficit (turning to the left while walking);

[0113] 3 points: moderate neurological deficit (leaning to the left);

[0114] 4 points: no spontaneous ambulation, decreased consciousness;

[0115] 5 points: death related to ischemia.

[0116] Depend on Figure 5 It can be seen that the severe nerve injury deficit scores of the model control group mice were 8 points and 5 points at 24 hours and 7 days after medication, respectively, while the severe nerve injury deficit scores of the model mice were reduced to 5 points and 2 points after injection of N15 polypeptide, respectively. The higher the nerve function deficit score, the more severe the nerve function damage. Compared with the model control group, the severe nerve injury deficit score of the mice after injection of N15 polypeptide was significantly reduced, indicating that N15 polypeptide can promote the recovery of the mouse's nerve function, especially the recovery of the mouse's motor ability.

[0117] ② Mouse rotarod test (rotarod test)

[0118] The mouse rotarod test is used to evaluate the mouse's athletic ability, physical fitness, body balance ability, etc. Specifically:

[0119] Seven days after the experimental animals were treated with the drug, each animal was placed on a rotating rod at a slow speed of 20 rpm and 40 rpm for 5 minutes, and the time the mouse remained on the uniform rotating rod was recorded. The results were as follows: Figure 6 and Figure 7 shown.

[0120] Note that all mice were given daily 30-min training trials during the week prior to the start of the experiment. During the trial, the animal was placed on the rotating rod; if the animal fell off the rod, it had to be immediately put back on the rod.

[0121] Figure 6 The results are recorded at 20 rpm. Figure 7 The results recorded at 40 rpm are given by Figure 6 , Figure 7 It can be seen that the same trend was shown at different rotation speeds of 20rpm and 40rpm, and there was no significant difference in the time that normal mice injected with normal saline and mice injected with N15 polypeptide stayed on the rod, indicating that N15 polypeptide would not cause any effect on the motor ability, physical strength and balance ability of normal group mice.

[0122] Compared with normal mice, the model mice stayed on the rod for a significantly shorter time, indicating that ischemic stroke would seriously affect the mice's motor ability, physical strength and balance ability. After the model mice were injected with N15 polypeptide, their stay on the rod was much longer than that of the model control group, indicating that N15 polypeptide can improve the physical fitness, body balance and coordination of stroke mice, and can promote the recovery of motor ability, physical strength and balance ability of stroke mice.

[0123] ③TTC staining to observe the volume of cerebral infarction in mice

[0124] TTC staining is a reaction between TTC and succinate dehydrogenase in the mitochondria of living cells to generate red formazan, which is used to indicate cell viability. In this example, the volume of cerebral infarction lesions in mice was observed by TTC staining, specifically:

[0125] After the mice were anesthetized and perfused with saline, the brains were removed and quickly frozen in a -20℃ refrigerator for about 20 minutes, and sliced. One slice was cut every 2 mm, with the first cut at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm; the second cut at the optic chiasm; the third cut at the infundibulum; the fourth cut between the infundibulum and the caudal pole of the posterior lobe; and the fifth cut at the middle of the last slice.

[0126] Place the slices in 2% TTC staining solution, cover with tin foil, and place in a 37°C incubator for 30 minutes. Turn the slices from time to time to ensure that they are evenly exposed to the staining solution. Label the stained slices, set a ruler, and take photos. The results are as follows: Figure 8 shown.

[0127] Depend on Figure 8 It can be seen that there is no significant difference in chromosome volume between normal mice injected with saline and mice injected with N15 polypeptide, indicating that N15 polypeptide will not have any effect on the brain chromosome volume of normal mice. In addition, the brain chromosome volume of mice in the model control group is significantly smaller than that of mice in the normal control group, and the volume of cerebral infarction lesions in mice in the model control group is significantly larger than that in mice in the normal control group, indicating that cerebral infarction in stroke mice can cause brain nerve damage and necrosis; while the brain chromosome volume of model mice injected with N15 polypeptide is significantly larger than that of mice in the model control group, and there is no significant difference in brain chromosome volume between them and normal mice, indicating that N15 polypeptide can inhibit brain nerve necrosis caused by cerebral infarction, showing obvious neuroprotective effects, and is conducive to promoting the repair of damaged nerves.

[0128] In summary, N15 polypeptide can promote the recovery of motor ability of stroke mice, improve balance and coordination, significantly reduce the area of ​​cerebral infarction, inhibit the necrosis and damage of nerve cells caused by cerebral infarction in stroke mice, and promote the repair of nerve cell damage and the recovery of nerve function. It can be seen that it has a significant therapeutic effect on mouse stroke and has great potential for the treatment of stroke. The present invention provides an important basis for the treatment of ischemic stroke with N15 polypeptide, and provides guidance for further research and development of new treatment methods.

[0129] Example 5 N15 polypeptide promotes the recovery of cognitive function and learning and memory ability in ischemic stroke mice

[0130] (1) Construction of animal model of ischemic stroke

[0131] Prepare 0.2 ml of 3.8% chloral hydrate and inject it subcutaneously into C57 mice. After 5 minutes, the mice were anesthetized and placed on an electric blanket. The teeth and limbs were fixed with thread and tape. A longitudinal skin incision of about 1 cm was made vertically on the left side of the neck, and the fascia was cut. The thyroid gland was peeled off, and the "Y"-shaped blood vessels were found under a 10X magnification microscope. Double knots were tied on the outside of the neck: a dead knot at the distal end and a slipknot at the proximal end. The proximal end was as low as possible, and the neck was tied with a dead knot. Ophthalmic scissors made a "V" incision on the outside of the neck, and the thread plug was inserted downward into the knot of the neck, and the slipknot on the outside of the neck was tied. The thread plug was turned down so that the thread plug originally in the neck was turned inside the neck. Continue to push the thrombus upward until there is a sense of resistance, and suture the skin of the mouse. The thread plug was pulled out after 1.5 hours, and behavioral tests were performed 24 hours later. The scores showed that the model was successfully established. Repeat the above method to model 28 C57 mice.

[0132] (2) N15 peptide drug treatment

[0133] Experimental groups: 7 normal mice (sham group) were taken as the normal control group, and the above 28 ischemic stroke model mice (MCAO group) were randomly divided into 4 groups, namely, model control group, N15 polypeptide low-dose group (0.5 mg N15 polypeptide), N15 polypeptide medium-dose group (1.0 mg N15 polypeptide) and N15 polypeptide high-dose group (1.5 mg N15 polypeptide), with 8 mice in each group.

[0134] Drug administration: The blank control group mice and the N15 polypeptide drug group mice were injected with an equal volume of normal saline through the tail vein at the same time, and each group was injected continuously for 7 days; 14 days after the successful modeling, the model control group was injected with an equal volume of normal saline (M+S) through the tail vein at the same time; the N15 polypeptide low-dose group was injected with 0.5 mg N15 polypeptide (M+LD) through the tail vein every day; the N15 polypeptide medium-dose group was injected with 1.0 mg N15 polypeptide (M+MD) through the tail vein every day; the N15 polypeptide high-dose group was injected with 1.5 mg N15 polypeptide (M+HD) through the tail vein every day, and each group was treated with medication continuously for 7 days.

[0135] (3) Evaluation of cognitive ability, learning and memory function of each group of mice

[0136] In this example, the Morris water maze experiment was used to test the learning and memory ability of mice for spatial position and direction, and to evaluate the cognitive ability and learning and memory function of mice, as follows:

[0137] Experimental Materials:

[0138] ① The rat circular pool was 100 cm in diameter, 50 cm in height, 30 cm in depth, with a black bottom and the water temperature maintained at 23±2℃. Four equidistant points N, E, S, and W were marked on the pool wall as the starting point of the test. The pool was divided into four quadrants, and a platform was placed in the center of any quadrant (the platform was equidistant from the center of the pool wall). The platform was black, 12 cm in diameter, 29 cm in height, and submerged 1 cm under water to make it invisible. Triangular reference clues were posted around the pool and remained unchanged for the rats to use to locate the platform.

[0139] ②The blackout curtain can effectively block stray light from entering the test area. Use blackout curtains and indirect light sources to avoid light interference during image acquisition.

[0140] ③ Automatic video recording system: including camera, monitor, computer (equipped with image acquisition card and analysis software). The camera is installed about 3 meters above the pool and connected to the monitor and computer.

[0141] ④Computer and analysis software: Extract the movement trajectory data of experimental animals in the Morris water maze experiment and analyze and process it using SPSS statistical software.

[0142] Experimental process:

[0143] ① Place the mouse in the water with its head facing the pool wall. Randomly place it in one of the four starting positions: east, west, south, or north. Record the time (s) it takes for each group of animals to find the underwater platform. The results are as follows: Fig. 9 As shown;

[0144] Note that in the first few training sessions, if this time exceeds 60 s, guide the animal to the platform and let it stay on the platform for 10 s.

[0145] ② Move the animal away, dry it, and put it back in the cage. Each animal is trained 4 times a day, with an interval of 15 to 20 minutes between two training sessions, and the training continues for 5 days. On the second day after the last acquisition training, the platform is removed and a 60-second exploration training is started. The animal is placed in the water from the opposite side of the original platform quadrant. The automatic video recording system records the time the mouse spends in the target quadrant (the quadrant where the platform was originally placed) and the number of times it enters the quadrant. This is used as a detection indicator of spatial memory. The results are as follows: Fig.10 As shown in the figure, the time statistics of the mouse positioning platform in the Morris water maze experiment are as follows: Fig.11 shown.

[0146] Depend on Fig. 9 It can be seen that the time taken by the mice in the model control group to find the underwater platform after 5 days of training was significantly longer than that of the mice in the normal control group, and the time taken was statistically different between the groups (p < 0.05), indicating that stroke can reduce the cognitive ability and learning and memory ability of mice; while the time taken by the mice in the high-dose N15 polypeptide group to find the underwater platform was significantly shorter than that of the model control group, and there was no significant difference in the time taken by the mice in the normal control group, indicating that the N15 polypeptide can promote the recovery of the cognitive ability and learning and memory ability of mice, among which the high-dose N15 polypeptide can restore the cognitive ability and learning and memory ability of mice to normal levels.

[0147] Depend on Fig.10 The automatic video recording system records the movement trajectory of mice and exports the data for analysis. Fig.10 From left to right in the figure are the analysis results of the time taken to find the underwater platform for the first time, the number of times the underwater platform is crossed within 60 seconds, and the time spent on the underwater platform within 60 seconds. The specific results are analyzed as follows:

[0148] The time taken by the normal control group (sham) to find the underwater platform for the first time was the shortest, and the time taken by the model control group (MCAO) was much longer than that of the normal control group (sham), and there was a statistical difference in the time taken between the groups (p < 0.01); while in the N15 polypeptide drug group, as the dosage of N15 polypeptide gradually increased, the time taken to find the underwater platform for the first time gradually decreased, and a dose effect was shown, and there was a statistical difference in the time taken between the groups (p < 0.05), especially the time taken by the high-dose N15 polypeptide group to find the underwater platform for the first time was much shorter than that of the model control group.

[0149] In addition, the number of times mice in the normal control group (sham) crossed the underwater platform within 60 seconds was much greater than that in the model control group (MCAO), and there was a statistical difference in the number of times the two groups crossed the underwater platform (p < 0.01); while in the N15 polypeptide drug group, as the dosage of N15 polypeptide gradually increased, the number of times each group crossed the underwater platform gradually increased, and a dose effect was shown, and there was a statistical difference in the number of times the groups crossed the underwater platform (p < 0.05), especially the high-dose N15 polypeptide group crossed the underwater platform within 60 seconds much more than the model control group.

[0150] The normal control group (sham) stayed on the underwater platform much longer than the model control group (MCAO), and there was a statistical difference in the underwater platform stay time between the two groups (p < 0.01); while in the N15 polypeptide drug group, as the dosage of N15 polypeptide gradually increased, the stay time of each group on the underwater platform gradually prolonged, showing a dose effect, and there was a statistical difference in the stay time on the underwater platform between the groups (p < 0.01), especially the high-dose N15 polypeptide group passed through the underwater platform many more times within 60 seconds than the model control group.

[0151] In summary, N15 peptide can inhibit the neural damage caused by cerebral embolism in stroke mice and promote the recovery of cognitive ability and learning and memory ability in ischemic stroke mice.

[0152] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a polypeptide in the preparation of a drug for preventing or treating stroke, characterized in that: The polypeptide comprises the amino acid sequence shown in SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in preparing a drug for preventing or treating stroke, characterized in that: The polypeptide is used for preparing medicine for preventing or treating ischemic stroke.

3. Use of the polypeptide according to claim 2 in the preparation of a drug for preventing or treating stroke, characterized in that: The polypeptide also includes an amino acid sequence expressing a cell-penetrating vector; The cell-penetrating vector comprises the cell-penetrating polypeptide TAT.

4. Use of the polypeptide according to claim 3 in preparing a drug for preventing or treating stroke, characterized in that: It is used to prepare drugs for repairing brain nerve damage caused by ischemic stroke.

5. A drug for preventing or treating stroke, characterized in that: The active ingredient of the drug is a polypeptide, which includes the amino acid sequence shown in SEQ ID NO: 1 and the amino acid sequence of the cell-penetrating vector.

6. The drug for preventing or treating stroke according to claim 5, characterized in that: The amino acid sequence of the expression cell transmembrane vector is shown in SEQ ID NO:

2.

7. The drug for preventing or treating stroke according to claim 6, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

3.

8. The drug for preventing or treating stroke according to claim 7, characterized in that: The content of the polypeptide in the stroke prevention drug is ≥10ug / ml.

9. The drug for preventing or treating stroke according to claim 7, characterized in that: The content of the polypeptide in the drug for treating stroke is ≥0.01%wt.

10. The drug for preventing or treating stroke according to any one of claims 5 to 9, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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