Application of polypeptide in preparation of medicine for treating cerebral apoplexy

Abstract

The invention belongs to the technical field of medicines, and provides application of polypeptide in preparation of a medicine for treating cerebral apoplexy. The amino acid sequence of the polypeptide is as follows: Ala-Glu-Pro-Val-Pro-Leu. The polypeptide has different degrees of significant improvement effects on death rate, neurobehavioral scoring and cerebral infarction volume of model rats, can significantly improve rat brain tissue hippocampal CA1 region vertebral cell injury, and also has significant improvement effects on brain microcirculation after bilateral carotid artery ligation of the model rats.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular, relates to the application of a polypeptide in the preparation of a drug for treating cerebral stroke. Background Art

[0002] Stroke, commonly known as stroke, is divided into two types: ischemic stroke and hemorrhagic stroke. It is a disease caused by various reasons that damage cerebral blood vessels and cause focal or global brain tissue damage. The incidence of the disease is higher in cold seasons, and the peak of the disease usually occurs near noon. Common symptoms of stroke include sudden numbness or weakness, especially abnormalities on one side of the body, language or comprehension problems, vision problems, headaches, dizziness, etc.

[0003] Stroke has the characteristics of high incidence, disability, recurrence and mortality, and is the leading cause of death among Chinese residents. According to statistics, the incidence of stroke in China is increasing year by year, from 0.40% in 1993 to 1.23% in 2013, and the average age of onset is also decreasing. The recurrence rate of acute stroke patients in China is as high as 17.7% within one year, and the cumulative recurrence rate in five years exceeds 30%. Among them, ischemic stroke accounts for 75% to 90% of all strokes, while hemorrhagic stroke accounts for 10% to 25%.

[0004] Currently, the treatment of ischemic stroke usually takes measures such as vascular dilation, endarterectomy or arterial bypass surgery. The treatment of hemorrhagic stroke usually focuses on the removal of hemangiomas and blood clots. Immediate treatment after a stroke may significantly improve the prognosis. The above operations are risky, with more postoperative complications and more difficult recovery. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of a polypeptide in the preparation of a drug for treating cerebral stroke; the polypeptide has a significant improvement effect to varying degrees on the mortality rate, neurobehavioral score, and cerebral infarction volume of model rats, and can significantly improve the vertebral cell damage in the CA1 region of the hippocampus of rat brain tissue, and also has a significant improvement effect on the cerebral microcirculation of model rats after bilateral carotid artery ligation.

[0006] In order to achieve the above object, the solution adopted by the present invention is:

[0007] A polypeptide is used in the preparation of a drug for treating cerebral stroke, wherein the amino acid sequence of the polypeptide is: Ala-Glu-Pro-Val-Pro-Leu.

[0008] Furthermore, in a preferred embodiment of the present invention, stroke includes ischemic stroke and hemorrhagic stroke.

[0009] Furthermore, in a preferred embodiment of the present invention, the dosage form of the drug includes one of oral liquid, tablets, granules, capsules, pills, injections, transdermal absorption preparations, external lotions and in vivo implant preparations.

[0010] The beneficial effects of the use of a polypeptide provided by the present invention in the preparation of a drug for treating stroke are:

[0011] (1) Under the experimental conditions of Example 2 of the present application, a rat model of middle cerebral artery occlusion ischemia-reperfusion was successfully established. The polypeptides provided in the present application have significantly improved the mortality rate, neurobehavioral scores, and cerebral infarction volume of the model rats to varying degrees. In terms of mortality, the mortality rate of the polypeptide group provided in the present application was as low as 5%, and in terms of neurobehavioral scores and cerebral infarction volume, the polypeptide group provided in the present application showed significant improvements. In summary, under the conditions of the present experiment, the polypeptide group provided in the present application showed a significant neuroprotective effect and showed a clear trend of reducing cerebral infarction volume.

[0012] (2) Under the experimental conditions of Example 3 of the present application, the bilateral carotid artery ligation model was used for rats. The experimental results showed that the polypeptide provided by the present application can significantly improve the damage of pyramidal cells in the CA1 region of the hippocampus of rat brain tissue, and has a significant improvement effect on the cerebral microcirculation of the model rats after bilateral carotid artery ligation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A schematic diagram showing the steps of solid phase synthesis of peptides. DETAILED DESCRIPTION

[0014] The following is an explanation of the present invention in combination with specific experiments, which is not intended to limit the scope of protection of the present invention.

[0015] Table 1 English names or abbreviations of reagents and solvents used in writing

[0016]

[0017]

[0018] Example 1: Chemical synthesis of polypeptides

[0019] The synthesis of polypeptide compounds adopts conventional solid phase synthesis methods, and goes through multiple cycles of resin swelling, substitution, deprotection, washing, amino acid dissolution, amino acid activation and condensation, washing, re-deprotection, and finally cleavage and side chain deprotection.

[0020] Schematic diagram of peptide solid phase synthesis steps. Figure 1 . Figure 1In the figure, Cl-2-Cl-Resin represents 2-chlorotrityl chloride resin (2-Chlorotrityl Chloride Resin); Fmoc-Aa(n) and the like represent 9-fluorenylmethoxycarbonyl amino acids; DIPEA is N,N-diisopropylethylamine; DCM is dichloromethane; PIP is piperidine; DMF is N,N-dimethylformamide; HOBt is 1-hydroxybenzotriazole; DIC is N,N'-diisopropylcarbodiimide; TFA is trifluoroacetic acid; and TIPS is triisopropylsilane.

[0021] The following is a method for the synthesis and purification of a polypeptide, comprising the steps of:

[0022] 1. Preparation of fully protected peptide resin; 2. Cleavage and deprotection; 3. Purification (salt exchange) and freeze-drying.

[0023] Step 1. Preparation of fully protected peptide resin

[0024] (1) Resin swelling: Weigh 2.0192 g of 2-Chlorotrityl Chloride Resin (S = 0.73 mmol / g), add it into a synthesis tube with a sieve plate, swell it with 40 ml of dichloromethane for 30 min, and remove the dichloromethane by suction filtration.

[0025] (2) Preparation of Fmoc-Asp(OtBu)-resin: Weigh Fmoc-Asp(OtBu)-OH and DIPEA at a molar ratio of 1:1.5:1.65, dissolve in 20 ml of dichloromethane, and add to a synthesis tube. Bubble with N2 at room temperature for 1-3 hours, directly add 2 ml of methanol to the reaction solution to seal for 30 minutes. Then wash with dimethylformamide 4 times, 25 ml / time, and drain the resin.

[0026] (3) Removal of Fmoc protecting group: 20 ml of 20% piperidine-DMF (v / v) solution was added to the reactor, N2 was bubbled for 20 min, and then the mixture was drained; then the mixture was washed with dimethylformamide for 6 times, 25 ml / time, 3 min / time, and the removal of Fmoc was detected by the ninhydrin method.

[0027] (4) Amino acid preactivation: Add 4.38 mmol of Fmoc-protected amino acid, 5.26 mmol of HOBt, and 4.60 mmol of DIC into a 250 ml round-bottom flask, dissolve in 20 ml of 1:1 DCM-DMF (v / v), and preactivate for 30-60 min under stirring at -5-0°C ice bath.

[0028] (5) Amino acid connection: Pour the activated protected amino acid solution into the reactor and add an appropriate amount of DCM to clean the utensils. Bubble N2 at room temperature for 1-3 hours. Use the ninhydrin method to check whether the amino acid connection is complete. If complete, drain. Wash the resin with dimethylformamide 4 times, 25 ml / time, 3 min / time, and drain. The amount of each amino acid, condensation agent and specific reaction time are shown in Table 2.

[0029] (6) After the first amino acid is condensed, steps (3) to (5) are repeated to extend the peptide chain in the order of amino acids until the last amino acid is coupled.

[0030] Table 2 Amount of amino acid and condensing agent

[0031] Amino acid name AA / eq Amino acid dosage / g HOBt / g DIPEA / g DIC / g Fmoc-L-Asp(OtBu)-OH 2.19 0.90 0 0.31 0 Fmoc-L-Gln(Trt)-OH 4.38 2.67 0.71 0.57 0.58 Fmoc-L-Lys(Boc)-OH 4.38 2.05 0.71 0.57 0.58 Fmoc-L-Val-OH 4.38 1.49 0.71 0.57 0.58 Fmoc-L-Leu-OH 4.38 1.55 0.71 0.57 0.58 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16 Fmoc-L-Val-OH 4.38 1.49 0.71 0.57 0.58 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16 <![CDATA[Fmoc-L-Glu(OtBu)-OH·H2O]]> 4.38 1.94 0.71 0.57 1.16 <![CDATA[Fmoc-L-Ala-OH·H2O]]> 4.38 1.44 0.71 0.57 1.16 <![CDATA[Fmoc-L-Ala-OH·H2O]]> 4.38 1.44 0.71 0.57 1.16 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16

[0032] Step 2: Cutting and deprotection

[0033] (1) Add 50 ml of cutting agent (TFA:TIPS:H2O=95:2.5:2.5, v / v) to the synthesis tube of step 1, and allow N2 to bubble for reaction for 1.5-3 hours.

[0034] (2) After the cleavage reaction is completed, the cleavage agent is filtered into a 250 ml round-bottom flask. After vacuum concentration to one-third of the original volume of the cleavage agent, 10 times the current volume of methyl tert-butyl ether is added and stirred for 30 minutes. The obtained mixed solvent is filtered and washed three times with 30 ml of methyl tert-butyl ether respectively, and the obtained crude peptide product is placed in a sand core funnel and blown dry with N2 in a fume hood to evaporate the solvent until the crude peptide is in powder form. 1.87 g of crude peptide is obtained, with a crude yield of 85.1%.

[0035] Step 3: Purification (salt exchange) and freeze-drying

[0036] The crude peptide obtained in step 2 was purified by HPLC using the following chromatographic parameter condition A. Specifically, the crude peptide obtained in step 2 was dissolved in water and / or acetonitrile and filtered through a 0.45 μm filter membrane; injected; gradient eluted with an acetonitrile-water mobile phase; the target peptide eluate was collected; and finally concentrated by rotary evaporation.

[0037] Chromatographic parameter conditions A:

[0038] Chromatographic column: YMC-Actus Triart C18 30*250mm;

[0039] Eluent A: 0.1% (v / v) TFA / H2O;

[0040] Eluent B: acetonitrile;

[0041] Flow rate: 25 ml / min;

[0042] UV detection wavelength 220nm;

[0043] Table 3 Gradient elution conditions

[0044] Time / min Eluent A (%) Eluent B (%) 0 90 10 30 75 25

[0045] Then, the product obtained in the above steps was salt-exchanged by HPLC using the following chromatographic parameter condition B to obtain the final polypeptide. Specifically, a 95% A1+5% B equilibrated chromatographic column was used; then the sample was injected; then a 95% A2+5% B equilibrated chromatographic column was used; gradient elution was performed using A1 and B; the target peptide eluate was collected; finally, the evaporation was concentrated by rotary evaporation and freeze-dried to obtain a polypeptide (purification yield 71.7%, purity 100%). The structure of the polypeptide was confirmed by MS and 1H-NMR.

[0046] Chromatographic parameter condition B:

[0047] Chromatographic column: YMC-Actus Triart C18 30*250mm

[0048] Eluent A1: 0.1 M acetic acid

[0049] Eluent A2: 0.025M acetic acid + 0.1M ammonium acetate

[0050] Eluent B: acetonitrile

[0051] Flow rate 25ml / min

[0052] UV detection wavelength 220nm

[0053] Table 4 Gradient elution conditions

[0054] Time / min Eluent A1 (%) Eluent B (%) 0 95 5 5 95 5 35 70 30

[0055] Table 5 Synthesized peptides

[0056] Compound sequence Amount of crude product (g) Purification yield purity MS Ala-Glu-Pro-Val-Pro-Leu 2.7 71.7 100％ 625.1

[0057] Peptide:Ala-Glu-Pro-Val-Pro-Leu acetate

[0058] 1H NMR(600MHz,DMSO)δ8.29(d,J=7.1Hz,1H),7.89(d,=8.6Hz,1H),7.79(d,J=8.0Hz,1H), 4.54(d,J=5.4Hz,1H),4.42-4.38(m,1H),4.37-4.28(m,2H),4.09(dd,J=14.2,8.5Hz,1 H),3.66-3.46(m,5H),2.28(t,J=7.4Hz,2H),2.13-1.71(m,16H,AcOH),1.71-1.58(m,2 H),1.53-1.32(m,2H),1.18(d,J=6.9Hz,3H),0.90-0.83(m,9H),0.80(d,J=6.6Hz,3H).

[0059] Example 2

[0060] Experimental animals: 60 male SD rats, SPF grade, room-fed, 6-7 weeks old, 180-220 g; Source: Sichuan Weitonglihua Experimental Animal Technology Co., Ltd., Experimental Animal Production License: SCXK (Sichuan) 2023-0040, Experimental Animal Quality Certificate Number: 511215600003638.

[0061] Model establishment: After rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (anesthetic volume of 3 mL / kg), they were fixed on the operating table in a supine position. After a midline incision in the neck, the muscle layer was bluntly separated to expose the right common carotid artery, and the external carotid artery, internal carotid artery and other related vascular branches were separated in turn. The external carotid artery was ligated with double lines, and a loose knot was tied with a single line at the stump of the external carotid artery. At the same time, the artery clamp temporarily clamped the proximal end of the common carotid artery and the internal carotid artery; a 45° small opening was cut between the two lines of the external carotid artery, and the line plug wrapped with silicone was carefully inserted from the external carotid artery to the internal carotid artery, the external carotid artery was cut, and the external carotid artery was pulled to insert the line plug in the direction of the carotid, the fixed line was tied, the internal carotid artery clamp was released, and the line plug was continued to be inserted into the middle cerebral artery until a slight resistance was felt. The line plug was inserted for about 1.8-2.2 cm, the fixed line plug was ligated, and the artery clamp of the common carotid artery was released. After 2 hours of ischemia, the line plug was pulled out for reperfusion, the stump of the external carotid artery was tied, and the skin was sutured.

[0062] Grouping information: Group design: model group and polypeptide group, 30 mice / group, male; Grouping method: random grouping according to the behavioral score of neurological function injury before ischemia-reperfusion.

[0063] Experimental methods: Dosage and route design basis: According to the information provided by the client, it is consistent with the proposed clinical route of the test substance; no drug was given to the model group; drug preparation of the peptide group: prepared with normal saline; route of administration of the peptide group: tail vein injection (iv); frequency of administration: 2 times, immediately during ischemia-reperfusion, and repeated once 6 hours after ischemia-reperfusion; dosing cycle: 1 day; administration volume: 5mL / kg;

[0064] Detection indicators:

[0065] (1) General observation

[0066] Observe animals: once a day;

[0067] Observation time and frequency: Starting from the day of surgery, observe the general condition of the animals once a day.

[0068] Observation content: including but not limited to the animal's physical appearance, general behavior, mental state, glandular secretions, respiratory status, fecal characteristics, dying and death conditions, etc.

[0069] (2)Weight

[0070] Test animals: surviving animals in each group;

[0071] Testing time and frequency: once before reperfusion;

[0072] (3) Neurobehavioral score

[0073] Test animals: surviving animals in each group;

[0074] Testing time and frequency: Longa's method neurobehavioral scores were performed on all surviving animals before reperfusion after ischemia, 1h and 24h after reperfusion (rats with scores of 1-3 points were judged as qualified models and were given medication). The scoring criteria are shown in Table 6:

[0075] Table 6 Neurobehavioral scoring standards

[0076] score standard 0 points Normal, no symptoms of nerve damage 1 point Inability to fully extend the contralateral forepaw, mild neurological deficit 2 points When walking, the rats circled toward the paralyzed side, with moderate neurological deficits 3 points When walking, the rats fell to the paralyzed side and had severe neurological deficits. 4 points Unable to walk spontaneously, loss of consciousness

[0077] (4) Cerebral infarction volume

[0078] Test animals: surviving animals in each group;

[0079] Detection time: 24h after reperfusion;

[0080] Detection method: After 24 hours of reperfusion, the surviving rats in each group were dissected, the hearts were perfused with pre-cooled saline, the brain tissue was taken, and brain slices were made at intervals of 2 mm. They were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC), fixed in 4% paraformaldehyde for 24 hours, and the brain slices were photographed to calculate the percentage of cerebral infarction volume. Cerebral infarction volume % = cerebral infarction volume / total brain volume × 100%.

[0081] Test results:

[0082] (1) General clinical observation

[0083] In this experiment, all the animals (100%) were found to have black secretions in the left eye 1.5-2 hours after ischemia, and symptoms such as salivation were observed. After 1 hour of reperfusion, the mortality rates of the model group and the polypeptide group were 50% (15 / 30) and 5% (6 / 30), respectively. Except for the above conditions, no obvious abnormalities were found in the appearance signs, general behavior activities, glandular secretions, respiratory status, feces shape, etc. of the animals in each group.

[0084] (2)Weight

[0085] There was no significant difference in body weight among the groups.

[0086] (3) Neurobehavioral score

[0087] The statistical results of behavioral scores are shown in Appendix 7.

[0088] Table 7 Statistical results of behavioral scores (Mean±SEM)

[0089] Group Before perfusion 1h after perfusion 24h after perfusion Model Group 2.6±0.2 2.2±0.1 2.3±0.2 Peptide Group 2.5±0.2 1.6±0.2 0.8±0.2*

[0090] Note: *, P<0.05, vs model group. .

[0091] In this experiment, the neurobehavioral scores of rats with middle cerebral artery occlusion at different time points showed that the neurobehavioral scores of the model group and the polypeptide group before ischemia-reperfusion (0h) were 2.6±0.2 and 2.5±0.2, respectively, with no significant difference between the groups; the neurobehavioral scores 1h after ischemia-reperfusion were 2.2±0.1 and 1.6±0.2, respectively, with no significant difference between the groups; the neurobehavioral scores 24h after ischemia-reperfusion were 2.3±0.2 and 0.8±0.2, respectively. Compared with the model group, the neurobehavioral score of the polypeptide group was significantly decreased (P<0.05).

[0092] (4) Cerebral infarction volume

[0093] The statistical results of cerebral infarction volume rate (%) are shown in Table 8.

[0094] Table 8 Cerebral infarction volume rate % and mortality %

[0095] Group <![CDATA[Total brain volume / mm 3 > <![CDATA[Total infarct volume / mm 3 > Infarct volume rate / % mortality rate / % Model Group 470068.12 144649.08 30.8％ 50％ Peptide Group 491345.24 28,290.12 <![CDATA[5.8% * ]]> <![CDATA[5% * ]]>

[0096] Note: *, P < 0.05, vs. model group.

[0097] In this experiment, 24 hours after ischemia-reperfusion, the percentages of cerebral infarction volume in the model group and the polypeptide group were 30.8% and 5.8% respectively. Among them, the average infarction volume in the polypeptide group was significantly lower than that in the model group.

[0098] Example 3

[0099] Experimental animals: 16 Sprague Dawley rats, ordinary grade, 8 weeks old, 188 - 230 g when entering the room; Source: Guangdong Vital River Laboratory Animal Technology Co., Ltd., Laboratory Animal Quality Certificate Number: SCXK(Yue)2022 - 0063.

[0100] Model establishment: Food was withheld 12 hours before the experiment, and water was withheld 4 hours before. The rats were induced to anesthesia by inhaling 3 - 4% isoflurane and maintained with 1 - 2% isoflurane at a flow rate of 0.6 - 0.8 L / min. The left and right common carotid arteries were isolated, and about 1 cm below the carotid artery bifurcation was ligated doubly with 4 - 0 silk thread. Then, the skin incision was sutured with normal suture, and the cerebral blood flow (CBF) in the midline of the rat head was measured.

[0101] Grouping information: Group design: Model group, polypeptide group, 8 rats / group, male; Grouping method: Grouped according to CBF after model establishment.

[0102] Experimental method: Design basis for dosage and administration route: According to the information provided by the client, administration started on the 15th day after model establishment, once a day by tail vein injection for 14 consecutive days; The model group was not given medicine; Drug preparation in the polypeptide group: Prepared with normal saline; Administration route in the polypeptide group: Tail vein injection (i.v.); Administration frequency: Once a day; Administration cycle: Continuous administration for 2 weeks; Administration volume: 5 mL / kg.

[0103] Detection indexes:

[0104] (1) General observation

[0105] Observation time and frequency: From after model establishment to animal dissection, the general condition of the animals and the reaction at the surgical site were observed once a day; Observation indexes or contents: Including but not limited to local reaction at the surgery, external appearance signs of the animals, general behavioral activities, mental state, glandular secretion, respiratory state, fecal traits and colors, genitalia, death and other situations and other toxic manifestations.

[0106] (2) Body weight

[0107] Inspection frequency: Once a week before model establishment and after the operation; Once a week after administration started.

[0108] Test animals: All living animals.

[0109] (3) Cerebral blood flow

[0110] Inspection time: before modeling, after modeling, 14 days after modeling, and 28 days after modeling;

[0111] Test animals: All living animals;

[0112] Detection methods and indicators: Laser speckle measurement of rat cerebral blood flow.

[0113] (4) Histopathological examination

[0114] At the end of the experiment, brain tissues were fixed and immunofluorescence double staining (CD31 / AQP4) was performed to detect brain microvessels.

[0115] HE staining of brain tissue was used for pathological grading assessment: the grading was determined based on the pathological changes in the neurons in the CA1 region of the hippocampus. The grading criteria are as follows:

[0116] Grade 0, no neuronal cell death was observed in the target area;

[0117] Grade 1, scattered neuronal cell death can be seen in the target area;

[0118] Grade 2, large areas of neuronal cell death can be seen in the target area;

[0119] Level 3: Almost all nerve cells in the target area die.

[0120] 2-4 sections were selected for each animal, and each tissue section was graded on both sides, and the average grade of both sides was used as the final result for statistics.

[0121] Test results:

[0122] (1) General clinical observation

[0123] The experimental animals in this project showed no abnormalities.

[0124] (2)Weight

[0125] There was no significant difference in body weight among the groups.

[0126] (3) Changes in cerebral blood flow (CBF) are shown in Table 9:

[0127] Table 9 Changes of CBF in rats in each group

[0128] Group Before modeling After modeling 2 weeks after modeling After 14 days of treatment Model Group 2626.24±30.97 1869.94±30.87 2134.65±25.63 2648.86±42.21 Peptide Group 2514.15±28.56 2488.02±26.54 1967.13±22.05 2965.56±15.14

[0129] Table 9 shows that the peptide group drug treatment was carried out 14 days after modeling, and the treatment was continued for 14 days. The CBF results showed that the model group almost recovered to the level before modeling, and the CBF of the peptide group increased by about 18% compared with that before modeling, indicating that the treatment with the peptide group of the present application can significantly increase the cerebral blood flow of rats.

[0130] (4) Immunofluorescence co-staining of specific marker molecules (CD31 and AQP4) to evaluate microvascular circulatory function

[0131] At the end of the experiment, the rats were perfused and brain tissues were obtained and fixed before embedding and sectioning. Immunofluorescence staining of CD31 and AQP4 was performed. The experimental results are shown in Table 10.

[0132] AQP4 and CD31 play an important role in maintaining the integrity and function of the blood-brain barrier. AQP4 is highly expressed in the end feet of vascular astrocytes around the blood-brain barrier. Studies have shown that oxidative stress can trigger the upregulation of AQP4, the disorder of blood-brain barrier integrity and the formation of edema. CD31 (PECAM-1) is a member of the immunoglobulin family of cell adhesion molecules. It plays an important role in the inflammatory mechanism related to the migration of leukocytes through the endothelium during neuroinflammation and neurodegeneration. Increased CD31 expression can lead to blood-brain barrier dysfunction and may also cause pathophysiological changes of neural damage and dysfunction.

[0133] Table 10 Positive expression area of ​​AQP4 and CD31 in the hippocampal CA1 region (μm 2 ), Mean ± SEM

[0134] Group AQP4 area vs. Model, P CD31 area vs. Model, P Model Group 2814.41±249.57 / 2352.08±205.88 / Peptide Group 4645.65±443.41 0.004 5208.97±401.32 0.002

[0135] In Table 9, the positive fluorescence area of ​​AQP4 in hippocampal CA1 of the polypeptide group (4645.65±443.41) increased by 65.1% compared with that of the model group (2814.41±249.57), which was significantly different (P=0.004).

[0136] The positive fluorescence area of ​​CD31 in the hippocampal CA1 region of the peptide group (5208.97±401.32) increased by 2.2 times compared with the model group (2352.08±205.88), which was significantly different (P=0.002).

[0137] (5) Morphological changes of neurons in the CA1 region of the hippocampus of rats in each group:

[0138] The brain tissue was fixed and embedded and then HE stained for pathological scoring. Global cerebral ischemia in the model group rats caused obvious neuronal damage in the hippocampal CA1 region. Under the microscope, large areas of neuronal cell destruction and death were observed. The cells were loosely arranged and disordered, with a large number of cell fragments scattered around. The remaining cells shrank and were irregular in shape. The pathological grading results are shown in Table 10:

[0139] Table 10 Pathological grading results, Mean ± SEM

[0140] Group HE pathological staining grading Model Group 1.91±0.18 Peptide Group 0.74±0.20

[0141] The results in Table 10 show that in the pathological grading score, the polypeptide group was significantly better than the model group.

[0142] In summary, the polypeptide provided by the present invention has a significant improvement effect on the mortality rate, neurobehavioral score and cerebral infarction volume of model rats to varying degrees, and can significantly improve the vertebral cell damage in the CA1 region of the hippocampus of rat brain tissue, and has a significant improvement effect on the cerebral microcirculation of model rats after bilateral carotid artery ligation. Therefore, the polypeptide provided by the present invention has an application prospect in the preparation of drugs for treating cerebral stroke.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a polypeptide in the preparation of a drug for treating stroke, characterized in that: The amino acid sequence of the polypeptide is: Ala-Glu-Pro-Val-Pro-Leu.

2. Use of the polypeptide according to claim 1 in the preparation of a drug for treating stroke, characterized in that: The stroke includes ischemic stroke and hemorrhagic stroke.

3. Use of the polypeptide according to claim 1 in the preparation of a drug for treating stroke, characterized in that: The dosage form of the drug includes one of oral liquid, tablet, granule, capsule, dripping pill, injection, transdermal absorption preparation, external lotion and in vivo implant preparation.

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