Application of a polypeptide in preparing a drug for treating ischemic stroke

Abstract

The present invention discloses the use of a polypeptide in the preparation of a drug for treating ischemic stroke, and belongs to the field of medical technology. The present invention establishes an ischemic stroke model of middle cerebral artery occlusion / reperfusion (MCAO / R) in rats, and finds that the polypeptide of the present application can significantly reduce the neurological deficit of ischemic stroke rats; in an in vitro cerebral ischemia model of human brain microvascular endothelial cells (hCMEC / D3) under a glucose-oxygen deprivation / reoxygenation-reglycosylation (OGD / R) model, it is found that the polypeptide of the present application can significantly improve the survival rate of cells.

Description

Technical Field

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

[0002] Ischemic stroke is a cerebrovascular disease with an increasing incidence rate year by year, often manifested by symptoms such as limb paralysis and cognitive impairment. Currently, alteplase (rtPA) is commonly used for thrombolytic therapy, but this method has a narrow therapeutic window and many contraindications to thrombolysis, which poses a challenge to the development of drugs for the treatment of ischemic stroke.

[0003] The rat middle cerebral artery occlusion / reperfusion (MCAO / R) model is generally considered to be a standard animal model of focal cerebral ischemia. Its basic principle is to occlude the origin of the middle cerebral artery with a suture, causing ischemia in the middle cerebral artery blood supply area, thereby causing focal cerebral ischemia in rats. This method has good stability, good repeatability, small damage, precise infarction site and high success rate. The glucose deprivation / reoxygenation (OGD / R) model is an in vitro ischemia model. The concentrations of CO2 and O2 in the incubator are precisely controlled by directly introducing N2, while depriving the cell culture medium of sugar, thereby creating an oxygen-glucose-deficient environment for the cells, simulating the in vivo ischemia. Summary of the invention

[0004] In view of the 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 ischemic stroke; in the process of in vivo studies on the pharmacological activity of the therapeutic effect on ischemic stroke, it was found that the polypeptide of the present invention can significantly improve the neurological function defects of rats, enhance motor coordination ability, reduce the area of ​​cerebral infarction and the number of apoptotic brain neuronal cells, and reduce the levels of brain inflammatory factors and oxidative stress; in the process of in vitro studies on the pharmacological activity of the therapeutic effect on ischemic stroke, it was found that the polypeptide of the present invention can increase the survival rate of hCMEC / D3 cells, enhance cell migration and repair ability, and reduce the level of cell active oxygen.

[0005] The technical solution adopted by the present invention to solve the technical problem is:

[0006] In a first aspect, the present invention provides a use of a polypeptide and related materials thereof in the preparation of a drug for treating ischemic stroke; the related materials are polynucleotides capable of encoding the polypeptide or vectors containing the polynucleotides; the polypeptide is selected from the following A) or B):

[0007] A) the amino acid sequence is NYEPPTVVPGGDL, as shown in SEQ ID NO.1;

[0008] B) a product obtained by conventionally modifying the amino acid side chain groups of the polypeptide described in A), the amino terminus or the carboxyl terminus of the polypeptide described in A), or a product obtained by attaching a tag for polypeptide or protein detection or purification to the polypeptide described in A);

[0009] The conventional modification is amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization or biotinylation;

[0010] The tag is His, GST, EGFP, MBP, Nus, HA, IgG, FLAG or c-Myc, etc.

[0011] The polypeptides described above are obtained by using methods known in the prior art, such as chemical synthesis using an automatic polypeptide synthesizer; deducing the nucleotide sequence from a short peptide sequence and then cloning it into a vector for biosynthesis; or extracting and purifying it in large quantities from existing organisms.

[0012] Preferably, the present invention protects the use of the above-mentioned polypeptide in the preparation of a drug for treating MCAO / R-induced ischemic stroke in rats and / or OGD / R-induced damage to human brain microvascular endothelial cells (hCMEC / D3 cells).

[0013] In a second aspect, the present invention provides a drug for preventing and treating ischemic stroke, comprising the polypeptide described above.

[0014] Preferably, the drug further comprises one or more pharmaceutically acceptable carriers.

[0015] More preferably, the pharmaceutically acceptable carrier is a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, an absorption carrier, a surfactant or a lubricant.

[0016] The medicine can be further prepared into tablets, granules, capsules, oral liquids or injections and other forms. Various acute medicines can be prepared according to conventional methods in the pharmaceutical field.

[0017] In a third aspect, the present invention provides a method for treating ischemic stroke by administering the polypeptide described above.

[0018] The method of administration can be determined based on the activity, toxicity and patient tolerance of the drug.

[0019] Beneficial Effects

[0020] The use of a polypeptide provided by the present invention in the preparation of a drug for treating ischemic stroke has the following advantages and effects:

[0021] (1) In the in vivo study of the pharmacological activity of the peptide NYEPPTVVPGGDL in the treatment of ischemic stroke, it was found that the peptide NYEPPTVVPGGDL helped rats gain weight, significantly improved rats' neurological deficits, enhanced motor coordination, reduced the area of ​​cerebral infarction and the number of apoptotic brain neurons, and reduced the levels of brain inflammatory factors and oxidative stress.

[0022] (2) In the in vitro study of the pharmacological activity of the peptide NYEPPTVVPGGDL in the treatment of ischemic stroke, it was found that the peptide NYEPPTVVPGGDL can increase the survival rate of hCMEC / D3 cells, enhance cell migration and repair capabilities, and reduce the level of cellular reactive oxygen species.

[0023] Through the above research, the pharmacological activity of the peptide NYEPPTVVPGGDL in treating MCAO / R-induced ischemic stroke and OGD / R-induced brain endothelial cell injury was systematically investigated both in vivo and in vitro, which promoted the development of the peptide NYEPPTVVPGGDL, which is expected to be developed as a therapeutic drug for ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The polypeptide NYEPPTVVPGGDL of the present application improves the body weight and behavior of rats with ischemic stroke under MCAO / R model, wherein: (A) body weight change; (B) behavioral score of neurological deficit; (C) latency of rotarod test;

[0025] Figure 2 The polypeptide NYEPPTVVPGGDL of the present application reduces the cerebral infarction area and neuronal cell apoptosis in rats with ischemic stroke under MCAO / R model, wherein (A, B) TTC staining images and percentage of cerebral infarction area; (C, D) brain neuronal cell apoptosis images and percentage of apoptosis;

[0026] Figure 3 The polypeptide NYEPPTVVPGGDL of the present application reduces the levels of brain inflammatory factors and oxidative stress in rats with ischemic stroke under MCAO / R model, including (A, B, C) brain IL-6, IL-1β, TNF-α levels; (D, E) brain MDA and SOD levels;

[0027] Figure 4 The polypeptide NYEPPTVVPGGDL of the present application protects hCMEC / D3 cells under OGD / R conditions, wherein: (A) cell survival rate; (B, C) cell scratch images and cell migration rate; (D, E) cell reactive oxygen fluorescence images and reactive oxygen levels. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and examples. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0029] The preparation and purification of the polypeptide NYEPPTVVPGGDL of the present invention are the same as those in CN112898385A.

[0030] Example 1: Peptide NYEPPTVVPGGDL for the treatment of ischemic stroke caused by MCAO / R

[0031] Step 1: Experimental grouping (6-7 week old SD male mice, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., n = 6)

[0032] i) Sham operation group: After sham operation, the mice were intraperitoneally injected with an equal volume of normal saline (0.5 mL / 200 g) for 7 days.

[0033] ii) Modeling group: After modeling, the mice were intraperitoneally injected with an equal volume of normal saline (0.5 mL / 200 g) for 7 days.

[0034] iii) Peptide 10 mg / kg group: After model establishment, an equal volume of peptide NYEPPTVVPGGDL (0.5 mL / 200 g) was intraperitoneally injected for 7 days.

[0035] Step 2: Establishment of rat middle cerebral artery occlusion / reperfusion (MCAO / R)-induced ischemic stroke model

[0036] i) A midline incision was made on the rat neck, and the surrounding tissues were separated to expose the common carotid artery and the internal and external carotid arteries.

[0037] ii) Separate the common carotid artery, separate the external carotid artery and internal carotid artery in sequence along the common carotid artery, tie a loose knot on the internal carotid artery and keep it for later use. Clamp the common carotid artery with an artery clamp. After double-ligating the external carotid artery, cut a 45° small incision on the external carotid artery, insert the thread plug in the direction of the carotid, free the stump of the external carotid artery, tie the fixation thread, and release the artery clamp.

[0038] iii) Carefully insert the silicone-coated suture from the external carotid artery into the internal carotid artery to a depth of 2 cm. After the suture is left in place for 2 hours of ischemia, remove the suture and allow the head to retreat to the external carotid artery to achieve reperfusion of the middle carotid artery.

[0039] iv) Suture the wound skin and put the animal back into the cage after it wakes up from anesthesia. Inject normal saline after surgery to reduce the mortality rate. Pay attention to keeping the animal warm and monitoring vital signs during and after the operation. It is advisable to maintain the room temperature at 25℃, and pay attention to postoperative care.

[0040] Step 3: Effects of the peptide NYEPPTVVPGGDL on body weight and behavior in rats with ischemic stroke

[0041] i) Body weight: The rats were weighed daily after administration, and a time course graph of the body weight difference was drawn.

[0042] ii) Behavioral scoring of neurological deficits: Neurological behavioral scoring was performed in combination with the Longa and Bederson scoring methods. The higher the score, the more severe the neurological deficit. The specific scoring method is as follows: no neurological deficit: 0 points; the forepaw on the paralyzed side cannot be fully extended when placed on the table, and after the tail is lifted, the forelimb on the paralyzed side is retracted and flexed under the abdomen: 1 point; turning in circles toward the paralyzed side when walking, and the resistance to pushing objects toward the paralyzed side is significantly lower than that on the normal side: 2 points; falling toward the paralyzed side when walking: 3 points; unable to walk automatically, with loss of consciousness: 4 points.

[0043] iii) Rotarod test: Before modeling, rats were trained on the rotarod instrument for 3 days, 3 times a day. One day before modeling, the rotarod program time was set to 300 s, and the speed was accelerated from 0 rpm to 40 rpm. The latency of the rats on the rotarod was recorded as the baseline value. Using the same conditions, unmodeled rats were screened out one day after modeling. The test was carried out 7 days after modeling, and the latency of the rats on the rotarod was recorded.

[0044] The experimental results showed that the weight of rats with ischemic stroke increased after administration ( Figure 1 .A), decreased behavioral scores of neurological deficits ( Figure 1 .B), the latency of the rotarod test increased ( Figure 1 .C), indicating that the peptide NYEPPTVVPGGDL can increase the body weight of rats with ischemic stroke, reduce neurological deficits, and improve motor coordination ability.

[0045] Step 4: Effects of the peptide NYEPPTVVPGGDL on cerebral infarction area and neuronal apoptosis in rats with ischemic stroke

[0046] i) Cerebral infarction area: Rats were killed 7 days after modeling, and brain tissue was taken and frozen at -80℃ for several minutes and then evenly cut into 2 mm slices. The first cut was made at the midpoint of the line connecting the anterior pole of the brain and the optic chiasm; the second cut was made at the optic chiasm; the third cut was made at the infundibulum handle; and the fourth cut was made between the infundibulum handle and the caudal pole of the posterior lobe. Subsequently, the five slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37℃ for 20-30min, and these brain slices were imaged with a camera. The infarcted area appeared white, while the non-infarcted tissue appeared red. ImageJ software was then used to analyze and calculate the infarct area.

[0047] ii) Level of apoptosis of brain neurons:

[0048] ① Sample collection and fixation: The rats were bled from the femoral artery, and the rats were perfused with normal saline and 4% paraformaldehyde, and the brain tissue was fixed in 4% paraformaldehyde overnight.

[0049] ② Dehydration: Dehydrate with 20% sucrose solution for 3 days, then with 30% sucrose solution for 3 days, changing the solution every day.

[0050] ③ Embedding and sectioning: Dehydrated tissue was embedded with OCT glue and cut into 50 μm brain slices using a freezing microtome. Wash with PBS three times, 5 min each time.

[0051] ④ Blocking: Use 0.3% Triton and 5% BSA solution as blocking solution to block the brain slices at room temperature for 1.5-2 hours.

[0052] ⑤ Primary antibody incubation: Prepare NeuN antibody at a ratio of 1:350 with blocking solution and incubate overnight at 4°C. Place at room temperature for 2 hours before incubating with secondary antibody.

[0053] ⑥ Secondary antibody incubation: remove the primary antibody, wash the brain slices 3 times with PBS, incubate with green fluorescent labeled secondary antibody at room temperature in the dark for 1 hour, and wash 3 times with PBS.

[0054] ⑦ Add TUNEL detection solution to the brain slices, incubate at 37℃ in the dark for 60min, and wash 3 times with PBS.

[0055] ⑧After sealing with anti-fluorescence quenching sealing solution, collect pictures under a laser confocal microscope, and use imageJ to count the number of apoptotic neurons in the field of view.

[0056] The experimental results showed that the peptide NYEPPTVVPGGDL could reduce the cerebral infarction area of ​​rats with ischemic stroke ( Figure 2 .A, Figure 2 .B), in addition, after administration, the number of NeuN-labeled neuronal cells increased, and the number of TUNEL-labeled apoptotic neuronal cells decreased ( Figure 2 .C, Figure 2 .D), indicating that the peptide NYEPPTVVPGGDL can reduce the level of neuronal apoptosis and promote neuronal repair.

[0057] Step 5: Effects of the peptide NYEPPTVVPGGDL on the levels of brain inflammatory factors and oxidative stress in rats with ischemic stroke

[0058] i) Brain inflammatory factors and oxidative stress levels: Rats were killed 7 days after modeling, and the cortical area on the ischemic side of the brain was obtained and homogenized with ice PBS at a ratio of 1:9 (w / v). The supernatant was collected after centrifugation and measured strictly according to the instructions of the inflammatory factor (IL-1β, IL-6 and TNF-α) kit and the oxidative stress (MDA, SOD) kit. After the reaction, the results were read using an enzyme reader and the results were calibrated with protein for statistical analysis.

[0059] The experimental results showed that the levels of IL-1β, IL-6 and TNF-α in the brain of rats with ischemic stroke decreased after administration ( Figure 3 .A, Figure 3 .B, Figure 3 .C), MDA level decreased, SOD level increased ( Figure 3 .D, Figure 3 .E), indicating that the peptide NYEPPTVVPGGDL can reduce the levels of inflammatory factors and oxidative stress in the brain of rats with ischemic stroke and enhance the antioxidant capacity.

[0060] Example 2: Peptide NYEPPTVVPGGDL for the treatment of brain endothelial cell damage caused by glucose deprivation / reoxygenation / re-glucose (OGD / R)

[0061] Step 1: Experimental grouping (human brain microvascular endothelial cells hCMEC / D3, purchased from ATCC cell bank, n = 6)

[0062] i) Control group: 8*10^3 / well were spotted on a 96-well plate. After 12 hours of adhesion, the medium was changed to serum-free medium and cultured under normal conditions for 24 hours. Then, the medium was changed to fresh serum-free medium and cultured under normal conditions for another 2 hours.

[0063] ii) Modeling group: 96-well plates were spotted with 8*10^3 / well. After 12 hours of adhesion, the medium was changed to sugar-free medium and cultured under OGD / R for 24 hours, and then the medium was changed to serum-free medium and cultured under normal conditions for another 2 hours.

[0064] iii) Peptide 5 μg / mL group: 8*10^3 / well were spotted on a 96-well plate. After 12 hours of adhesion, the medium was changed to sugar-free medium and cultured under OGD / R for 24 hours. Then, the solution of polypeptide (5 μg / mL) prepared in serum-free medium was changed to continue culture under normal conditions for 2 hours.

[0065] iv) Peptide 1 μg / mL group: 8*10^3 / well were spotted on a 96-well plate. After 12 hours of adhesion, the medium was changed to sugar-free medium and cultured under OGD / R for 24 hours. Then, the solution of polypeptide (1 μg / mL) prepared in serum-free medium was changed to continue culture under normal conditions for 2 hours.

[0066] v) Peptide 0.2 μg / mL group: 8*10^3 / well were spotted on a 96-well plate. After 12 hours of adhesion, the medium was changed to sugar-free medium and cultured under OGD / R for 24 hours. Then, the peptide (0.2 μg / mL) solution prepared in serum-free medium was changed to continue culture under normal conditions for 2 hours.

[0067] Step 2: Establishment of a model of brain endothelial cell injury induced by glucose deprivation / reoxygenation (OGD / R)

[0068] i) Add 1.5 mL of sterile 0.25% trypsin to hCMEC / D3 cells in good condition, place them at 37°C for 1 min, add 1 mL of culture medium containing 10% fetal bovine serum to terminate digestion, transfer to a 15 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the culture medium, add 1 mL of fresh culture medium and gently resuspend. Spot 96-well plates at 8*10^3 / well, wait for them to adhere to the wall for 12 hours, change to sugar-free culture medium and culture for 24 hours at 37°C, 5% CO2, 1.5% O2, and then change to serum-free culture medium and continue to culture for 2 hours under normal conditions.

[0069] Step 3: Study on the pharmacological activity of peptide NYEPPTVVPGGDL on brain endothelial cell injury induced by OGD / R

[0070] i) Cell survival rate experiment: hCMEC / D3 cells were spotted on 96-well plates at 8*10^3 / well. After 12 hours of attachment, the cells were cultured in sugar-free medium under OGD / R for 24 hours, and each group was treated with the corresponding treatment for another 2 hours. 110 μl of diluted CCK-8 solution (serum-free medium: CCK-8 solution = 10:1) was added, and the cells were incubated in a 37°C incubator for 1 hour, and the absorbance at 450 nm was measured.

[0071] The experimental results showed that under OGD / R conditions, the survival rate of hCMEC / D3 cells was significantly reduced. After the peptide was administered, the cell damage caused by OGD / R was significantly improved and the survival rate of hCMEC / D3 was increased ( Figure 4 .A). This indicates that the peptide NYEPPTVVPGGDL can improve the hCMEC / D3 cell damage caused by OGD / R and significantly increase the cell survival rate.

[0072] ii) Cell scratch assay: hCMEC / D3 cells were seeded in 6-well plates. When the cell confluence reached more than 90%, a straight line was drawn at the bottom of each well using a yellow gun tip along a ruler. 37°C PBS was added along the wall to gently wash twice to remove cell debris. Bright field images of cell scratches were taken at 0 h. Sugar-free medium was changed to culture under OGD / R for 24 h. Each group was given the corresponding treatment for another 2 h. 37°C PBS was added along the wall to gently wash twice to remove cell debris. Bright field images of cell scratches were taken at 24 h. The cell migration rate at 24 h was calculated using imageJ.

[0073] The experimental results showed that under OGD / R conditions, the cell migration rate of hCMEC / D3 decreased significantly, and after the peptide was administered, the cell migration rate was significantly increased ( Figure 4 .B, Figure 4 .C), indicating that the peptide NYEPPTVVPGGDL can improve the decreased cell migration rate caused by OGD / R and enhance the cell migration and repair ability.

[0074] iii) Cellular reactive oxygen species (ROS) determination: hCMEC / D3 cells were spotted on 96-well plates at 8*10^3 / well. After 12 hours of attachment, the cells were cultured in sugar-free medium for 24 hours under OGD / R, and each group was treated accordingly for another 2 hours. DCFH-DA fluorescent probe diluted in serum-free medium at 1:1000 was added to the cells and incubated in the dark at 37°C for 30 minutes for probe loading. The cells were then washed three times with serum-free medium to fully remove DCFH-DA that did not enter the cells. Green fluorescence images were taken with a live cell imager, and the readings were detected at an excitation wavelength of 488nm and an emission wavelength of 525nm.

[0075] The experimental results showed that under OGD / R conditions, the ROS level of hCMEC / D3 cells increased significantly, and after the peptide was administered, the cellular ROS level was significantly reduced ( Figure 4 .D, Figure 4 .E), indicating that the peptide NYEPPTVVPGGDL can improve the increased oxidative stress level of hCMEC / D3 cells caused by OGD / R.

[0076] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. Use of a polypeptide and related materials thereof in the preparation of a drug for treating ischemic stroke; the related materials are a polynucleotide capable of encoding the polypeptide or a vector containing the polynucleotide; The polypeptide is selected from the following A) or B): A) The amino acid sequence is shown in SEQ ID NO.1; B) a product obtained by conventionally modifying the amino acid side chain groups of the polypeptide described in A), the amino terminus or the carboxyl terminus of the polypeptide described in A), or a product obtained by attaching a tag for polypeptide or protein detection or purification to the polypeptide described in A); The conventional modifications are amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation or biotinylation.

2. The use according to claim 1, characterized in that: Use of the polypeptide in preparing a drug for treating MCAO / R-induced ischemic stroke in rats and / or OGD / R-induced ischemic stroke.

3. The use according to claim 1, characterized in that: The medicament further comprises a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that: The pharmaceutically acceptable carrier is a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, an absorption carrier, a surfactant or a lubricant.

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