Polypeptide and application thereof

Abstract

Relates to a polypeptide and application thereof in treatment of nerve injury related diseases such as cerebral apoplexy or application of the polypeptide in health care products.

Description

A polypeptide and its application Technical Field

[0001] The present invention relates to a polypeptide and its use in treating diseases related to nerve damage such as stroke or in health care products. Background Art

[0002] Neural damage repair and brain microangiogenesis are of great significance in the treatment of neurological diseases. With the aging population and environmental changes, the incidence of neurological diseases such as stroke, Alzheimer's disease, and Parkinson's disease is increasing year by year, leading to nerve cell damage and death, seriously affecting patients' cognitive and motor functions. Because the regenerative capacity of neurons in the adult brain is limited, repairing damaged neurons and reestablishing neural connections are key to improving these diseases.

[0003] The role of endogenous neurogenesis in the repair of nerve damage cannot be ignored. Studies have shown that after ischemic stroke, neural stem cells can migrate to the damaged area and differentiate into new neurons. This process is crucial for restoring brain function. In addition, angiogenesis and neurogenesis are mutually regulated, sharing some molecular mechanisms to promote the survival and functional recovery of nerve cells. Angiogenesis not only provides essential nutrients and oxygen to nerve cells, but also improves the microenvironment, reduces the formation of collagen fibers, and provides pathways for new axons, thereby promoting nerve regeneration.

[0004] Improved vascularization can reduce stroke recurrence and improve patients' quality of life. Therefore, therapeutic strategies for neurological injury should prioritize the integration of neurological repair and cerebral microangiogenesis. Promoting endogenous neurogenesis and angiogenesis offers promising prospects for the development of novel therapeutic agents to improve cognitive and motor function in patients with neurological diseases.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a polypeptide that can be used for nerve damage repair and brain microangiogenesis, thereby treating degenerative neurological diseases such as stroke.

[0007] In a first aspect, the present invention provides a polypeptide or a physiologically compatible salt thereof, wherein the amino acid sequence of the polypeptide is selected from the group consisting of:

[0008] H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 1);

[0009] HD-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 2);

[0010] H-Ala-D-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.3);

[0011] H-Ala-Leu-D-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.4);

[0012] H-Ala-Leu-Pro-D-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.5);

[0013] H-Ala-Leu-Pro-Ala-D-Pro-Gly-Thr-Leu-OH(SEQ ID No.6);

[0014] H-Ala-Leu-Pro-Ala-Pro-Gly-D-Thr-Leu-OH(SEQ ID No.7);

[0015] H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-D-Leu-OH(SEQ ID No.8);

[0016] Ac-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.9);

[0017] H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-NH2(SEQ ID No.10);

[0018] Ac-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-NH2(SEQ ID No.11);

[0019] H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Ala-OH(SEQ ID No.12);

[0020] H-Ala-Leu-Ala-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.13);

[0021] H-Ala-Leu-Gly-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.14);

[0022] H-Ala-Leu-Pro-Ala-Gly-Gly-Thr-Leu-OH(SEQ ID No.15);

[0023] H-Ala-Ala-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.16);

[0024] H-Ala-Leu-Pro-Ala-Ala-Gly-Thr-Leu-OH(SEQ ID No.17);

[0025] H-Ala-Leu-Pro-Ala-Pro-Gly-Ala-Leu-OH(SEQ ID No.18);

[0026] H-Leu-Ala-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.19);

[0027] H-Leu-Ala-Pro-Ala-Pro-Gly-Thr-Ile-OH(SEQ ID No.20);

[0028] H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Ile-OH(SEQ ID No.21);

[0029] H-Gln-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.22);

[0030] H-Pro-Ile-Ala-Ser-Gln-His-Leu-Asn-Leu-Ala-Pro-Ala-Pro-Gly-Thr-Ile-OH(SEQ ID No.23);

[0031] H-Ala-Lys-Asn-Gln-Leu-Pro-Ala-Pro-Gly-Thr-Leu-Gln-His-Phe-Cys-OH(SEQ ID No.24);

[0032] H-Gln-Leu-Pro-Ala-Pro-Gly-Thr-Leu-Gln-His-Phe-OH(SEQ ID No.25);

[0033] H-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.26);

[0034] H-Pro-Ala-Pro-Gly-Thr-OH(SEQ ID No.27);

[0035] H-Pro-Gly-Thr-Leu-OH(SEQ ID No.28);

[0036] H-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.29);

[0037] H-Ala-Leu-Pro-Ala-Pro-OH(SEQ ID No.30);

[0038] H-Leu-Pro-Ala-Pro-OH(SEQ ID No.31);

[0039] H-Leu-Pro-Ala-Pro-Gly-Thr-OH(SEQ ID No.32);

[0040] H-Leu-Pro-Ala-Pro-Gly-OH(SEQ ID No.33);

[0041] H-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.34);

[0042] H-Ala-Leu-Pro-Ala-OH(SEQ ID No.35);

[0043] H-Pro-Ala-Pro-Gly-OH(SEQ ID No.36);

[0044] H-Ala-Pro-Gly-Thr-OH(SEQ ID No.37);

[0045] H-Gly-Thr-Leu-OH(SEQ ID No.38);

[0046] H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-OH(SEQ ID No.39);

[0047] H-Ala-Leu-Pro-Ala-Pro-Gly-Oh(SEQ ID No.40);

[0048] Ac-Ala-Leu-Pro-Ala-D-Pro-Gly-Thr-Leu-NH2(SEQ ID No.41);

[0049] Ac-Ala-Leu-Pro-D-Ala-Pro-Gly-Thr-Leu-NH2(SEQ ID No.42)。

[0050] In some embodiments, the polypeptide is selected from: Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 10, Compound 11, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 30, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36, Compound 37, Compound 38, Compound 39, Compound 40, Compound 41, Compound 42, or a physiologically compatible salt thereof.

[0051] In a second aspect, the present invention provides the use of the polypeptide or a physiologically compatible salt thereof in the preparation of a medicament for treating or preventing a disease associated with nerve damage; or a method for treating or preventing a disease associated with nerve damage in a subject, the method comprising administering to the subject a therapeutically effective amount of the polypeptide or a physiologically compatible salt thereof; or the polypeptide or a physiologically compatible salt thereof, which is used to treat or prevent a disease associated with nerve damage.

[0052] In some embodiments, the disease associated with nerve damage involves abnormal brain microangiogenesis.

[0053] Furthermore, diseases related to nerve damage include stroke, diabetic neuropathy, brain injury, neurodegenerative diseases, vascular dementia, cerebrovascular disease, and epilepsy. Furthermore, neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and HIV-1-related dementia. Furthermore, brain injury includes chronic traumatic encephalopathy.

[0054] Furthermore, stroke includes ischemic stroke, hemorrhagic stroke and stroke sequelae.

[0055] Furthermore, brain injury includes cerebral ischemia-reperfusion brain injury.

[0056] In some embodiments, the polypeptides of the present invention improve neurological dysfunction. Further, the polypeptides of the present invention improve neurological dysfunction in stroke.

[0057] In some embodiments, the polypeptides of the present invention promote the number of hippocampal neurons, glial cells and new blood vessels in the late stage of stroke.

[0058] In some embodiments, the polypeptides of the present invention promote stroke neurogenesis and angiogenesis.

[0059] Furthermore, stroke sequelae include limb dysfunction, mental and intellectual impairment, speech dysfunction, and swallowing dysfunction. Limb dysfunction includes paralysis, which severely impacts patients' ability to live daily lives; speech dysfunction includes aphasia, which impacts their ability to communicate; and mental and intellectual impairment includes depression, which places an additional burden on patients' mental health. The polypeptides of the present invention can effectively improve stroke sequelae.

[0060] In a third aspect, the present invention provides a composition comprising a polypeptide of the present invention or a physiologically compatible salt thereof and a physiologically compatible excipient. In some embodiments, the composition of the present invention is a pharmaceutical composition, which may be in the form of an oral solution, tablet, granules, capsule, pill, injection, transdermal absorption preparation, external lotion, or in vivo implant preparation. In some embodiments, the composition of the present invention is a health care product composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 shows a partial MS-MS spectrum of the polypeptide of the present invention;

[0062] FIG2 shows the effects of the polypeptide of the present invention at different solubilities on promoting the proliferation and differentiation of neural stem cells;

[0063] FIG3 shows the proliferation-promoting effect of the polypeptide of the present invention on neural stem cell line C17.2 cells;

[0064] FIG4 shows the differentiation-promoting effect of the polypeptide of the present invention on primary neural stem cells NSCs;

[0065] FIG5 shows the angiogenesis-promoting effect of the polypeptide of the present invention;

[0066] FIG6 is a TTC staining result diagram (left) and a statistical diagram of cerebral infarction volume (right) showing the effect of the polypeptide of the present invention on cerebral infarction volume in stroke rats;

[0067] FIG7 is a diagram showing the HE staining results of the effect of the polypeptide of the present invention on pathological damage of hippocampal and cortical cells in stroke rats;

[0068] FIG8 is a statistical diagram of the mNSS scores of the effects of the polypeptide of the present invention on the neurological function of stroke rats;

[0069] FIG9 is a diagram showing the statistical results of the fatigue meter rotarod test on the effects of the polypeptide of the present invention on the balance ability and endurance of stroke rats;

[0070] FIG10 is an immunofluorescence staining result showing the effect of the polypeptide of the present invention on cell proliferation in the hippocampus of stroke rats;

[0071] FIG11 is an immunofluorescence staining result showing the effect of the polypeptide of the present invention on the differentiation of neural stem cells in the hippocampus of stroke rats (DCX and BrdU immunofluorescence double-labeling staining (left), GFAP and BrdU immunofluorescence double-labeling staining (right));

[0072] FIG12 is an immunofluorescence staining diagram showing the effect of the polypeptide of the present invention on the expression of vascular marker protein CD31 in the hippocampus and cortex of stroke rats;

[0073] FIG13 shows that the polypeptide of the present invention improves pathological tissue damage and alleviates cortical tissue atrophy in rats;

[0074] FIG14 shows the effects of the polypeptides of the present invention on the expression of PSD95 and SYP in the hippocampus and cortex of rats. DETAILED DESCRIPTION

[0075] The following is an illustrative description of the present invention with reference to specific embodiments, but does not limit the scope of protection of the present invention.

[0076] As used in this application, including the appended claims, singular forms such as "a," "an," and "the" include their plural referents unless the context clearly dictates otherwise.

[0077] Unless the context clearly indicates otherwise, the term "or" is used to mean, and is used interchangeably with, the term "and / or."

[0078] The term "effective amount" or "therapeutically effective amount" refers to an amount of an active ingredient (such as a compound) that is sufficient to affect such treatment of the disease, disorder or symptom when administered to a subject to treat a disease or at least one clinical symptom of a disease or disorder. A "therapeutically effective amount" can vary with the compound, the disease, disorder and / or the symptoms of the disease or disorder, the severity of the disease, disorder and / or the symptoms of the disease or disorder, the age of the subject to be treated and / or the weight of the subject to be treated. In any given case, the appropriate amount will be clear to those skilled in the art or can be determined by routine experimentation. In some embodiments, a "therapeutically effective amount" is an amount that is effective for the disease or disorder of a subject of "treating" (as defined above) a compound disclosed herein and / or at least one of its stereoisomers and / or at least one of its pharmaceutically acceptable salts. In the case of combination therapy, a "therapeutically effective amount" refers to the total amount of the combination subject used to effectively treat a disease, disorder or condition.

[0079] The term "physiologically compatible salt" refers to a salt form that is physiologically compatible (i.e., pharmacologically acceptable) and substantially non-toxic to the individual to whom the compound of the invention is administered. Physiologically compatible salts of the compounds of the invention include conventional and stoichiometric acid addition salts or base addition salts formed from suitable, non-toxic organic or inorganic acids or inorganic bases. In some embodiments, physiologically compatible salts include hydrochlorides, trifluoromethanesulfonates, and the like.

[0080] The term "physiologically compatible excipient" refers to any component that is compatible with the in vivo environment and does not cause adverse reactions (e.g., a vehicle that can suspend, form a complex, or dissolve the polypeptide of the present invention). Excipients include, but are not limited to, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, pigments, softeners, emulsifiers, fillers, film formers or coatings, flavorings, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water.

[0081] The term "pharmaceutical composition" refers to a formulation comprising a polypeptide of the present invention and a physiologically compatible excipient, the purpose of which is to promote the polypeptide to exert its therapeutic or preventive effect in vivo to alleviate the corresponding symptoms. The composition of the present invention can be in any form suitable for any route of administration or conventional use.

[0082] The term "nutraceutical composition" refers to any composition comprising, in addition to physiologically compatible excipients, food ingredients such as macronutrients, micronutrients, plants or plant extracts or substances having a nutritional or physiological effect, with the purpose of supplementing the human diet in order to improve the latter's nutritional status and thereby promote good health.

[0083] The term "subject" includes mammals, such as humans, rats, mice, dogs, monkeys, etc., typically humans.

[0084] The term "neurovascular disease" includes a variety of conditions, such as stroke, diabetic neuropathy, brain injury, neurodegenerative diseases, and epilepsy, among which stroke is the most common and serious one.

[0085] Stroke is an acute neurological dysfunction caused by cerebrovascular problems and is generally divided into ischemic stroke and hemorrhagic stroke. Ischemic stroke accounts for 87% of all cases and is mainly caused by blood vessel blockage or stenosis, resulting in reduced blood flow; while hemorrhagic stroke is caused by bleeding due to blood vessel rupture[3].

[0086] Diabetic neuropathy is a common complication in patients with diabetes, characterized by nerve damage leading to sensory loss, pain, and motor dysfunction.

[0087] Brain damage can be traumatic (such as traumatic brain injury) or acquired (such as brain damage caused by stroke, tumors, etc.). Brain damage can cause loss of consciousness, changes in thinking, and movement problems. The specific symptoms depend on the location and extent of the damage.

[0088] Neurodegenerative diseases are a class of disorders characterized by the gradual loss of function and death of nerve cells. These diseases typically affect movement, cognition, and autonomic function, leading to a significant decline in patients' quality of life. Common neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, and HIV-1-associated dementia.

[0089] Epilepsy is a common neurological disorder characterized by abnormal discharges of neurons in the brain, leading to recurring seizures. Seizures can occur in a variety of types and can affect a patient's consciousness, movement, sensation, and behavior.

[0090] The amino acid abbreviations used in this application are as follows

[0091] Table 1 English names or abbreviations and Chinese names of reagents and solvents used in writing

[0092] English and its abbreviated Chinese name

[0093] Ac acetyl

[0094] 2-Cl-CTC 2-Chlorotrityl chloride resin

[0095] DMF N,,N-dimethylformamide

[0096] Rink Amide Resin

[0097] Fmoc 9-fluorenylmethoxycarbonyl

[0098] DCM methyl chloride

[0099] PIP piperidine

[0100] HOBt 1-Hydroxybenzotriazole

[0101] DIEA N,N-Diisopropylethylamine

[0102] MeOH methanol

[0103] TFA trifluoroacetic acid

[0104] TIS triisopropylsilane

[0105] DIC N,N′-diisopropylcarbodiimide

[0106] Example 1: Chemical synthesis of polypeptides

[0107] The polypeptides of the present invention are synthesized by conventional Fmoc solid phase synthesis methods. The polypeptides with carboxyl groups at the carbon end are synthesized using 2-chlorotrityl chloride resin, while the polypeptides with amide groups at the carbon end are synthesized using Rink amine resin.

[0108] The process is generally divided into three steps: 1) preparation of fully protected peptide resin, i.e., linking the amino acids in the peptide sequence, including D-amino acids, in sequence (for peptides with acetylated N-terminus, acetylation is performed after linking the peptide sequence and removing Fmoc protection); 2) cleavage, i.e., cleaving the peptide from the resin and removing the amino acid side chain protecting groups; 3) purification by reversed-phase preparative liquid chromatography (HPLC) and lyophilization.

[0109] Step 1. Preparation of fully protected peptide resin

[0110] 1.1 Preparation of peptide resin with carboxyl-terminated peptides

[0111] The steps for linking peptide sequences in carboxyl peptide resin are shown in Table 2:

[0112] Table 2 Links to peptide sequences

[0113] 1.2 Preparation of peptide resin with amide-terminated peptides

[0114] The steps for linking peptide sequences in amide peptide resin are shown in Table 3:

[0115] Table 3 Links to peptide sequences

[0116] Acetylation: Flush the peptide resin from the synthesis tube with DMF, filter under reduced pressure, and collect the resin. Add 40 mL of DMF:acetic anhydride:DIEA = 85:10:5 (v / v / v) and allow to react under nitrogen bubbling for 30 min. After the reaction is complete, wash the peptide resin three times with 100 mL of DMF, each time for 1-2 min. Then, wash the peptide resin five times with 100 mL of DCM, each time for 1-2 min. Air dry the peptide resin.

[0117] Step 2: Cutting

[0118] To the naturally dried peptide resin, add 50-100 mL of TFA:TIS:H2O = 94:3:3 (v / v / v), bubbling with nitrogen, and react for 1-2 hours. Collect the liquid by filtration; wash the resin once with 10-20 mL of TFA. Combine the filtrates and concentrate under reduced pressure at 40°C until the volume is reduced to 1 / 3 of its original volume. Add 200 mL of pre-frozen isopropyl ether, stir, and precipitate. Filter and wash the solid three times with 30 mL of isopropyl ether each time. Place the white solid in a vacuum oven and dry it at -0.08 MPa at 40°C to obtain the crude product, which is then analyzed by LC-MS.

[0119] Step 3, reverse phase preparative liquid phase purification and freeze drying

[0120] Reverse-phase preparative liquid chromatography (HPLC) purification is divided into two stages: the first stage is purification and separation for the purpose of removing impurities, and the second stage is purification for the purpose of salt conversion (i.e., replacement with acetate). Generally speaking, when the purity of the crude product is greater than 90%, direct salt conversion can be used for one-step purification.

[0121] (1) Peptide HPLC purification and preparation

[0122] A. Chromatographic parameters

[0123] Chromatographic column: YMC-Actus Triart C18, 30*250mm

[0124] Eluent A: 10 mM ammonium bicarbonate aqueous solution

[0125] Eluent B: acetonitrile

[0126] Flow rate: 25ml / min

[0127] UV detection wavelength: 220nm

[0128] B. Operation steps

[0129] a) Dissolve the crude peptide in water and / or acetonitrile and filter through a 0.45 μm filter membrane

[0130] b) Injection

[0131] c) Gradient elution with eluents A and B

[0132] d) Collect the target peptide eluate

[0133] e) Rotary evaporation concentration

[0134] (2) Peptide HPLC salt change (acetate)

[0135] A. Chromatographic parameters

[0136] Chromatographic column: YMC-Actus Triart C18, 30*250mm

[0137] Eluent A: 0.1% acetic acid

[0138] Eluent B: acetonitrile

[0139] Flow rate: 25ml / min

[0140] UV detection wavelength: 220nm

[0141] B. Operation steps

[0142] a) 95% A + 5% B balanced column

[0143] b) Injection

[0144] c) Gradient elution with eluents A and B

[0145] d) Collect the target peptide eluate

[0146] e) Rotary evaporation concentration

[0147] The concentrate was lyophilized to obtain the pure target peptide. The sample was weighed and tested for purity, moisture, and acetic acid content. The structure was then confirmed by high-resolution mass spectrometry (HRMS) and secondary mass spectrometry (MS-MS). The results are shown in Table 4.

[0148] Table 4 Structural sequence and precise molecular weight of the polypeptide synthesized in the present invention

[0149] The deviation of the accurate molecular weight of all the polypeptides of the present invention measured by high-resolution mass spectrometry (HRMS) in the above table is ≤-2.8 ppm, which is consistent with the theoretical molecular weight of the polypeptides of the present invention with the corresponding structure; the fragment ions of the polypeptide samples analyzed by secondary mass spectrometry (MS-MS) have a high degree of match with the theory (see Figure 1), which can confirm that the polypeptide sequence is consistent with the corresponding polypeptide structure of the present invention.

[0150] Example 2: Study on the proliferation and differentiation of neural stem cells at different solubilities of the polypeptide of the present invention

[0151] This part of the experiment requires raising male and female rats in advance, then extracting hippocampal neural stem cells from SD rats within 24 hours of birth, culturing them to P3 for identification, and then using them for experiments.

[0152] Isolation and Culture of Primary Neural Stem Cells (NSCs): Hippocampal NSCs were isolated from 24-hour-old SD rat pups using the following procedure: Pups were first disinfected by soaking in 75% alcohol and then transferred to a biosafety cabinet for sterile manipulation. The entire brain was removed from the pups and immediately placed in ice-cold D-Hanks solution to remove non-neural tissue. Under a dissecting microscope, the meninges were carefully removed, and the hippocampal structures on both sides were isolated. The tissue was mechanically fragmented with a scalpel and pipetted. After pipetting, the tissue sample was transferred to a 15 mL centrifuge tube and centrifuged at 800 rpm for 5 minutes. After centrifugation, the supernatant was aspirated, and 100 μL of Accutas digestive enzyme was added per pupal brain. The tissue was incubated in a 37°C incubator for 5 minutes to facilitate tissue digestion, followed by the addition of complete DMEM / F12 medium to terminate digestion. Filter the cell suspension through a 200-mesh sieve and centrifuge again at 800 rpm for 5 minutes. Discard the supernatant, add complete DMEM / F12 medium, and gently pipette to mix. Resuspend the cells. Inoculate into T25 culture flasks. Incubate the flasks in a 37°C incubator with 5% CO2. Avoid moving the flasks for the first 3 days. Thereafter, change half the medium every 2 to 3 days, and subculture every 6 to 7 days.

[0153] 2.1 Primary neural stem cells (NSCs) were used to evaluate the proliferation-promoting activity of the polypeptide of the present invention.

[0154] Medium-sized neurospheres formed by culture at passages P3 to P4 were taken and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded, and a small amount of complete culture medium was added and pipetted to a single-cell suspension. The cells were resuspended and counted. The cells were seeded at 1.0x105 cells / mL in a 24-well plate coated with 25 μg / mL PLL. The cells were treated with polypeptide compound 1 at different concentrations (0.02, 0.1, and 0.5 mg / mL) and cultured in an incubator for 48 hours. BrdU was incorporated at 44 hours of treatment to a final concentration of 10 μM. Immunofluorescence staining was performed 4 hours later, and the cells were observed and photographed under a fluorescence microscope. The BrdU-positive cell rate was statistically analyzed.

[0155] 2.2 Evaluation of the differentiation-promoting activity of the polypeptide of the present invention using primary neural stem cells NSCs

[0156] Medium-sized neurospheres formed at passages P3-P4 were collected and centrifuged at 800 rpm for 5 minutes. The supernatant was discarded, and a small amount of differentiation medium (DMEM / F12 differentiation medium supplemented with 2% fetal bovine serum) was added and pipetted to a single-cell suspension. The cells were resuspended, counted, and plated at 2.0 × 105 cells / mL in 24-well plates coated with 25 μg / mL PLL. After overnight adherence, the cells were treated with different concentrations of polypeptide compound 1 (0.02, 0.1, and 0.5 mg / mL). Differentiation was continued for 4 days. After differentiation, fluorescence staining was performed. Primary antibodies were MAP-2 (Rabbit, 1:200) and GFAP (Mouse, 1:200); secondary antibodies were Cy3-conjugated goat anti-rabbit (1:200) and FITC-conjugated goat anti-mouse (1:200). The cells were then observed under a fluorescence microscope and photographed. The mean fluorescence intensity of MAP-2 and GFAP was statistically analyzed.

[0157] Test results: The test results are shown in Figure 2. Compared with the control group, all tested concentrations of polypeptide compound 1 can promote the proliferation and differentiation of primary neural stem cells NSCs, specifically, the BrdU-positive cell rate increased significantly, and the average fluorescence intensity of MAP-2 and the average fluorescence intensity of GFAP increased significantly. Overall, 0.1 mg / mL (100 μg / mL) is better.

[0158] Example 3: Study on the Effect of the Peptide of the Present Invention on Promoting the Proliferation and Differentiation of Neural Stem Cells

[0159] 1.1 The proliferation-promoting activity of the polypeptide of the present invention was evaluated using the neural stem cell line C17.2.

[0160] (1) C17.2 cells were seeded in a 96-well plate at a concentration of 1500 cells / 100 μL / well and cultured overnight to adhere before drug treatment.

[0161] (2) Drug treatment was given, with the final concentration of the polypeptide of the present invention being 100 μg / mL, and butylphthalide (NBP, 5 μM) was set as a positive control, and the cells were placed in an incubator and cultured for 48 h;

[0162] (3) BrdU was incorporated at 44 h after treatment to a final concentration of 10 μM. Immunofluorescence staining was performed 4 h later, and the BrdU-positive cell rate was statistically analyzed using a high-content cell imaging analysis system.

[0163] 1.2 Evaluation of the differentiation-promoting activity of the polypeptide of the present invention using primary neural stem cells NSCs

[0164] This part of the experiment requires raising male and female rats in advance, then extracting hippocampal neural stem cells from SD rats within 24 hours of birth, culturing them to P3 for identification, and then using them for experiments.

[0165] (1) Extraction and culture of primary neural stem cells (NSCs)

[0166] (2) NSCs cells that were passaged to P3 and had good vitality were seeded into 48-well plates with differentiation medium (2% FBS + DMEM / F12 1:1 medium) at a cell concentration of 5 × 104 cells / mL per well. After overnight culture, the cells were allowed to adhere to the wall and then treated with drugs.

[0167] (3) Drug treatment was given, with the final concentration of the polypeptide of the present invention being 100 μg / mL, and nerve growth factor (NGF, 50 ng / mL) was set as a positive control, and the cells were placed in an incubator and cultured for 4 days;

[0168] (4) After 4 days of treatment, immunofluorescence staining (primary antibody βIII-tubulin) was performed, and the mean fluorescence intensity analysis of βIII-tubulin and Neurite Outgrowth Assay were performed using a high-content cell imaging analysis system.

[0169] Experimental result 1: The polypeptide of the present invention promotes the proliferation of neural stem cell line C17.2 cells.

[0170] The effects of the polypeptide of the present invention on the proliferation of C17.2 cells were analyzed using a high-content cell imaging analysis system. The results are shown in Table 5:

[0171] Table 5 The results of the polypeptides of the present invention promoting the proliferation of neural stem cell line C17.2 cells

[0172] Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001; all three batches of experiments of the positive drug NBP showed significant differences; " / " indicates no significant difference, and some have cytotoxicity.

[0173] Compared with the blank control group, polypeptide compounds 5-8, compound 10, compound 11, compounds 15-20, compound 28, compound 32, compound 36, compounds 37 and 40 of the present invention can significantly promote the proliferation of C17.2 cells, as shown by a significant increase in the number of BrdU-positive cells; in addition, compound 3 also has a trend of promoting the increase in the number of BrdU-positive cells (Figure 3A-B).

[0174] Experimental result 2: The polypeptide of the present invention promotes differentiation of primary neural stem cells NSCs.

[0175] Images were collected and statistically analyzed using a high-content cell imaging analysis system. The results of the polypeptide of the present invention promoting differentiation of primary neural stem cells (NSCs) are shown in Table 6:

[0176] Table 6 The results of the polypeptides of the present invention promoting differentiation of primary neural stem cells NSCs

[0177] Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001; all three batches of experiments with the positive drug NGF showed significant differences; " / " indicates no significant difference, and a small number of them showed cytotoxicity.

[0178] Compared with the blank control group, the polypeptide compounds 1, 3, 16, 24, 25, 30, 34, 35, 40 and 42 of the present invention can promote the differentiation of NSCs into neuronal cells, as shown by a significant increase in the mean fluorescence intensity of the neuronal marker protein βIII-tubulin, and the polypeptide compounds 10, 17, 20, 23, 26, 29, 31 and 38 of the present invention also have a trend of increasing the mean fluorescence intensity of βIII-tubulin (Figure 4A); In addition, Neurite Outgrowth The assay results also confirmed that the polypeptide compounds 1, 2-5, 11, 22, 27, 34 and 35 of the present invention all showed the effect of promoting axon growth, specifically, the increase in total neurite length, the increase in the longest neurite length, the increase in the number of neurite endings, and the increase in neurite starting points (branches). The polypeptide compounds 6, 7, 10, 15, 20, 23, 28, 30, 36 and 41 of the present invention also showed a trend of promoting axon growth in the four evaluation indicators (Figure 4B). Combining the results of the two analysis methods, it can be concluded that the polypeptide compounds 1, 3, 34 and 35 of the present invention can promote the differentiation of NSCs into neurons and the effect is stable.

[0179] Example 4: Study on the Angiogenesis Promotion of the Polypeptide of the Present Invention

[0180] First, differential centrifugation was used to obtain mouse brain microvessel sediments, which were digested with collagenase II to obtain brain microvascular endothelial cells (BMECs) and brain microvascular smooth muscle cells (BMVSMCs), which were cultured in a cell culture incubator at 37°C and 5% CO2. The 3rd to 6th generation cells with good growth status and logarithmic growth were selected for in vitro angiogenesis experiments.

[0181] The night before the experiment, transfer the high-concentration matrix gel from a -20°C or -80°C refrigerator to a 4°C refrigerator and allow the matrix gel to slowly melt overnight in a 4°C refrigerator. Sterilize the pipette tips needed for the formal experiment in advance and place them in a 4°C refrigerator for pre-cooling in preparation for subsequent use.

[0182] On the day of the experiment, Matrigel was spread in a 96-well plate 1 hour in advance and placed in a 37°C cell culture incubator to solidify. The cells were then washed twice with 2 mL of PBS buffer and digested with 0.25% trypsin to obtain ECs and VSMCs pellets, and the cells were counted. Then, 5 × 10 cells were added to each well. 3 BMVSMCs and 1×10 3 A uniform cell suspension of BMECs was seeded into a 96-well plate coated with Matrigel and cultured in a cell culture incubator at 37°C and 5% CO2 for 3 days.

[0183] During the culture period, the tube formation was observed every day. The medium was changed on the 4th day, and then 0.1 mg / L of the polypeptide of the present invention or 10 / 15 / 20 ng / L VEGFA was added for 48 hours.

[0184] On the fifth day, Host 33342 and calcein AM were used to label cell nuclei and live cells, respectively. High-content brightfield and fluorescence images were then captured using a high-content cell analysis system. Angiogenesis Analyzer (ImageJ) plug-in was used to analyze angiogenesis in each treatment group, and statistical analysis was performed using GraphPad Prism 10.

[0185] Test results: The results of the angiogenesis-promoting test of the polypeptide of the present invention are shown in Table 7:

[0186] Table 7 Results of the angiogenesis-promoting experiment with the polypeptide of the present invention

[0187] Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001; all three batches of experiments of the positive drug VEGFA showed significant differences; " / " indicates no significant difference, and some have cytotoxicity.

[0188] Compared with the normal group, the polypeptide compounds 1, 2, 6, 8, 11, 18, 19, 26, 33, 34, 38, 39 and 41 of the present invention showed a pro-angiogenic effect; the polypeptide compounds 5, 14, 15 and 20 of the present invention had a promoting effect on the number of intersections but had no significant effect on branch length, while the polypeptide compounds 9, 22, 30-32, 36 and 42 of the present invention had a promoting effect on branch length but had no significant effect on the number of intersections, and the polypeptide compounds 3, 4, 7, 10, 17, 25, 35 and 40 of the present invention only had a promoting effect on cell proliferation. Overall, they did not have a significant pro-angiogenic effect (Figure 5).

[0189] Example 5: Effect of the polypeptide of the present invention on cerebral infarction volume in stroke rats

[0190] Model establishment: SD male rats, weighing 230-260 g.

[0191] A rat model of transient middle cerebral artery occlusion (tMCAO) was established using a suture method. Male Sprague-Dawley rats were anesthetized and fixed in the supine position on a surgical board. The neck was shaved with a razor and disinfected with iodine. The superficial fascia was incised slightly left of the midline of the neck. The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were isolated, taking care not to injure the vagus nerve. The CCA and ECA were ligated with 6-0 surgical sutures, and the ICA was temporarily clamped with a small artery clamp. A sterile needle was used to make an incision in the ECA. A 0.36 mm nylon suture was inserted into the ICA and slowly advanced, aiming to align it parallel to the internal carotid artery until slight resistance was felt. The silk slipknot and artery clamp ligated on the ICA were loosened, and a ligature was placed on the ECA incision to secure the suture and prevent blood flow from the internal carotid artery. The subcutaneous tissue and skin were sutured, and surgical blood was cleaned with sterile cotton balls and then wiped with iodine for disinfection. After 2 hours of occlusion, the nylon suture was slowly removed to complete the cerebral ischemia-reperfusion injury model. The animal's body temperature was maintained at 37±1°C during and after the operation until it regained its consciousness and resumed activity.

[0192] The criteria for judging the success of the model are: (1) the left limb is slow to retract painfully or does not retract painfully; (2) the left forelimb cannot be extended downward when hanging upside down by the tail; (3) the patient falls to the left or turns to the left in place when walking.

[0193] Grouping and Dosing: The polypeptide compound 1 of the present invention (10 mg / kg) was prepared by mixing the polypeptide with physiological saline, filtering through a 0.22 μm filter, and then setting aside. SD rats were randomly divided into the following groups: sham group (animals received a sham operation and an equal volume of physiological saline); vehicle group (animals received tMCAO and an equal volume of physiological saline); and drug group (animals received tMCAO and were injected with the polypeptide compound 1 of the present invention via the tail vein.

[0194] Experimental methods:

[0195] TTC (2,3,5-triphenyltetrazolium chloride) is the most commonly used ischemic infarction dye, which can react with dehydrogenase to appear red (normal tissue) and pale (ischemic tissue).

[0196] The volume of cerebral infarction was determined by TTC staining. Seven days after surgery, the rats were anesthetized and decapitated to remove the brain. Five 2-mm-thick coronal brain slices were cut equidistantly from the frontal pole and placed in 0.5% TTC staining solution. The slices were incubated at 37°C in the dark for 30 minutes, and the slices were turned over every 15 minutes. After the slices were evenly stained, they were transferred to 4% paraformaldehyde solution and fixed for at least 12 hours. They were then stored in a 4°C refrigerator. The white area of ​​the brain tissue indicated cortical infarction.

[0197] The infarct area was calculated as follows: ischemic area volume ratio = (sum of the white ischemic area of ​​each slice) / (sum of the brain area of ​​each slice) * 100%. The results are shown in FIG6 .

[0198] Experimental results: The results in FIG6 show that, compared with the model group, the cerebral infarction volume in the polypeptide-administered group of the present invention was significantly reduced.

[0199] Example 6: Effects of the polypeptide of the present invention on pathological damage in the hippocampus and cortex of stroke rats

[0200] The experimental animals, model establishment, grouping and drug administration were the same as those in Example 5.

[0201] Experimental methods:

[0202] Rats were deeply anesthetized and perfused with 4% paraformaldehyde transcardially to obtain the brain. The perfusion procedure was as follows: the rat was fixed in a supine position, the abdominal line was cut open to expose the heart, and then the perfusion needle was inserted into the right ventricle (approximately 3-5 mm) along the aortic orifice at the left apex. The right atrial appendage was pierced and perfused with heparinized phosphate-buffered saline (PBS) until the rat liver turned white and the outflowing fluid was bloodless. The solution was then replaced with 4% paraformaldehyde solution until the rat's body became rigid, and then the brain was removed by decapitation. The removed brain was paraffin-embedded and cut into 5-8 μm thick slices. After dewaxing, the slices were stained with eosin staining solution and then mounted with neutral resin. The mounted slices were observed under an upright microscope for the cells of the hippocampus on the injured side and images were collected.

[0203] Experimental results: Figure 7 (black arrows indicate damaged cells) shows that on the 14th day after modeling, more cells in the hippocampus and cortex of the model group showed damage compared with the drug-treated group: cell nuclear condensation, darker staining, damaged cell morphology, etc., indicating that the polypeptide of the present invention can reduce the damage and death of hippocampal and cortical cells in stroke rats.

[0204] Example 7: Effects of the polypeptide of the present invention on neuromotor function in stroke rats

[0205] The experimental animals, model establishment, grouping and drug administration were the same as those in Example 5.

[0206] Experimental method: After model establishment, the drug was injected into the tail vein, and neurological function scores (mNSS scores) were performed on the 1st, 7th, and 14th days of administration.

[0207] Experimental results: As shown in Figure 8, the results show that compared with the model control group, the mNSS scores of rats in the polypeptide group of the present invention were significantly reduced on days 7 and 14, indicating that the rats' neurological dysfunction, including sensation, reflexes, movement, and balance, were improved. As shown in Figure 9, the fatigue meter rotarod test showed that the time spent on the rotarod in the polypeptide group of the present invention was significantly increased compared with the model group, indicating that their balance ability and endurance were restored. The above results all demonstrate that the polypeptide of the present invention can significantly improve the late-stage neuromotor dysfunction in rats after cerebral ischemia reperfusion.

[0208] Example 8: Effect of the polypeptide of the present invention on cell proliferation in the hippocampus of rats with cerebral ischemia-reperfusion

[0209] The experimental animals, model establishment, grouping and drug administration were the same as those in Example 5.

[0210] Experimental methods:

[0211] 5-Brodeoxyuridine (BrdU) was administered twice daily. After treatment, the rat brain was harvested by cardiac perfusion. The brain tissue was fixed in 4% paraformaldehyde (PFA) and dehydrated with 20% and 30% sucrose solutions until it sank to the bottom. After dehydration, the tissue surface was carefully wiped clean of liquid, embedded, and frozen at a thickness of 10 μm. Frozen sections were then stained for BrdU immunofluorescence.

[0212] Experimental results: As shown in Figure 10 (arrows indicate newborn cells), the number of BrdU-positive cells in the SGZ of the polypeptide group of the present invention increased significantly compared with the model group, indicating that the polypeptide of the present invention can increase the number of newborn cells in the DG of rats after stroke.

[0213] Example 9: Effect of the polypeptide of the present invention on the differentiation of hippocampal neural stem cells in rats with cerebral ischemia-reperfusion

[0214] The experimental animals, model establishment, grouping and drug administration were the same as those in Example 5.

[0215] Experimental methods:

[0216] DCX (immature neuron marker protein) / BrdU, GFAP (astrocyte marker protein) / BrdU immunofluorescence double labeling.

[0217] Experimental results: The results are shown in Figure 11 (arrows indicate newly formed neurons and astrocytes). Compared with the model group, the number of DCX / BrdU and GFAP / BrdU double-positive cells in the SGZ area of ​​the polypeptide drug group was significant, indicating that the number of newly formed neurons and astrocytes increased, indicating that the polypeptide of the present invention can promote the differentiation of neural stem cells into neurons and astrocytes after stroke, and has the effect of promoting the differentiation of neural stem cells.

[0218] Example 10: Effect of the polypeptide of the present invention on the expression of the vascular marker protein CD31 in the hippocampus of rats with cerebral ischemia

[0219] The experimental animals, model establishment, grouping and drug administration were the same as those in Example 5.

[0220] Experimental methods:

[0221] Frozen sections of brain tissue from rats with cerebral ischemia-reperfusion were subjected to CD31 (vascular marker protein) / BrdU double immunofluorescence staining.

[0222] Experimental results: As shown in Figure 12 (arrows indicate newly formed blood vessels), compared with the model group, the number of CD31 / BrdU double-positive cells in the SGZ and cortex of the polypeptide group of the present invention increased significantly, indicating that the number of newly formed blood vessels increased significantly, indicating that the polypeptide of the present invention can promote angiogenesis in stroke rats.

[0223] Example 11: Effects of the polypeptide of the present invention on the expression of PSD95 and SYP in the hippocampus and cortex of rats with cerebral ischemia

[0224] The experimental animals, model establishment, grouping and drug administration were the same as those in Example 5.

[0225] Experimental methods:

[0226] The cortical index of rats in each group was calculated after 14 days of ischemia-reperfusion injury. tMCAO rats were treated with the polypeptide compound 1 of the present invention for 14 days, and the expression of PSD95 and SYP was detected by Western blot.

[0227] Experimental Results 1: The experimental results showed that 14 days of ischemia-reperfusion injury caused significant atrophy and collapse of rat cortical tissue (P < 0.01). However, after 14 days of treatment with the polypeptide compound 1 of the present invention, the cortical index increased significantly (P < 0.01), and the depression of the cortex on the injured side was significantly improved (Figure 13). These data indicate that the polypeptide compound 1 of the present invention has a significant effect on improving pathological tissue damage in tMCAO rats and alleviating cortical tissue atrophy.

[0228] Experimental result 2: As shown in Figure 14, compared with the Sham group, the expression of PSD95 and SYP in the hippocampus and cortex of the rats in the Vehicle group was significantly reduced (P < 0.05), while treatment with the polypeptide compound 1 of the present invention significantly increased the expression of PSD95 and SYP (P < 0.05). These results indicate that cerebral ischemia-reperfusion injury inhibits the expression of synaptic proteins in the hippocampus of SD rats and affects the formation of synaptic structures, which may be an important cause of neurological dysfunction. After treatment with the polypeptide compound 1 of the present invention, the expression of synaptic proteins in the hippocampus of tMCAO rats was significantly increased. This suggests that the polypeptide compound 1 of the present invention may restore rat neural function through synaptic remodeling.

[0229] In summary, the polypeptide of the present invention has the effect of promoting the cell proliferation of the neural stem cell line C17.2 and the cell differentiation of the primary neural stem cell NSCs, as well as the effect of promoting the regeneration of the in vitro cerebrovascular model. At the same time, it can effectively improve the neuromotor dysfunction of stroke rats, significantly reduce the volume of cerebral infarction, and reduce the pathological damage of the hippocampus and cortical areas, such as reducing the damage and death of hippocampal and cortical cells in stroke rats. Further, the polypeptide of the present invention also promotes the proliferation and differentiation of neural stem cells in stroke rats, increases the number of hippocampal neurons and glial cells and the number of new blood vessels in the stroke recovery period, promotes neurogenesis and angiogenesis in stroke rats, and increases the expression of synaptic proteins in the hippocampus to restore rat neural function through synaptic remodeling. This shows that the polypeptide of the present invention has a good therapeutic effect in treating stroke.

[0230] Although the present invention discloses the above embodiments, the implementation of the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the present invention should be considered as equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. [Corrected on 22.11.2024 in accordance with Rule 26] A polypeptide or a physiologically compatible salt thereof, wherein the amino acid sequence of the polypeptide is selected from the group consisting of: H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 1); HD-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 2); H-Ala-D-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 3); H-Ala-Leu-D-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 4); H-Ala-Leu-Pro-D-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 5); H-Ala-Leu-Pro-Ala-D-Pro-Gly-Thr-Leu-OH (SEQ ID No. 6); H-Ala-Leu-Pro-Ala-Pro-Gly-D-Thr-Leu-OH (SEQ ID No. 7); H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-D-Leu-OH (SEQ ID No. 8); Ac-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 9); H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-NH2 (SEQ ID No. 10); Ac-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Leu-NH2 (SEQ ID No. 11); H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Ala-OH (SEQ ID No. 12); H-Ala-Leu-Ala-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 13); H-Ala-Leu-Gly-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 14); H-Ala-Leu-Pro-Ala-Gly-Gly-Thr-Leu-OH (SEQ ID No. 15); H-Ala-Ala-Pro-Ala-Pro-Gly-Thr-Leu-OH (SEQ ID No. 16); H-Ala-Leu-Pro-Ala-Ala-Gly-Thr-Leu-OH (SEQ ID No. 17); H-Ala-Leu-Pro-Ala-Pro-Gly-Ala-Leu-OH (SEQ ID No. 18); H-Leu-Ala-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.19); H-Leu-Ala-Pro-Ala-Pro-Gly-Thr-Ile-OH(SEQ ID No.20); H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-Ile-OH(SEQ ID No.21); H-Gln-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.22); H-Pro-Ile-Ala-Ser-Gln-His-Leu-Asn-Leu-Ala-Pro-Ala-Pro-Gly-Thr-Ile-OH(SEQ ID No.23); H-Ala-Lys-Asn-Gln-Leu-Pro-Ala-Pro-Gly-Thr-Leu-Gln-His-Phe-Cys-OH(SEQ ID No.24); H-Gln-Leu-Pro-Ala-Pro-Gly-Thr-Leu-Gln-His-Phe-OH(SEQ ID No.25); H-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.26); H-Pro-Ala-Pro-Gly-Thr-OH(SEQ ID No.27); H-Pro-Gly-Thr-Leu-OH(SEQ ID No.28); H-Leu-Pro-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.29); H-Ala-Leu-Pro-Ala-Pro-OH(SEQ ID No.30); H-Leu-Pro-Ala-Pro-OH(SEQ ID No.31); H-Leu-Pro-Ala-Pro-Gly-Thr-OH(SEQ ID No.32); H-Leu-Pro-Ala-Pro-Gly-OH(SEQ ID No.33); H-Ala-Pro-Gly-Thr-Leu-OH(SEQ ID No.34); H-Ala-Leu-Pro-Ala-OH(SEQ ID No.35); H-Pro-Ala-Pro-Gly-OH(SEQ ID No.36); H-Ala-Pro-Gly-Thr-OH(SEQ ID No.37); H-Gly-Thr-Leu-OH(SEQ ID No.38); H-Ala-Leu-Pro-Ala-Pro-Gly-Thr-OH(SEQ ID No.39); H-Ala-Leu-Pro-Ala-Pro-Gly-Oh (SEQ ID No. 40); Ac-Ala-Leu-Pro-Ala-D-Pro-Gly-Thr-Leu-NH2 (SEQ ID No. 41); and Ac-Ala-Leu-Pro-D-Ala-Pro-Gly-Thr-Leu-NH2 (SEQ ID No. 42).

2. The polypeptide of claim 1, wherein the polypeptide is selected from: Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 10, Compound 11, Compound 15, Compound 16, Compound 17, Compound 18, Compound 19, Compound 20, Compound 22, Compound 23, Compound 24, Compound 25, Compound 26, Compound 27, Compound 28, Compound 29, Compound 30, Compound 31, Compound 32, Compound 33, Compound 34, Compound 35, Compound 36, Compound 37, Compound 38, Compound 39, Compound 40, Compound 41, Compound 42 or a physiologically compatible salt thereof.

3. Use of the polypeptide of claim 1 or 2 or a physiologically compatible salt thereof in the preparation of a medicament for treating or preventing diseases associated with nerve damage.

4. The use according to claim 3, wherein the disease associated with nerve damage involves abnormal cerebral microangiogenesis.

5. The use of claim 4, wherein the diseases associated with nerve damage include stroke, diabetic neuropathy, brain injury, neurodegenerative diseases, vascular dementia, cerebrovascular disease and epilepsy.

6. The use of claim 5, wherein the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis and HIV-1 related dementia.

7. The use according to claim 5, wherein the brain injury comprises chronic traumatic encephalopathy and cerebral ischemia-reperfusion brain injury.

8. The method of claim 5, wherein the stroke comprises ischemic stroke, hemorrhagic stroke and sequelae of stroke.

9. The use according to claim 8, wherein the polypeptide improves neurological dysfunction in stroke.

10. The use according to claim 8, wherein the polypeptide promotes the number of hippocampal neurons, glial cells and new blood vessels in the late stage of stroke.

11. The use according to claim 8, wherein the polypeptide promotes neurogenesis and angiogenesis in stroke patients.

12. The use of claim 8, wherein the sequelae of stroke include limb dysfunction, mental and intellectual disability, speech dysfunction and swallowing dysfunction.

13. A composition comprising the polypeptide of claim 1 or 2 or a physiologically compatible salt thereof and a physiologically compatible excipient.

14. The composition of claim 13, wherein the composition is a pharmaceutical composition, which can be in the form of oral liquid, tablet, granule, capsule, dripping pill, injection, transdermal absorption preparation, external lotion and in vivo implant preparation.

15. The composition of claim 13, wherein the composition is a nutraceutical composition.

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