Beta-hairpin structure polypeptide and application thereof in treatment of Alzheimer's disease

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a polypeptide with a beta-hairpin structure and application of the polypeptide to treatment of Alzheimer's disease, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. A novel protein skeleton sequence designed by the invention is based on a beta-hairpin structure and can specifically bind and inhibit a key hydrophobic core region (KLVFF, 16-21 amino acids) in an Abeta42 peptide fragment, and through optimal design of the sequence, the sequence can be stably bound with a key site of the Abeta42 peptide fragment, so that aggregation of Abeta42 is inhibited, and the Abeta42 peptide fragment is inhibited. Further, the pathological progress of the Alzheimer's disease is slowed down.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a β-hairpin structure polypeptide and its application in the treatment of Alzheimer's disease. Background Art

[0002] Alzheimer's Disease (AD) is a serious neurodegenerative disease that mainly affects neurons in the brain, leading to a gradual decline in the patient's memory, thinking, and behavioral abilities. The occurrence and development of Alzheimer's disease are affected by multiple factors, including genetics, environment, lifestyle, etc. Currently, the pathogenesis of Alzheimer's disease has not been fully elucidated. Although scientists have proposed various hypotheses, such as the amyloid hypothesis, neurofibrillary tangle hypothesis, etc., no conclusive evidence has been found to determine its root cause. This makes it extremely difficult to develop precise treatment strategies targeting the pathogenesis.

[0003] Currently, the drugs on the market mainly relieve the symptoms of Alzheimer's disease rather than treat the root cause. For example, cholinesterase inhibitors (such as donepezil) and NMDA receptor antagonists (such as memantine) can only relieve symptoms to a certain extent. In addition, the drug R & D cycle is long, the cost is high, and the clinical trial results are often not satisfactory, resulting in slow progress in drug R & D. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a β-hairpin structure polypeptide, which is designed to specifically bind to and inhibit the key hydrophobic core region (KLVFF, amino acids 16 - 21) in the Aβ42 peptide segment, thereby inhibiting the aggregation of Aβ42 and slowing down the pathological progression of Alzheimer's disease.

[0005] To achieve the above purpose, the technical solutions adopted by the present invention include:

[0006] In the first aspect, the present invention provides a polypeptide, and the amino acid sequence of the polypeptide is as shown in (a) or (b):

[0007] (a) The amino acid sequence is as shown in SEQ ID NO: 1;

[0008] (b) An amino acid sequence obtained by substituting, deleting, or adding at least one amino acid to the sequence shown in SEQ ID NO: 1 and having the same function as the polypeptide in (a).

[0009] Preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1; the C-terminus of the polypeptide is amidated, and the structural formula of the polypeptide is as follows:

[0010]

[0011] Preferably, the polypeptide is a β - hairpin structure.

[0012] The present invention proposes a novel protein backbone sequence, aiming to specifically bind to and inhibit the key hydrophobic core region (KLVFF, amino acids 16 - 21) of the Aβ42 peptide segment, thereby slowing down the pathological progression of Alzheimer's disease (AD). The design of this polypeptide sequence is based on an in - depth understanding of the key pathological mechanisms of Alzheimer's disease, especially the research on the aggregation behavior of the Aβ42 peptide segment. The KLVFF sequence is widely regarded as the key driving factor for Aβ42 to form toxic oligomers and fibrils. Therefore, specific binding to and inhibition of this region are the core strategies for designing novel therapeutic molecules.

[0013] To achieve this goal, the present invention uses a β - hairpin structure as the backbone and introduces multiple hydrophobic and aromatic amino acids (such as leucine and valine) so that the polypeptide can effectively embed and bind to the hydrophobic core region of Aβ42. The choice of the β - hairpin structure enables the polypeptide to have good spatial adaptability, which helps to embed and stably bind to the key sites of Aβ42. Through computer simulation, structure optimization, and experimental verification, it is ensured that the binding of this polypeptide sequence to Aβ42 has high affinity and specificity.

[0014] In addition, the polypeptide sequence of the present invention has undergone multiple rounds of candidate sequence screening, comparative studies, and experimental debugging, and finally obtained this polypeptide with both stability and effectiveness. This design not only provides a new idea for the treatment of Alzheimer's disease but also lays a foundation for the development of related drugs. By inhibiting the aggregation of Aβ42, this polypeptide sequence is expected to slow down the pathological progression, improve the cognitive function of Alzheimer's disease patients, and provide new treatment means for future clinical applications.

[0015] In a second aspect, the present invention provides the use of the polypeptide in the preparation of a drug for preventing or treating neurodegenerative diseases.

[0016] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0017] The present invention evaluates the therapeutic effect on a mouse model of Alzheimer's disease through pharmacological activity experiments and finds that the novel β - hairpin structure polypeptide can significantly slow down the memory decline of Alzheimer's disease mice, and thus can be used for the drug development and research of Alzheimer's disease.

[0018] Preferably, the polypeptide targets and inhibits the aggregation of Aβ42 and / or Aβ40.

[0019] Preferably, the polypeptide targets the central hydrophobic core region (KLVFF, amino acids 16 - 21) in the Aβ42 peptide segment, thereby inhibiting the aggregation of Aβ42.

[0020] Preferably, the effective concentration of the polypeptide is 1 - 5 μM.

[0021] Preferably, the effective concentration of the polypeptide is 5 μM.

[0022] When the designed novel β - hairpin structure polypeptide of the present invention was used to explore the ThT inhibition of Aβ40 or Aβ42, it was found that the ThT inhibition effect of the polypeptide on Aβ40 or Aβ42 was concentration - dependent. When the effective concentration range was 1 μM to 5 μM, the aggregation of Aβ40 or Aβ42 was effectively reduced, and the inhibition effect was the best at a concentration of 5 μM.

[0023] In a third aspect, the present invention provides a drug for preventing or treating neurodegenerative diseases, and the drug comprises the polypeptide described above.

[0024] Preferably, the drug is a pharmaceutically acceptable lyophilized product and / or solvate.

[0025] After lyophilizing the extracted novel β - hairpin structure polypeptide of the present invention, it was formulated into solutions with different concentrations for administration with solvents. The solvents include but are not limited to water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0026] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] In order to further enhance the therapeutic effect of Alzheimer's disease (AD), the present invention designed a new protein backbone sequence. Based on the β - hairpin structure, it can specifically bind to and inhibit the key hydrophobic core region (KLVFF, amino acids 16 - 21) in the Aβ42 peptide segment. Through the optimized design of this sequence, it can form a stable binding with the key sites of the Aβ42 peptide segment, thereby inhibiting the aggregation of Aβ42 and then slowing down the pathological progression of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 For the mass spectrometry analysis chart ( Figure 1 A) and HPLC chart ( Figure 1 B) of the novel β - hairpin structure polypeptide;

[0030] Figure 2 For the circular dichroism (CD) spectrum chart of the novel β - hairpin structure polypeptide;

[0031] Figure 3 The inhibitory effect diagram of the novel β-hairpin structure polypeptide on the aggregation of Aβ40 and Aβ42 at different concentrations (1 μM to 5 μM), with the percentage of fluorescence intensity as the index;

[0032] Figure 4 The result diagram of the inhibitory effect of the novel β-hairpin structure polypeptide on the aggregation of Aβ40 or Aβ42 in HEK293T cells. Detailed implementation manners

[0033] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and the test materials used in the following embodiments are all commercially available through conventional channels unless otherwise specified.

[0035] Embodiment 1

[0036] This embodiment provides a preparation method of the β-hairpin structure polypeptide, and the specific method is as follows:

[0037] 1. Plasmid construction and transfection

[0038] Use standard molecular biology techniques to construct a plasmid expressing the novel β-hairpin structure polypeptide and transfect Escherichia coli.

[0039] 1.1 Plasmid construction

[0040] 1. Vector selection: Select the pET-28a plasmid vector containing the T7 promoter and His tag for high-efficiency expression in Escherichia coli.

[0041] 2. Polypeptide sequence insertion:

[0042] (a) Design a DNA fragment containing the β-hairpin structure polypeptide sequence and enhance its expression in Escherichia coli through codon optimization. The nucleotide sequence of the DNA is shown in SEQ ID NO: 2.

[0043] (b) Introduce NdeI and XhoI restriction sites at both ends of the polypeptide coding sequence for enzymatic digestion and insertion.

[0044] 3. Ligation reaction: After double-digesting the pET-28a plasmid and the polypeptide coding sequence with NdeI and XhoI, use T4 DNA ligase to ligate overnight at 16°C.

[0045] 1.2 Transfect Escherichia coli

[0046] 1. Preparation and transformation of competent cells: Use BL21(DE3) Escherichia coli competent cells, add the ligation product into the cells and perform heat shock (42 °C, 45 seconds), then immediately place on ice for 2 minutes.

[0047] 2. Cultivation and screening:

[0048] (a) Add 1 mL of LB medium without antibiotics, resuscitate at 37 °C for 1 hour. Spread on an LB plate containing kanamycin (50 μg / mL) and culture overnight at 37 °C.

[0049] (b) Pick a single colony and inoculate it into 5 mL of LB liquid medium containing kanamycin, and culture with shaking overnight at 37 °C.

[0050] 1.3 Screening of positive clones

[0051] 1. Plasmid extraction and identification:

[0052] (a) Use a plasmid extraction kit to extract the plasmid, and perform NdeI and XhoI double digestion to identify the inserted fragment.

[0053] (b) Confirm the successful construction of the plasmid by agarose gel electrophoresis.

[0054] 2. Protein extraction and lyophilization

[0055] Extract the novel β-hairpin structure polypeptide using lysis and purification techniques, and store it after lyophilization.

[0056] 2.1 Protein expression

[0057] 1. Culture: Inoculate the positive clone into 50 mL of LB medium containing kanamycin, and culture with shaking at 37 °C until the OD600 reaches 0.6 - 0.8.

[0058] 2. Induced expression: Add IPTG to a final concentration of 0.5 mM, lower the temperature to 16 °C, and culture for 16 hours.

[0059] 2.2 Cell lysis

[0060] 1. Collect bacteria: Centrifuge at 4000 rpm for 10 minutes at 4 °C to collect the bacterial cells.

[0061] 2. Preparation of lysis buffer: Prepare the buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10 mM Imidazole, 1 mM PMSF).

[0062] 3. Ultrasonic disruption: Resuspend the bacterial cells in 10 mL of lysis buffer, and perform ultrasonic disruption in an ice bath, with a 10-second interval for 20 seconds, repeating 20 times.

[0063] 4. Centrifugation: 12,000 g, 4 °C, 30 minutes. Collect the supernatant for subsequent purification.

[0064] 2.3 Protein purification

[0065] 1. Nickel affinity chromatography:

[0066] (a) Pass the supernatant through a pre-equilibrated Ni-NTA affinity chromatography column and pre-wash it with an equilibration buffer containing 10 mM Imidazole.

[0067] (b) After washing away non-specifically bound proteins, elute the target polypeptide with 250 mM Imidazole and collect the purified polypeptide fraction.

[0068] 2.4 Freeze-drying and storage

[0069] 1. Dialysis and buffer replacement: Use a dialysis bag (molecular weight cut-off 3.5 kDa) to remove Imidazole and replace it with 20 mM Tris-HCl (pH 7.5).

[0070] 2. Freeze-drying treatment: Aliquot the dialyzed protein, first freeze it at -80 °C for 4 hours, and then place it in a freeze-dryer for vacuum freeze-drying, usually for 24 - 48 hours.

[0071] 3. Storage conditions: Store the freeze-dried polypeptide powder at -20 °C.

[0072] Effect Example 1

[0073] This effect example conducts mass spectrometry (MS) identification analysis and HPLC separation on the β-hairpin structure polypeptide obtained in Example 1. The specific methods are as follows:

[0074] For the HPLC separation, an ODS chromatographic column is used for polypeptide separation. Eluent A is acetonitrile with 0.1% trifluoroacetic acid, and eluent B is water with 0.1% trifluoroacetic acid. The gradient elution program is adjusted as follows: starting at 95% B, gradually increasing to 50% B within 25 minutes, with a flow rate of 1 mL / min, a detection wavelength of 214 nm, and a column temperature of 25 °C; dissolve the freeze-dried polypeptide in water / acetonitrile (1:1, containing 0.1% TFA) to prepare a concentration of 0.1 mg / mL, take 20 μL of the sample and inject it into the HPLC system to analyze the retention time and peak shape of the sample. In addition, dissolve the freeze-dried polypeptide in a water / methanol solution (1:1, containing 0.1% formic acid) to prepare a concentration of 0.01 mg / mL, and directly inject the sample into the mass spectrometer for analysis after sampling.

[0075] Figure 1A is the mass spectrometry analysis chart of the novel β-hairpin structure polypeptide. The X-axis represents the mass-to-charge ratio (m / z), and the Y-axis represents the relative abundance (%). The peak value of the molecular ion peak in the chart, 2337.4154 m / z, corresponds to the theoretical mass-to-charge ratio of the novel β-hairpin structure polypeptide (M = 2336.4152), verifying the correct molecular mass of the polypeptide. Figure 1 B is the high performance liquid chromatography (HPLC) separation chart of the novel β-hairpin structure polypeptide. The X-axis represents time (minutes), and the Y-axis represents the detector response (mV). The picture shows that the retention time of the target polypeptide is 13.880 minutes, indicating good separation effect and high purity in HPLC.

[0076] Effect Example 2

[0077] In this effect example, the circular dichroism (CD) method was used to explore the structural characteristics of the β-hairpin structure polypeptide prepared in Example 1. The specific experimental method is as follows:

[0078] 1. Sample preparation

[0079] Prepare a solution containing the target β-hairpin polypeptide with a concentration of 0.1 mg / mL, using deionized water as the solvent, and filter the solution (pore size 0.22 μm) to remove insoluble particles.

[0080] 2. Experimental conditions

[0081] (a) Instrument: Use a circular dichroism spectrometer (Jasco J-1500).

[0082] (b) Optical path: Select a 0.1 cm quartz cell.

[0083] (c) Scanning wavelength range: 190 nm to 250 nm.

[0084] (d) Scanning speed: 50 nm / min.

[0085] (e) Step size: 1 nm.

[0086] 3. Measurement steps

[0087] (a) Load the sample into the quartz cell, ensuring that all bubbles are completely removed;

[0088] (b) Use a blank control (deionized water) for baseline correction;

[0089] (c) Record the ellipticity data ([θ], unit: deg·cm 2 / dmol) at different wavelengths;

[0090] (e) Each sample is scanned three times and the average value is taken to ensure data reliability.

[0091] 4. Data Analysis

[0092] The ellipticity data is converted into secondary structure characteristics to determine the secondary structure composition of the target polypeptide.

[0093] Figure 2 It is the circular dichroism (CD) spectrum of the β-hairpin structure polypeptide. The spectral results show that the polypeptide has a significant positive peak at around 195 nm, which is a characteristic of the typical β-sheet structure, and a negative peak is observed near 218 nm, further indicating that the polypeptide has a significant β-sheet secondary structure. The characteristics of the positive and negative peaks in the spectrum are consistent with the circular dichroism characteristics of the known β-sheet, indicating that the target polypeptide successfully forms the expected β-hairpin structure. The formation of this structure supports its ability to bind to the key hydrophobic core region (KLVFF, amino acids 16 - 21) of Aβ42, thus laying a structural foundation for inhibiting the aggregation of Aβ42 and providing a structural basis for its functional design in the treatment of Alzheimer's disease.

[0094] Effect Example 3

[0095] This effect example explored the ThT inhibition experiment of the β-hairpin structure polypeptide obtained in Example 1 on Aβ40 and Aβ42. The specific method is as follows:

[0096] 1. Experimental materials:

[0097] (a) Aβ40 peptide segment: Purchased from Shanghai Sibio Biotechnology Co., Ltd., with a purity ≥ 95%, and the final concentration used is 5 μM.

[0098] (b) Aβ42 peptide segment: Purchased from Beijing Biotech Co., Ltd., with a purity ≥ 95%, and the final concentration is prepared to be 5 μM.

[0099] (c) Novel β-hairpin structure polypeptide: The polypeptide prepared in Example 1 is formulated into solutions with different concentrations (1 μM, 2 μM, 3 μM, 4 μM, 5 μM) with sterile water or DMSO solvent.

[0100] (d) Thioflavin T (ThT): Purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with a final concentration of 20 μM.

[0101] (e) Dimethyl sulfoxide (DMSO): Purchased from Sinopharm Chemical Reagent Co., Ltd., analytically pure, used to dissolve Aβ40 and Aβ42 peptide segments.

[0102] 2. Experimental steps:

[0103] (a) Dissolve Aβ40 and Aβ42 in DMSO respectively and dilute to a concentration of 5 μM.

[0104] (b) Prepare the novel β-hairpin structure polypeptide at concentrations of 1 μM, 2 μM, 3 μM, 4 μM, and 5 μM, and mix it with Aβ40 / Aβ42 and ThT solution.

[0105] (c) Incubate at 37 °C for 96 hours, and detect the fluorescence intensity using an excitation wavelength of 430 nm and an emission wavelength of 485 nm.

[0106] 3. Experimental results: According to the fluorescence intensity, record the inhibitory effects of polypeptides at different concentrations on the aggregation of Aβ40 or Aβ42. Figure 3 The results show that as the polypeptide concentration increases, the fluorescence intensity gradually decreases, indicating the effective inhibitory effect of the polypeptide on the aggregation of Aβ40 and Aβ42. Through dose-dependent experiments, the preferred effective concentration of the novel β-hairpin structure polypeptide is confirmed to be 1 μM to 5 μM. Within this range, the polypeptide can significantly inhibit the aggregation of Aβ40 and Aβ42, and the inhibitory effect is the best at a concentration of 5 μM.

[0107] Effect Example 4

[0108] This effect example explored the inhibitory effect of the β-hairpin structure polypeptide obtained in Example 1 on the aggregation of Aβ40 and Aβ42 at the cellular level. The specific method is as follows:

[0109] 1. Experimental materials:

[0110] (a) HEK293T cells: Purchased from the China Center for Type Culture Collection (CCTCC), numbered CCTCC-HEK293T.

[0111] (b) DAPI dye: Purchased from Beyotime Biotechnology Co., Ltd., with a concentration of 1 μg / mL, used for nuclear staining.

[0112] (c) Novel β-hairpin structure polypeptide: The β-hairpin structure polypeptide obtained in Example 1, with the final concentrations set at 2 μM and 5 μM.

[0113] (d) Aβ40 gene vector: Purchased from Shanghai Genechem Co., Ltd., formulated in the pEGFP-N1 vector, used for cell transfection.

[0114] (e) Aβ42 gene vector: Purchased from Vigene Biosciences (Nanjing) Co., Ltd. and expressed using the pEGFP-N1 vector.

[0115] 2. Experimental procedures:

[0116] (a) Culture HEK293T cells in DMEM medium containing 10% fetal bovine serum until 80% confluence.

[0117] (b) Transfect the HEK293T cells with the Aβ40 and Aβ42 gene vectors respectively to express Aβ40 and Aβ42 respectively, and then add the novel polypeptide to both, with concentrations of 2 μM and 5 μM, and observe by staining after culturing for 24 hours.

[0118] (c) Perform DAPI staining to show the cell nuclei, and observe the aggregation state of Aβ40 or Aβ42 in the cells through a confocal microscope.

[0119] 3. Experimental results: Figure 4 The fluorescence signals showed that with the increase in the concentration of the polypeptide, the aggregation of Aβ40 or Aβ42 in the cells decreased significantly, indicating that the novel polypeptide has an inhibitory effect on the aggregation of Aβ40 or Aβ42 at the cellular level and shows a concentration-dependent effect.

[0120] Effect Example 5

[0121] This effect example explored the pharmacological activity experiment of the β-hairpin structure polypeptide obtained in Example 1. Eight-month-old male APP mice (weight 30 ± 2 g), purchased from the Model Animal Research Institute of Nanjing University (NBRI), were intraperitoneally injected with 1.0 mg / kg of the novel β-hairpin structure polypeptide once a day for 7 consecutive days. At the same time, Morris water maze test, Y maze test and novel object recognition test were carried out. The specific test methods are as follows:

[0122] 1. Morris water maze test (Morris Water Maze, MWM)

[0123] 1. Experimental purpose: To test the spatial learning and memory ability of mice.

[0124] 2. Experimental method:

[0125] 2.1 Experimental grouping

[0126] (a) Blank group: Normal mice, not receiving any treatment.

[0127] (b) AD group: Alzheimer's disease model mice, purchased from the Model Animal Research Institute of Nanjing University (NBRI).

[0128] (c) AD polypeptide treatment group: Mice treated with the novel β-hairpin structure polypeptide described in Example 1 after establishing the Alzheimer's disease model.

[0129] 2.2 Equipment

[0130] (a) The water maze (1.2 meters in diameter, 30 centimeters in depth) is filled with milky white water, and the water temperature is maintained at 22 ± 1 °C.

[0131] (b) The hidden platform (10 centimeters in diameter) is placed 1 centimeter below the water surface.

[0132] 2.3 Experimental procedures

[0133] (a) Adaptation period: Each mouse was allowed to swim freely in the maze for 2 minutes without a platform.

[0134] (b) Training period (5 days, 4 trials per day): The mice were placed at different positions on the edge of the maze, and the latency and path length required to find the hidden platform were recorded. The maximum time for each trial was 60 seconds. If the mouse did not find the platform, it was guided to the platform and stayed there for 10 seconds.

[0135] (c) Probe test (day 6): The platform was removed, and the residence time (60 seconds) of the mouse in the quadrant where the platform was previously located was recorded. This test was used to evaluate the retention of spatial memory.

[0136] 2.4 Data recording and analysis

[0137] (a) Latency: The time required for the mouse to find the platform in each training session. The shorter the time, the stronger the learning ability.

[0138] (b) Path length: The total length of the path the mouse took to reach the platform.

[0139] (c) Residence time in the target quadrant: The time the mouse stayed in the target quadrant during the probe test.

[0140] 2. Y - Maze Test

[0141] 1. Experimental purpose: To test the short - term memory and spontaneous alternation behavior of mice.

[0142] 2. Experimental methods:

[0143] (a) Control group: Normal mice without any treatment.

[0144] (b) AD group: Alzheimer's disease model mice purchased from the Model Animal Research Institute of Nanjing University (NBRI).

[0145] (c) AD polypeptide treatment group: Mice that were injected with the novel β - hairpin structure polypeptide described in Example 1 after establishing the Alzheimer's disease model.

[0146] 2.2 Equipment

[0147] Y - shaped maze (each arm is 40 cm long, 10 cm wide, and 12 cm high), and the angle between the three arms is 120°.

[0148] 2.3 Experimental procedures

[0149] (a) Adaptation period: Each mouse was allowed to freely explore the Y - maze for 5 minutes, and the exploration behavior was recorded.

[0150] (b) Testing period: Place the mouse at the end of one of the arms and allow it to freely explore for 5 minutes. Record the order in which the mouse enters different arms each time.

[0151] 2.4 Data recording and analysis

[0152] (a) Spontaneous alternation rate: Calculate the proportion of the mouse continuously choosing different arms among the three arms. Spontaneous alternation rate = number of spontaneous alternations / (total number of arm entries - 2) × 100%.

[0153] (b) Number of arm entries: Record the number of times the mouse enters each arm during the testing period.

[0154] 3. Novel Object Recognition (NOR) test

[0155] 1. Experimental purpose: To test the recognition memory of mice.

[0156] 2. Experimental method:

[0157] 2.1 Experimental grouping

[0158] (a) Blank group: Normal mice without any treatment.

[0159] (b) AD group: Alzheimer's disease model mice purchased from the Model Animal Research Institute of Nanjing University (NBRI).

[0160] (c) AD polypeptide treatment group: Mice treated with the novel β-hairpin structure polypeptide described in Example 1 after establishing the Alzheimer's disease model.

[0161] 2.2 Equipment

[0162] Open field (40 cm × 40 cm × 40 cm) and two objects (different in shape, size and color).

[0163] 2.3 Experimental procedure

[0164] (a) Adaptation period: The mouse freely explores in the empty open field for 10 minutes to reduce the sense of unfamiliarity with the environment.

[0165] (b) Training period: Place two identical objects in the open field and let the mouse explore for 10 minutes. Record the exploration time of the mouse for the objects.

[0166] (c) Testing period (24 hours later): Replace one object with a new object and allow the mouse to explore for 5 minutes. Record the exploration time of the mouse for the new object and the old object.

[0167] 2.4 Data recording and analysis

[0168] (a) Discrimination index: Calculate the discrimination index, where discrimination index = (new object exploration time - old object exploration time) / (new object exploration time + old object exploration time) × 100%.

[0169] (b) Exploration time: Record the exploration time of the mouse for each object.

[0170] 4. Experimental results

[0171] The specific results of the Morris water maze test, Y maze test, and novel object recognition test are shown in Tables 1 - 3. Among them, the residence time in the target quadrant is the time that the mouse stays in the target quadrant during the exploration experiment.

[0172] Table 1 Results of the Morris water maze experiment

[0173]

[0174] The results in Table 1 show that the mice in the blank group exhibited good learning and memory abilities. As the number of training days increased, the latency and path length were significantly shortened, and the residence time in the target quadrant was longer. The mice in the AD group had impaired learning and memory abilities, manifested as longer latency and path length, and a significantly reduced residence time in the target quadrant. The mice in the AD polypeptide treatment group showed improvement in learning and memory, with a reduction in latency and path length, and the residence time in the target quadrant approaching that of the blank group.

[0175] Table 2 Results of the Y maze experiment

[0176] Group Spontaneous alternation rate (%) Arm entry times (times) Blank group 73±4 32±2 AD group 51±5 24±3 AD polypeptide treatment group 66±4 29±2

[0177] The results in Table 2 show that the mice in the blank group exhibited a high spontaneous alternation rate and frequent arm entry behavior, indicating normal short - term memory function. The mice in the AD group had a significantly reduced spontaneous alternation rate, indicating impaired short - term memory. The mice in the AD polypeptide treatment group had an increased spontaneous alternation rate, and the short - term memory function was partially restored.

[0178] Table 3 Results of the novel object recognition experiment

[0179]

[0180] The results in Table 3 show that the mice in the blank group showed an obvious preference for new objects and had a high discrimination index, indicating normal recognition memory function. The mice in the AD group had a decreased ability to discriminate new objects, and the discrimination index was significantly reduced, indicating impaired recognition memory. The discrimination index of the mice in the AD polypeptide treatment group was significantly higher than that of the AD group, indicating that the novel β - hairpin structure polypeptide had a certain restorative effect on the recognition memory function.

[0181] In summary, the results of the simulation experiment showed that the mice in the blank group performed best in all cognitive tests, the cognitive function of the Alzheimer's disease model group decreased significantly, while the mice treated with the novel β-hairpin structure polypeptide improved in all tests, indicating that the novel β-hairpin structure polypeptide described in the present invention has potential protective and restorative effects on the cognitive function of Alzheimer's disease model mice and can provide new treatment means for future clinical applications.

[0182] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in (a) or (b): (a) the amino acid sequence is shown in SEQ ID NO: 1; (b) An amino acid sequence having the same function as the polypeptide described in (a) obtained by substituting, deleting or adding at least one amino acid in the sequence shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; the C-terminus of the polypeptide is amidated with amino group, and the structural formula of the polypeptide is as follows:

3. The polypeptide according to claim 1 or 2, characterized in that The polypeptide is a β-hairpin structure.

4. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for preventing or treating a neurodegenerative disease.

5. The use according to claim 4, characterized in that The neurodegenerative disease is Alzheimer's disease.

6. The use according to claim 4, characterized in that The polypeptide targets and inhibits the aggregation of Aβ42 and / or Aβ40.

7. The use according to claim 4, characterized in that The effective concentration of the polypeptide is 1-5 μM.

8. The use according to claim 7, characterized in that The effective concentration of the polypeptide is 5 μM.

9. A drug for preventing or treating neurodegenerative diseases, characterized in that: The drug comprises the polypeptide according to any one of claims 1 to 3.

10. The drug according to claim 9, characterized in that The drug is a pharmaceutically acceptable lyophilized agent and / or solvate.

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