Polypeptides targeting mst1 and p53 binding and their use in the treatment of alzheimer's disease

Abstract

The application discloses a polypeptide targeting MST1 and p53 combination and application thereof in treatment of Alzheimer's disease, and relates to the field of biological medicine. The polypeptide provided by the embodiment of the application can block the neurodegenerative process from the upstream by targeting the MST1 kinase, has not only a direct neuroprotective effect, but also can improve synaptic plasticity, and is expected to realize substantial intervention on the disease process, rather than being limited to relieving symptoms. As a polypeptide drug, the polypeptide has a small molecular weight, better potential tissue penetration, lower immunogenicity, better safety and tolerance. The polypeptide can more effectively intervene in the AD disease process, improve cognitive impairment and memory dysfunction of patients, and provides a new way for AD treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to peptides that target the binding of MST1 and p53 and their application in the treatment of Alzheimer's disease. Background Technology

[0002] Currently, the drugs used clinically to treat Alzheimer's disease (AD) can be mainly divided into the following two categories: (I) Symptom relief drugs. These drugs aim to improve cognitive, behavioral and functional symptoms, but cannot change the course of the disease. They mainly include: (1) Cholinesterase inhibitors: such as donepezil, rivastigmine, and galantamine. Their mechanism of action is to increase the level of acetylcholine in the brain to partially compensate for the neurotransmitter deficiency caused by neuronal loss, thereby improving the cognition and daily function of patients with mild to moderate AD. (2) Glutamate receptor antagonists: mainly memantine. It reduces excitatory toxicity by regulating the glutamatergic system and is used to treat the symptoms of moderate to severe AD. It can be used alone or in combination with cholinesterase inhibitors. (II) Disease modification therapy drugs. These drugs aim to intervene in the core pathophysiological process of AD (such as β-amyloid deposition) in order to delay or stop the progression of the disease. Representative drugs are: anti-Aβ monoclonal antibodies: such as lencanemab and aducanumab. They work by directly targeting and clearing β-amyloid plaques in the brain.

[0003] Existing technologies have the following objective drawbacks: 1. Insufficient target specificity and disease progression intervention: Existing drugs (such as cholinesterase inhibitors) mainly act on downstream symptoms and cannot precisely intervene in the core signaling pathways leading to neuronal death (such as MST1-mediated neuronal apoptosis). 2. Lack of neuroprotective and repair functions: Conventional therapies cannot effectively protect surviving neurons or promote neuronal repair. 3. Difficulty in delivering large molecule drugs and risks of side effects: Although novel antibody drugs can target pathological proteins, they are large molecules, making it difficult to efficiently penetrate the blood-brain barrier, and there are risks of side effects such as immunogenicity and cerebral edema. 4. Tolerance and safety issues in long-term treatment: Long-term use of existing drugs may lead to reduced efficacy or intolerable side effects (such as gastrointestinal reactions and mental and behavioral abnormalities).

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide that targets the binding of MST1 and p53 and its application in the treatment of Alzheimer's disease.

[0006] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a polypeptide having the amino acid sequence shown in SEQ ID NO:1 (ITSAGGD).

[0007] Secondly, embodiments of the present invention provide an isolated nucleic acid molecule that encodes the polypeptide described in the foregoing embodiments.

[0008] Thirdly, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in the foregoing embodiments.

[0009] Fourthly, embodiments of the present invention provide a recombinant cell containing the recombinant vector described in the foregoing embodiments.

[0010] Fifthly, embodiments of the present invention provide a method for preparing the polypeptide as described in the foregoing embodiments, comprising: artificially synthesizing the polypeptide as described in the foregoing embodiments or culturing the recombinant cells as described in the foregoing embodiments.

[0011] In a sixth aspect, embodiments of the present invention provide a pharmaceutical composition whose active ingredient includes the polypeptide described in the foregoing embodiments.

[0012] In a seventh aspect, embodiments of the present invention provide the use of the polypeptides described in the foregoing embodiments, the isolated nucleic acid molecules described in the foregoing embodiments, the recombinant vectors described in the foregoing embodiments, the recombinant cells described in the foregoing embodiments, or the preparation methods described in the foregoing embodiments in the preparation of medicaments for treating or improving at least one of neuronal dysfunction, memory impairment, and cognitive impairment.

[0013] Eighthly, embodiments of the present invention provide the application of the polypeptides, isolated nucleic acid molecules, recombinant vectors, recombinant cells, or preparation methods described in the foregoing embodiments in the preparation of foods that help improve memory.

[0014] Ninthly, embodiments of the present invention provide the application of the polypeptides, isolated nucleic acid molecules, recombinant vectors, recombinant cells, or preparation methods described in the foregoing embodiments in the preparation of health products that help improve memory.

[0015] The present invention has the following beneficial effects: This invention provides a polypeptide that targets MST1 kinase and inhibits the MST1-p53 pathway, blocking neurodegenerative processes upstream. This not only provides direct neuroprotection but also improves synaptic plasticity, potentially enabling substantial intervention in disease progression beyond just symptom relief. As a polypeptide drug, it has a small molecular weight, better potential tissue penetration, and lower immunogenicity, resulting in better safety and tolerability. This polypeptide can more effectively intervene in the progression of Alzheimer's disease (AD), improving patients' cognitive and memory impairments, and providing a new approach for AD treatment. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A schematic diagram of the process for screening peptides that specifically inhibit the binding of MST1 and P53 using phage display technology; Figure 2 The results are as follows: (a), (b), and (c) show the cytotoxicity of three candidate peptides T1, T2, and T3 at different concentrations on SH-SY5Y cells as detected by CCK-8 assay; (d), (e), and (f) show the effects of different concentrations of T1, T2, and T3 peptides on cell viability after SH-SY5Y cells were modeled using Aβ. Figure 3 The results of immunofluorescence and co-immunoprecipitation on the inhibition of MST1 and p53 binding by the three selected peptides are shown. Among them, (a) is the immunofluorescence image of intracellular colocalization of the candidate peptides and MST1, and (b) is the result of co-immunoprecipitation (Co-IP) to verify the effect of T1, T2 and T3 on the interaction between protein MST1 and protein p53. Figure 4 The results show the expression of synaptic-related proteins in the hippocampus. Among them, (a) shows the protein expression levels of N-methyl-D-aspartate receptor subunit 2B (NR2B), postsynaptic density protein 95 (PSD95, postsynaptic scaffold protein), and presynaptic capsule protein SYP in four groups of hippocampus; (b) shows the statistical graph of the relative protein expression of N-methyl-NR2B / β-actin; (c) shows the statistical graph of the relative protein expression of PSD95 / β-actin; and (d) shows the statistical graph of the relative protein expression of SYP / β-actin. Figure 5 The results show the expression of apoptosis-related proteins in the hippocampus; (a) shows the protein expression levels of B-cell lymphoma-2 protein (Bcl-2), Bcl-2-related X protein (Bax), and cleaved cysteine ​​protease 3 in the hippocampus of the four groups; (b) shows the statistical graph of the relative protein expression of Bcl-2 / Bax; and (c) shows the statistical graph of the relative protein expression of Cleaved Caspase 3 / β-actin. Figure 6The results of the Y-maze experiment are shown below; (a) is a schematic diagram of the mouse Y-maze test, (b) is the spontaneous alternation rate of the Y-maze for each group of mice, and (c) is the total number of times each group of mice entered the Y-maze. Figure 7 The results of the novel object recognition experiment are shown below; (a) is a schematic diagram of the novel object recognition test for mice, (b) is the discrimination index (%) of each group of mice at 0h, (c) is the discrimination index (%) of each group of mice at 2h, and (d) is the discrimination index (%) of each group of mice at 24h. Figure 8 The results are shown in the water maze experiment. Among them, (a) is a schematic diagram of the Morris water maze test for mice, (b) is the escape latency of mice in the Morris water maze from day 1 to day 5, (c) is the percentage of time mice in the target area on day 6, and (d) is the number of times mice in the target platform crossed on day 6. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0019] Definition of noun The term "MST1" stands for Mammalian Sterile 20-like Kinase 1.

[0020] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0021] The term "recombinant vector" refers to a molecule containing all the elements necessary for the replication, integration, amplification, and / or expression of the contained nucleic acid molecule in recombinant cells. These elements include, but are not limited to, promoters, polyadenylation sites, terminators, replication initiation sites, transcription initiation sequences, enhancers, selection elements, and reporter genes. For example, antibiotic resistance genes or selection marker genes (such as the ampicillin resistance gene AmpR, the thymidine kinase gene TK, etc.) and the coding sequence for the target protein can be inserted into the multiple cloning site (MCS). Optional types of recombinant vectors include plasmids, bacteriophages, lentiviruses, adenoviruses, and adeno-associated viruses.

[0022] The term "recombinant cell" refers to a cell in which nucleic acid molecules are replicated, integrated, amplified, and / or expressed. Recombinant cells include, but are not limited to, bacteria, fungi, insect cells, and animal cells. Bacteria can be, for example, *Escherichia coli* or *Bacillus subtilis*; fungi can be, for example, yeast or *Aspergillus*; insect cells can be, for example, S2 *Drosophila* cells or Sf9 cells; and animal cells can be, for example, mammalian cells such as those from mice, rats, pigs, sheep, monkeys, chimpanzees, or humans, specifically such as 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, and HEK293 cells. It is understood that these recombinant cells can all be non-reproductive materials.

[0023] The term "composition" as used herein can be formulated in any possible dosage form, including but not limited to tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. "Pharmaceutical" and "pharmaceutical composition" can be substituted for equivalents. The pharmaceutical or pharmaceutical composition as used herein can also be formulated as a suspension in an aqueous, anhydrous, or mixed medium. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including but not limited to sodium carboxymethyl cellulose, sorbitol, and / or dextran. Suspensions may also contain stabilizers. In addition to the active ingredients of this invention, the pharmaceutical composition may also include pharmaceutically acceptable carriers. These carriers include, but are not limited to, diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorbents.

[0024] In this article, “treatment” includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0025] To overcome the limitations of existing MST1 peptide inhibitors, including their scarcity, insufficient targeting, and instability, this application first utilizes phage display technology combined with computer-aided design (such as molecular docking and kinetic simulation) based on the structural characteristics of MST1 to design and screen peptide candidate molecules with high affinity and specificity. Subsequently, the efficacy of the peptide drug in an AD model was systematically evaluated. Validation in cell and animal models successfully demonstrated that the peptide drug can effectively inhibit neuronal apoptosis, improve synaptic plasticity, and significantly enhance cognitive function, thus resolving the issue of the effectiveness of peptide drugs in AD treatment. This application fills the research gap in the field of peptide drug targets for AD treatment, aiming to develop a novel therapeutic drug targeting MST1 with its specific peptide inhibitor as the core component, in order to more effectively intervene in the progression of AD and improve patients' cognitive function.

[0026] Specific technical solutions On one hand, embodiments of the present invention provide a polypeptide having an amino acid sequence as shown in SEQ ID NO:1, 2 or 3.

[0027] On the other hand, embodiments of the present invention provide an isolated nucleic acid molecule that encodes the polypeptide described in the foregoing embodiments.

[0028] On the other hand, embodiments of the present invention provide a recombinant vector containing the isolated nucleic acid molecules described in the foregoing embodiments.

[0029] In optional embodiments, when linking the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the aforementioned nucleic acid molecules. The vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. The vector may also include components that facilitate its entry into cells, including but not limited to viral particles, liposomes, or protein coats. The vector can be an expression vector or a cloning vector. The expression vector can be derived directly from the vector itself or it can be exogenous, i.e., not derived from the vector itself. Of course, the aforementioned nucleic acid molecules and control elements only need to be operatively linked. In this document, "operatively linked" means linking a foreign gene to a vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene.

[0030] In an optional implementation, the aforementioned recombinant vector is a plasmid expression vector.

[0031] On the other hand, embodiments of the present invention provide a recombinant cell containing the recombinant vector described in the foregoing embodiments.

[0032] On the other hand, embodiments of the present invention provide a method for preparing the polypeptide as described in the foregoing embodiments, which includes: artificially synthesizing the polypeptide as described in the foregoing embodiments or culturing the recombinant cells as described in the foregoing embodiments.

[0033] On the other hand, embodiments of the present invention provide a pharmaceutical composition whose active ingredient includes the polypeptide described in the foregoing embodiments.

[0034] On the other hand, embodiments of the present invention provide the use of the polypeptides described in the foregoing embodiments, or the isolated nucleic acid molecules described in the foregoing embodiments, or the recombinant vectors described in the foregoing embodiments, or the recombinant cells described in the foregoing embodiments, or the preparation methods described in the foregoing embodiments, in the preparation of medicaments for treating or improving at least one of neuronal dysfunction, memory impairment, and cognitive impairment.

[0035] In an optional embodiment, the disease includes Alzheimer's disease.

[0036] On the other hand, embodiments of the present invention provide the application of the polypeptides, isolated nucleic acid molecules, recombinant vectors, recombinant cells, or preparation methods described in the foregoing embodiments in the preparation of reagents that improve neuronal apoptosis or damage.

[0037] On the other hand, embodiments of the present invention provide the application of the polypeptides, isolated nucleic acid molecules, recombinant vectors, recombinant cells, or preparation methods described in the foregoing embodiments in the preparation of foods that help improve memory.

[0038] On the other hand, embodiments of the present invention provide the application of the polypeptides, isolated nucleic acid molecules, recombinant vectors, recombinant cells, or preparation methods described in the foregoing embodiments in the preparation of health products that help improve memory.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Example 1 First, this embodiment uses phage display technology to screen for peptides that can specifically bind to MST1, thereby inhibiting the binding of MST1 to P53. The specific operation of phage display technology is as follows: Step 1: Phage amplification: Take 20 mL of LB liquid medium and add 200 μL of frozen E. coli (more can be added as needed), amplify for 4-6 hours (the time is not fixed, until turbidity occurs, 37 ℃, 150 rpm). Add 200 mL of LB culture medium to a large Erlenmeyer flask, pour in the amplified E. coli, and amplify overnight (37 ℃, 250 rpm) using a 1 mL LB:1 μL phage ratio. Step 2: Purification: Pour the liquid from the Erlenmeyer flask into a large centrifuge tube, centrifuge at 4500 rpm for ten minutes, and collect the supernatant. (The precipitate is fragments of E. coli). Put the supernatant into a new small Erlenmeyer flask, add NaCl / PEG 8000 at a volume ratio of 4:1, mix well, and let stand for one hour or overnight at 4 ℃. Pour into a new large centrifuge tube, centrifuge at 12000 rpm for 15 minutes, and collect the precipitate. Resuspend in 1 mL TBS and transfer to an EP tube. Centrifuge at 8000 rpm and 4 °C for 5 minutes, and collect the supernatant to remove cell debris and impurities. Transfer the supernatant to a new EP tube, add 200 μL NaCl / PEG 8000, and incubate on ice for 15-60 minutes. Centrifuge at 12000 rpm for 10 minutes, and collect the precipitate. Add 200 μL TBS, centrifuge at 8000 rpm for 5 minutes, and collect the supernatant. Add glycerol at a 1:1 volume ratio and freeze at -20 °C. Third step: concentration titration: 20 mL LB (Tet) + 200 μL E. coli, shake at 37 °C and 250 rpm for 4-5 hours (until turbid). Pour an appropriate amount of bottom culture medium, not too thick, and seal by inverting after solidification. Determine the dilution gradient and prepare the corresponding number of 5mL tubes. Add 3mL of top-layer culture medium to each tube and place the rack in a 48℃ water bath. 100-fold dilution: 990μL TBS + 10μL phage; 10-fold dilution: 900μL TBS + 100μL phage. Clearly write the dilution factor on the tube. Take a new tube, add 200μL E. coli + 10μL phage, and mix well. Clearly write the dilution factor on the tube. Take 200μL of the liquid, add it to the top-layer culture medium, mix quickly, and pour it onto the bottom plate. Clearly write the dilution gradient. After solidification, seal and invert, incubate overnight at 37℃. The next day, count the blue spots and calculate the concentration (number per μL). Step 4: Target Binding: Prepare three plates. Plate 1 contains BSA and NaHCO3; Plate 2 contains MST1, p53, and NaHCO3; Plate 3 contains MST1 and NaHCO3. First, add bacteriophage to Plate 1 and incubate at room temperature for 1 hour. Then, elute the unbound bacteriophages in Plate 1 and add them to Plate 2. After incubation at room temperature for 1 hour, elute the unbound bacteriophages in Plate 2 and add them to Plate 3. Incubate at room temperature for 1.5 hours. Repeat this process 3-5 times to obtain bacteriophages that can bind to MST1 and block the binding of MST1 to p53.Subsequently, using molecular docking, molecular simulation, and binding energy calculations, after screening and docking candidate peptides, three peptides (T1, T2, and T3) with good binding effects were obtained. The amino acid sequence of T1 is shown in SEQ ID NO:2 (CRGLFVR), the amino acid sequence of T2 is shown in SEQ ID NO:3 (VSCTSLC), and the amino acid sequence of T3 (same as ITS) is shown in SEQ ID NO:1 (ITSAGGD). See [link / reference needed]. Figure 1 .

[0041] Example 2 The effect of different peptide drugs at different concentrations on the viability of SH-SY5Y cells was detected using CCK-8 assay. The specific procedure was as follows: SH-SY5Y cell suspension was seeded into 96-well plates, 100 μl per well, and incubated for a period of time (37℃, 5% CO2). Seven gradient concentrations of peptide drugs (T1, T2, T3) of 0, 1, 5, 10, 20, 50, and 100 μM were added to each well of the plate, and incubation continued. Medium containing 10% CCK-8 was prepared and added to the wells via medium exchange. Air bubbles were avoided during sample addition to prevent affecting OD readings. After incubation and color development, the absorbance (OD) at 450 nm was measured using a microplate reader, and the viable cell rate was calculated. The experimental results showed that (…). Figure 2 (a), (b) and (c) in the text) The polypeptide drug ITS has no toxic effect on cells.

[0042] To evaluate the effect of different concentrations of peptide drugs on improving the survival of Aβ-induced SH-SY5Y cells, SH-SY5Y cell suspension was first seeded into 96-well plates, with 100 μL added to each well, and incubated for a period of time (37 ℃, 5% CO2). Aβ powder was prepared into a 40 μM Aβ solution using pure water. Eight treatment groups were set up for each peptide drug, with three replicates per group. One group without Aβ and peptide drugs served as a control. In the seven groups with Aβ, seven gradient concentrations of peptide drugs (T1, T2, and T3) of 0, 1, 5, 10, 20, 50, and 100 μM were added, respectively. After incubation for 12 hours, the absorbance (OD) at 450 nm was measured using a microplate reader, and the viable cell rate was calculated. The experimental results showed that (…). Figure 2 Of the (d), (e), and (f) results, the polypeptide drug T3 showed the most significant protective effect on cells.

[0043] Example 3 Immunofluorescence assays were performed at the cellular level to further validate the efficacy of the three candidate peptides and confirm the optimal peptide. Six-well plates containing SH-SY5Y cell samples were washed 2-3 times with pre-chilled PBS for 5 min each time. Fixed with 4% PFA at 37°C for 1 h. After removing the supernatant, excess formaldehyde was quenched with 0.1 M glycine dissolved in PBS for 5 min, followed by two more washes with chilled PBS for 5 min each. Cell membranes were permeated with 0.1% Triton × 100 for 0.5 h, followed by two washes with warm PBS for 5 min each time. Blocking was performed overnight with 1% goat serum. The gel slides were incubated overnight with MST1 primary antibody at 37°C. Washed twice with warm PBS at 37°C for 1 h each time. Incubated with goat anti-rabbit secondary antibody at 37°C for 4 h and wrapped in aluminum foil to prevent light exposure. Washed with chilled PBS for 1 h. Stained with DAPI for 2 min, followed by three washes with PBS for 5 min each time. Finally, anti-fluorescence quenching mounting solution was added. Images were acquired using confocal microscopy. Results of intracellular colocalization experiments of peptide and MST1 ( Figure 3 (a) shows that all three drugs can enter cells and bind to the MST1 protein, but the effect of T1 is not as significant as that of T2 and T3.

[0044] In SH The three peptides T1, T2, and T3 obtained through screening were validated in SY5Y cells. SH was collected. SY5Y cells were lysed on ice for 30 minutes with occasional agitation after adding pre-chilled non-denaturing lysis buffer. The cells were then centrifuged at 4°C (12000 rpm, 15 minutes) and the supernatant (total protein sample) was collected. A portion of the protein supernatant was collected and mixed with normal IgG and Protein A / G agarose beads of the same species as those used in subsequent experiments. The mixture was incubated at 4°C for 1-2 hours, and the supernatant was collected after centrifugation. The protein supernatant was divided into four aliquots: experimental group (containing specific antibodies against T1, T2, and T3 target proteins) and negative control group (containing normal IgG of the same species). The agarose beads were incubated overnight at 4°C to allow the antibodies to fully bind to the target proteins. Protein A / G agarose beads were added, and the mixture was incubated at 4°C for 2-4 hours. The antibody-protein complex will bind to the beads. The agarose beads were collected by centrifugation, and the supernatant (non-specific unbound protein) was carefully aspirated. The beads were washed 3-5 times with pre-chilled lysis buffer, each time at 4°C for 5-10 minutes, followed by centrifugation to collect the supernatant. The more thorough the washing, the cleaner the background. Add protein loading buffer (containing SDS and reducing agent) to the washed beads and heat at 95-100 °C for 5-10 minutes to denature the protein and dissociate it from the beads. Centrifuge and collect the supernatant for Western blotting. CO-IP experiments showed that, compared with the control group, T3 cells inhibited the binding of MST1 and p53, while T1 and T2 cells had no inhibitory effect.

[0045] Based on the above results, T3 (hereinafter referred to as ITS) was selected as the research object for subsequent experiments.

[0046] Example 4 To examine the expression of proteins closely related to ITS and synaptic function and cognitive ability.

[0047] In this study, Western blot was used to detect the expression levels of N-methyl-D-aspartate receptor subunit (2NR2B), postsynaptic density protein 95 (PSD95), and presynaptic membrane protein (SYP) in the hippocampus of mice in each group. Decreased levels of NR2B, PSD95, and SYP all impair synaptic function and activate apoptosis signals through pathways affecting calcium signaling, mitochondrial function, and oxidative stress, ultimately leading to neuronal death and impaired learning and memory abilities.

[0048] Six-month-old wild-type mice were divided into two groups: WT and WT+ITS. Age-matched 5×FAD model mice were divided into two groups: AD and AD+ITS, for a total of four groups, with eight mice in each group. The WT+ITS and AD+ITS groups were administered an ITS peptide inhibitor (5 mg / kg / 2 days) via tail vein injection for one month, while the WT and AD groups were simultaneously administered an equal volume of saline via tail vein injection. Behavioral tests were performed after the administration period, and samples were collected for subsequent experiments.

[0049] Mouse hippocampal tissue was collected, and proteins were extracted after tissue disruption for protein concentration determination. A 10% separating gel SDS-PAGE gel was prepared, loaded with the sample, and subjected to electrophoresis. After electrophoresis, the membrane was transferred at 100V for 90 min. After transfer, the PVDF membrane was removed, washed in TBST for 5 min, and then transferred to rapid blocking buffer for 20 min at room temperature. Based on the molecular weight of the target protein, the membrane was cut into appropriate bands, placed in an incubation chamber, and incubated overnight at 4°C with diluted primary antibody. β-actin was used as an internal control for Western blotting. The next day, the membrane was washed with TBST for 5 min × 5 times, and then incubated with diluted secondary antibody for 1 h at room temperature. After incubation, the membrane was washed with TBST for 7 min × 4 times. A developing solution was prepared, and the membrane was exposed and photographed.

[0050] The results are as follows Figure 4 As shown, after administration of peptide inhibitors via tail vein injection to model mice, the expression levels of NR2B, SYP, and PSD95 proteins in the hippocampus were all increased. Figure 4 (a)-(d)). The results showed that the peptide inhibitor ITS upregulated the expression of hippocampal synapse-related proteins in model animals and exhibited a good restorative effect under conditions of impaired synaptic function.

[0051] Example 5 The expression of apoptosis-related proteins that are closely related to ITS and neuronal apoptosis and cognitive ability was confirmed.

[0052] In this embodiment, Western blot was used to detect the expression of B-cell lymphoma-2 protein (Bcl-2), Bcl-2-associated X protein (Bax), and cleaved caspase 9. Cleaved caspase 9 is a key active protein that initiates apoptosis, leading to cell death by activating downstream caspases. In neurodegenerative diseases, it is associated with neuronal loss and cognitive decline. The anti-apoptotic protein Bcl-2 prevents caspase 9 activation by inhibiting the mitochondrial pathway. Decreased Bcl-2 expression relieves the inhibition of the pro-apoptotic protein Bax, thereby triggering mitochondrial dysfunction, the apoptotic cascade, and neuronal damage.

[0053] like Figure 5 As shown, tail vein injection of a peptide inhibitor into 5×FAD mice increased the Bcl-2 / Bax level in the hippocampus of mice. Figure 5 (a) and (b) in the text reduce the level of Cleaved Caspase 9. Figure 5 (a) and (c) in the figure). The results show that the peptide inhibitor ITS exhibits excellent recovery ability against apoptosis.

[0054] Example 6 Y-maze test.

[0055] The Y-maze consists of three identical arms (length × width × height: 35 cm × 10 cm × 15 cm) connected at a 120° angle, made of opaque gray plastic. Figure 6 (a) A camera was installed above the maze, connected to a computer and behavioral analysis software (such as Any-Maze) to track the mouse's movement in real time. The experimental environment was kept quiet with uniform lighting (50 lux), and odor neutralizers were used to eliminate residual odor interference if necessary. The mouse was placed from the center of the maze or a designated starting arm (such as Arm A) and allowed to explore freely for 5 minutes. The order, number of times, and duration of the mouse entering each arm were recorded. The mice were tested on day 31 after a 30-day follow-up intravenous injection of the drug.

[0056] The results are as follows Figure 6 As shown, the Y-maze test results indicate that, compared with the AD group, the spontaneous alternation rate of the AD+ITS group was significantly higher, and its level was similar to that of the WT group and the WT+ITS group. Figure 6 (b)). There was no significant difference in the number of arm-entry attempts among the groups of mice, indicating that the basic motor function and exploratory activity of each group were within the normal range, and interference from differences in physical strength or exploratory motivation on memory assessment could be ruled out. Figure 6 (c)). Therefore, the spontaneous alternation rate can serve as a specific indicator reflecting the function of spatial working memory.

[0057] Example 7 Experiments on the identification of novel objects ( Figure 7 (a) in the middle.

[0058] The experiment consisted of three phases: a training phase, a short-term memory test phase (Test 1), and a long-term memory test phase (Test 2). During the training phase, two identical objects were placed equidistantly at opposite ends of one arm of a box. The mice were placed facing the box wall and allowed to explore freely for 10 minutes, recording the time spent exploring each object. Two hours after the training phase ended, the short-term memory test (Test 1) was conducted. One object was replaced with a new object of a different shape, and the mice were allowed to explore freely for 5 minutes, recording the time spent exploring both the old and new objects. Twenty-four hours after the training phase ended, the long-term memory test (Test 2) was conducted. The new object was replaced with another new object of a different shape, and the mice were allowed to explore freely for 5 minutes, recording the time spent exploring both the old and new objects. The novelty object recognition index was calculated as: Recognition Index = (Time spent exploring the new object / Total time spent exploring both the old and new objects) × 100%. Mice were administered the drug via tail vein injection for 30 days, and the tests were conducted on days 32-33 following the same procedure.

[0059] The results are as follows Figure 7 As shown, the results of novel object exploration indicate that during the adaptation period (0 h), there was no difference in the exploration of new and old objects among the four groups of mice. Figure 7 (a) and (b) in the text). At 2h and 24h, compared to the AD group, treatment with the peptide inhibitor enhanced the ability to recognize and remember novel objects, indicating improved short-term and long-term learning and memory functions (especially recognition memory and exploratory preference). Figure 7 (c) and (d) in the text.

[0060] Example 8 Assess the spatial learning and memory abilities of mice.

[0061] Morris water maze test was conducted ( Figure 8(a) The training was conducted in a circular pool (90 cm in diameter, 60 cm in height) filled with opaque water at a temperature maintained between 20 and 22°C. A top-mounted camera connected to a computer and tracking software tracked the mice's movements. The pool was divided into four quadrants of equal area. During the initial training phase, a 10 cm diameter platform was located in the center of the northwest quadrant, submerged 1 cm below the water surface. The training phase lasted 5 days, starting randomly from four points (northeast, east, south, and southwest) each day. The time required for the mice to find the platform (escape latency) was recorded. In the spatial exploration test phase (day 6), the platform was removed, and the mice were allowed to explore the pool starting from the southeast quadrant. The test phase examined the time the mice spent in the target quadrant and the number of times they crossed the platform. Mice were administered the drug via tail vein injection for 30 days, and were trained using the same method from days 34 to 38. The platform was removed on day 39, and the test was conducted.

[0062] During the initial training phase, the escape latency of the AD+ITS group was shorter than that of the AD group, especially around days 3-5, approaching the escape latency of the WT and WT+ITS groups. Figure 8 (b)). The AD+ITS group had significantly better target quadrant dwell time and platform crossing times than the AD group ( Figure 8 (c) and (d) in the text. This indicates that peptide inhibitors can significantly improve the learning ability of AD mice.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. An isolated nucleic acid molecule, characterized in that, It encodes the polypeptide described in claim 1.

3. A recombinant vector, characterized in that, It contains the isolated nucleic acid molecules as described in claim 1.

4. A recombinant cell, characterized in that, It contains the recombinant vector as described in claim 3.

5. The method for preparing the polypeptide according to claim 1, characterized in that, It includes: The polypeptide of claim 1 is synthesized artificially or the recombinant cells of claim 4 are cultured.

6. A pharmaceutical composition, characterized in that, Its active ingredient includes the polypeptide described in claim 1.

7. The use of the polypeptide of claim 1, the isolated nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, or the preparation method of claim 5 in the preparation of a medicament for treating or improving a disease accompanied by at least one of neuronal dysfunction, memory impairment, and cognitive impairment.

8. The application according to claim 7, characterized in that, The disease in question is Alzheimer's disease.

9. The application of the polypeptide of claim 1, the isolated nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, or the preparation method of claim 5 in the preparation of food that helps improve memory.

10. The application of the polypeptide of claim 1, the isolated nucleic acid molecule of claim 2, the recombinant vector of claim 3, the recombinant cell of claim 4, or the preparation method of claim 5 in the preparation of health products that help improve memory.

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