A pharmaceutical composition for treating Alzheimer's disease

Abstract

The invention belongs to the field of biomedical technology, and specifically relates to a pharmaceutical composition for preventing / treating Alzheimer's disease. The present invention proposes a pharmaceutical composition based on the NPAFP protein, which can play a role at multiple pathological levels by utilizing the synergistic effect of natural and synthetic compounds, and significantly improve the symptoms of AD. The composition has significant antioxidant, anti-inflammatory, promoting Aβ clearance, improving mitochondrial quality, reducing the pathological state caused by Aβ42 secretion and Tau PFF, etc., and can protect neurons by regulating the metabolic process. By screening and optimizing compounds with synergistic effects and further clarifying their mechanism of action, a new safe and efficient potential solution for the treatment of AD is provided, with broad clinical application prospects. The drug has high safety, strong specificity, a simple preparation method, and can be mass-produced.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical composition for treating Alzheimer's disease. Background Art

[0002] Alzheimer's Disease (AD) is a chronic neurodegenerative disease that mainly affects the cognitive function of the elderly and is one of the most common types of dementia, mainly manifested as memory loss, decline in cognitive function, and behavioral changes. The etiology of Alzheimer's disease is complex, involving the combined effects of genetic and environmental factors, and its pathogenesis has not been fully understood, but it is generally believed to be closely related to multiple pathological processes such as β-amyloid (Aβ) deposition, neurofibrillary tangles (NFTs), neuronal synaptic dysfunction, metabolic abnormalities, neuroinflammation, and oxidative stress. Among them, the overproduction and clearance disorder of β-amyloid (Aβ) lead to its deposition in the cerebral cortex and hippocampus to form plaques, resulting in neuronal dysfunction (Teeba Athar, K. Al Balushi et al. “Recent advances on drug development and emerging therapeutic agents for Alzheimer’s disease.” Molecular Biology Reports(2021).). Neurofibrillary tangles (NFTs) are caused by the aggregation of abnormally phosphorylated tau protein. In the brains of AD patients, tau protein is hyperphosphorylated, resulting in its loss of the ability to bind to microtubules, thereby destroying the microtubule structure and triggering neuronal dysfunction (Wang Jianzhi, Tian Qing. The mechanism of tau protein hyperphosphorylation and its role in neuronal degeneration in Alzheimer's disease [J]. Progress in Biochemistry and Biophysics, 2012.). With the intensification of global aging, the prevalence of AD has been increasing year by year, bringing great life troubles to patients and also causing huge economic pressure on the global medical system.

[0003] Currently, the treatment methods for AD mainly include two categories: drug treatment and non-drug treatment. Drug treatment mainly includes cholinesterase inhibitors, NMDA receptor antagonists, and anti-β-amyloid drugs: Cholinesterase inhibitors (such as donepezil, galantamine, rivastigmine, etc.) are one of the main drugs currently approved for the treatment of AD in clinical practice, which improve the cognitive function of patients by increasing the concentration of acetylcholine in the brain (R. Malik, Sunishtha Kalra et al. “Overview of therapeutic targets in management of dementia..” Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie(2022).); The NMDA receptor antagonist memantine is a low-affinity NMDA receptor antagonist that protects neurons by reducing excitotoxicity and is suitable for patients with moderate to severe AD (M. Prince, D. Acosta et al. “Packages of Care for Dementia in Low- and Middle-Income Countries.” PLoS Medicine(2009).. Martin J. Prince, Daisy Acosta.); Drugs targeting Aβ pathology include monoclonal antibodies (such as aducanumab, solanezumab, etc.), which delay disease progression by clearing Aβ plaques in the brain (Z. Fišar. “Linking the Amyloid, Tau, and Mitochondrial Hypotheses of Alzheimer’s Disease and Identifying Promising Drug Targets.” Biomolecules(2022).). Non-drug interventions include lifestyle interventions such as exercise training, dietary regulation, and sleep management; cognitive training, psychotherapy, and music and aromatherapy (Victoria García-Morales, Anabel Gónzalez-Acedo et al. “Current Understanding of the Physiopathology, Diagnosis and Therapeutic Approach to Alzheimer’s Disease.” Biomedicines(2021).).These non-pharmacological intervention methods have been proven to improve sleep, mood, and behavior in AD patients (M. Prince, D. Acosta et al. “Packages of Care for Dementia in Low- and Middle-Income Countries.” PLoS Medicine(2009).).

[0004] Recent research has shown that some natural and synthetic compounds can act on the pathological process of AD through multiple biological pathways, exerting anti-inflammatory, antioxidant, and promoting Aβ clearance effects. For example, natural compounds such as curcumin and resveratrol reduce neuroinflammation and improve nerve function by activating the AMPK pathway or inhibiting the NF-κB signaling pathway; rhodiola rosea and ginsenoside Rg1 show potential therapeutic effects through antioxidant, anti-inflammatory, and promoting nerve regeneration effects (Z. Fišar. “Linking the Amyloid, Tau, and Mitochondrial Hypotheses of Alzheimer’s Disease and Identifying Promising Drug Targets.” Biomolecules(2022).); polyphenols can inhibit Aβ aggregation by crossing the blood-brain barrier; and some synthetic compounds (such as BACE1 inhibitors and metal ion chelators) show potential in preventing Aβ production. In addition, research on the gut-brain axis has also revealed that gut microbial metabolites (such as short-chain fatty acids) may further delay the progression of AD by regulating neuroimmune responses and brain metabolism.

[0005] However, the pathological mechanism of AD is complex, with significant differences among different patients, resulting in varying treatment effects. Existing treatment regimens generally have limitations such as limited efficacy, single target, and obvious side effects, making it difficult to fundamentally improve the symptoms of AD and delay the disease progression (A. E. Abdallah. “Review on anti-alzheimer drug development: approaches, challenges and perspectives.” RSC Advances(2024).). Therefore, there is an urgent need to develop innovative treatment methods with multi-target effects, higher efficacy, and better safety.

[0006] In previous research ("Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict", Deng et al. PNAS, 2010.), it was first discovered and proven that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein AFPIII through neofunctionalization during the process of escaping adaptive conflict, and it was named LdAFPII. This protein evolved from SAS-B and there are repeats of multiple functional domains (such as LdAFPIII-1 with one repeat functional domain, LdAFPIII-4 with four repeat functional domains, LdAFPIII-12 with twelve repeat functional domains, etc.). Each AFPIII domain contains 62 amino acids. The sequence of the LdAFPII protein was disclosed in a previous study ("Cloning and evolutionary analysis of the polymeric type III antifreeze protein gene of Antarctic eel Pout (Lycodichthys dearborni)", Yu Jing et al., Acta Genetica Sinica, 2005, 32 (8): 789-794.). Each AFPIII domain contains 62 amino acids. Using alphafold3 for protein structure prediction and optimization, the amino acids at positions 36 and 37 were changed from KL to RI, and the amino acid at position 50 was changed from D to E to increase the flexibility of the protein structure. To explore its application in neurological diseases, it was named NeuroProtection AFPIII (such as NPAFP1, NPAFP4, NPAFP12, etc.). NPAFP proteins with different domains have different antifreeze capabilities. Among them, NPAFP1 contains one AFPIII domain, NPAFP4 contains four AFPIII domains, and NPAFP12 contains twelve AFPIII domains. HYPERLINK "https: / / doi.org / 10.1039 / d3ra08333k" \t "https: / / metaso.cn / search / _blank"

[0007] There is currently no research on the treatment of Alzheimer's disease with NPAFP proteins. HYPERLINK "https: / / doi.org / 10.3390 / biom12111676" \t "https: / / metaso.cn / search / _blank" Summary of the Invention

[0008] In the present application, it has been found that the NPAFP protein can improve the pathological characteristics of Alzheimer's disease through multiple mechanisms such as antioxidant, improving mitochondrial quality, reducing Aβ42 secretion, and Tau PFF, and has the effect of treating Alzheimer's disease. Based on this, the present invention has been completed.

[0009] In a first aspect, the present invention provides a pharmaceutical composition for treating Alzheimer's disease, the pharmaceutical composition containing the NPAFP protein, and the NPAFP protein being selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11, and / or NPAFP12.

[0010] Further, the NPAFP protein is preferably one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are respectively as shown in SEQ ID NO: 1-5; and their amino acid sequences are respectively as shown in SEQ ID NO: 6-10.

[0011] Further, the NPAFP protein can improve the antioxidant stress ability.

[0012] Even further, the NPAFP protein can improve cell viability.

[0013] Even further, the NPAFP protein can reduce the ROS level.

[0014] Further, the NPAFP protein can reduce the Aβ42 secretion of a subject.

[0015] Further, the NPAFP protein can reduce the pathological conditions of Alzheimer's disease caused by Tau PFF.

[0016] Further, the pharmaceutical composition may further contain other active ingredients for treating Alzheimer's disease.

[0017] Further, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0018] Further, the pharmaceutical composition can be made into various forms such as tablets, powders, granules, capsules, oral liquids, injectable preparations, or aerosols; the drugs in the above various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

[0019] Further, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0020] Further, the tablets can widely use various carriers well-known in the art, including one or more of a diluent and absorbent, a wetting agent and binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, and / or a lubricant.

[0021] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0022] Further, the wetting agent and binder include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and / or polyvinylpyrrolidone.

[0023] Further, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, laminarin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, and / or ethyl cellulose.

[0024] Further, the disintegration inhibitor includes, but is not limited to, sucrose, glyceryl tristearate, cocoa butter, and / or hydrogenated oil, etc.

[0025] Further, the absorption enhancer includes, but is not limited to, one or more of quaternary ammonium salts and / or sodium dodecyl sulfate.

[0026] Further, the lubricant includes, but is not limited to, one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0027] Further, the tablets can also be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets, and multi-layer tablets.

[0028] Further, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a freeze-dried powder injection, and / or a suspension.

[0029] Furthermore, the injectable preparation can use all common diluents in the art, including, but not limited to, one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, and / or polyoxyethylene sorbitan fatty acid ester.

[0030] Furthermore, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose, glycerol, conventional solubilizers, buffers and / or pH regulators can be added to the injectable preparation.

[0031] Furthermore, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

[0032] Furthermore, the pharmaceutical composition can be introduced into the body by physically or chemically mediated methods such as intramuscular, intradermal, subcutaneous or intravenous.

[0033] In a second aspect, the present invention provides the use of NPAFP protein in the preparation of a drug for the treatment of Alzheimer's disease; the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0034] Furthermore, the NPAFP protein is preferably one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and / or NPAFP12 proteins; the nucleotide sequences of NPAFP1, NPAFP2, NPAFP3, NPAFP4 and NPAFP12 proteins are shown in SEQ ID NO: 1-5 respectively; their amino acid sequences are shown in SEQ ID NO: 6-10 respectively.

[0035] Furthermore, the NPAFP protein enhances the antioxidant stress ability.

[0036] Even further, the NPAFP protein increases cell viability and / or reduces the ROS level.

[0037] Furthermore, the NPAFP protein reduces the secretion of Aβ42 in the subject.

[0038] Furthermore, the NPAFP protein reduces the pathological conditions of Alzheimer's disease caused by Tau PFF.

[0039] Furthermore, the pharmaceutical composition can also contain other active ingredients for the treatment of Alzheimer's disease.

[0040] Furthermore, one or more pharmaceutically acceptable carriers can also be added to the pharmaceutical composition.

[0041] Furthermore, the pharmaceutical composition can be made into various forms such as tablets, powders, granules, capsules, oral liquids, injectable preparations or aerosols, etc.; the drugs of the above various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

[0042] Furthermore, the preparation may be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation, and / or various particulate drug delivery systems.

[0043] Furthermore, the tablets may widely use various carriers well-known in the art, including one or more of a diluent and absorbent, a wetting agent and binder, a disintegrant, a disintegration inhibitor, an absorption enhancer, and / or a lubricant.

[0044] Still further, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0045] Furthermore, the wetting agent and binder include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and / or polyvinylpyrrolidone.

[0046] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, laminarin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose, and / or ethyl cellulose.

[0047] Furthermore, the disintegration inhibitor includes, but is not limited to, sucrose, glyceryl tristearate, cocoa butter, and / or hydrogenated oil, etc.

[0048] Furthermore, the absorption enhancer includes, but is not limited to, one or more of quaternary ammonium salts and / or sodium dodecyl sulfate.

[0049] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silica, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0050] Furthermore, the tablets may further be made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, bilayer tablets, and multilayer tablets.

[0051] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder for injection, and / or a suspension.

[0052] Still further, the injectable preparation may use all diluents commonly used in the art, including, but not limited to, one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, and / or polyoxyethylene sorbitan fatty acid ester.

[0053] Furthermore, in order to prepare an isotonic injection solution for the injectable preparation, an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional solubilizers, buffers, and / or pH regulators may be added to the injectable preparation.

[0054] Furthermore, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners, or other materials may also be added to the pharmaceutical preparation for various preparations.

[0055] Furthermore, the pharmaceutical composition can be introduced into the body, such as muscle, intradermal, subcutaneous, or intravenous, by physical or chemically mediated methods.

[0056] In a third aspect, the present invention provides a product for enhancing antioxidant stress ability, the product containing NPAFP protein; the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11, and / or NPAFP12.

[0057] Furthermore, the NPAFP protein is preferably one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are respectively shown as SEQ ID NO: 1-5; their amino acid sequences are respectively shown as SEQ ID NO: 6-10.

[0058] Furthermore, the product includes health products.

[0059] In a fourth aspect, the present invention provides the use of NPAFP protein in the preparation of a product for enhancing antioxidant stress ability, the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11, and / or NPAFP12.

[0060] Furthermore, the NPAFP protein is preferably one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are respectively shown as SEQ ID NO: 1-5; their amino acid sequences are respectively shown as SEQ ID NO: 6-10.

[0061] Further, the product includes health care products.

[0062] In a fifth aspect, the present invention provides the use of NPAFP protein in constructing a biological model for anti-Alzheimer's disease, and the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11, and / or NPAFP12.

[0063] Further, the NPAFP protein is preferably one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are shown as SEQ ID NO: 1-5 respectively; and their amino acid sequences are shown as SEQ ID NO: 6-10 respectively.

[0064] Further, the biological model includes but is not limited to mammalian models and aquatic biological models.

[0065] Further, the NPAFP protein can improve the antioxidant stress ability.

[0066] Furthermore, the NPAFP protein can improve cell viability.

[0067] Furthermore, the NPAFP protein can reduce the ROS level.

[0068] Further, the NPAFP protein can reduce the Aβ42 secretion of the subject.

[0069] Further, the NPAFP protein can reduce the pathological conditions of Alzheimer's disease caused by Tau PFF.

[0070] Beneficial effects

[0071] The present invention provides a drug composition based on NPAFP protein. By utilizing the synergistic effect of natural and synthetic compounds, it can act on multiple pathological levels and significantly improve AD symptoms. This composition has significant antioxidant, anti-inflammatory, promoting Aβ clearance, improving mitochondrial quality, reducing Aβ42 secretion, and reducing the pathological state caused by Tau PFF, etc., and can protect neurons by regulating the metabolic process. By screening and optimizing compounds with synergistic effects and further clarifying its mechanism of action, it provides a new safe and efficient potential solution for the treatment of AD and has broad clinical application prospects. This drug has high safety, strong specificity, a simple preparation method, and can be mass-produced. Brief description of the drawings

[0072] Figure 1 The NPAFP protein enhances the antioxidant stress tolerance of 293T cells.

[0073] Note: (A) Plasmid structure diagram of the NPAFP gene; (B) CCK8 assay for cell viability; (C) Detection of relative ROS content.

[0074] Figure 2 The NPAFP protein improves the mitochondrial mass of APP cells.

[0075] Note: (A) Mitochondrial TOM20 staining of wild-type SY5Y cells; (B) Mitochondrial TOM20 staining of cells transfected with the NPAFP gene and empty vector plasmids; (C) Statistical analysis of relative fluorescence intensity after TOM20 staining.

[0076] Figure 3 The NPAFP protein reduces Aβ secretion in APP cells.

[0077] Figure 4 The NPAFP protein alleviates the pathological state of Alzheimer's disease caused by Tau PFF. Specific implementation manners

[0078] The following further describes the specific implementation manners of the present invention. It should be noted here that the description of these implementation manners is for helping to understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the following described implementation manners can be combined with each other as long as they do not conflict with each other.

[0079] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0080] Term explanation

[0081] SY5Y cells: A human neuroblastoma cell line derived from human malignant neuroblastoma of the brain, with high proliferative ability and differentiation potential. The cell surface expresses various receptors and proteins, such as TrkA, BDNF receptor, etc. These receptors play important roles in neuronal function and signal transduction; during cell differentiation, neuronal markers (such as neurofilament protein, synaptophysin, etc.) can be expressed, and certain neuroblastoma-like characteristics can be shown, which are widely used in neuroscience research.

[0082] 293T cells: They are derived from the 293 cell line (HEK293), which was established by introducing the SV40 T antigen gene into human embryonic kidney cells. 293T cells retain many characteristics of the 293 cell line, including high-efficiency protein expression ability and an easily manipulable genetic background. It is a mammalian cell line widely used in biomedical research. It is derived from human embryonic kidney cells and transformed by an adenovirus vector. This cell line is widely used because of its high transfection efficiency, easy cultivation, and flexibility in genetic manipulation.

[0083] DNA sequence of NPAFP

[0084] DNA sequence of NPAFP1:

[0085] ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA

[0086] DNA sequence of NPAFP2:

[0087] ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA

[0088] DNA sequence of NPAFP3:

[0089] ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAA

[0090] DNA sequence of NPAFP4:

[0091] ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA

[0092] DNA sequence of NPAFP12:

[0093]

[0094] Amino acid sequence of NPAFP

[0095] Amino acid sequence of NPAFP1:

[0096] MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE

[0097] Amino acid sequence of NPAFP2:

[0098] MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE

[0099] Amino acid sequence of NPAFP3:

[0100] MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE

[0101] Amino acid sequence of NPAFP4:

[0102] MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE

[0103] Amino acid sequence of NPAFP12:

[0104] MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE

[0105] Example 1 Detection of the antioxidant capacity of NPAFP protein by H2O2 treatment

[0106] 1.1 Test method

[0107] Pretreatment

[0108] The constructed NPAFP expression plasmid was transfected into 293T cells using a chemical transfection reagent; after transfection, the cells were treated with hydrogen peroxide (H2O2), and then the fresh medium was replaced and the cells were cultured continuously.

[0109] CCK8 was used to detect the cell viability.

[0110] After replacing the cells treated with H2O2 with fresh medium and continuing the culture, CCK8 reagent was added and the cells were incubated in an incubator; then, the OD value was read; the OD value of the cells treated with H2O2 was ratio-processed with the OD value of the untreated cells, and the survival rate was statistically analyzed.

[0111] Detection of intracellular ROS level

[0112] CM-H2DCFDA was diluted with serum-free culture medium to a final concentration of 5 μmol / L. After the cells were collected, they were suspended in the diluted CM-H2DCFDA for incubation; mixed well and washed to fully remove the CM-H2DCFDA that did not enter the cells; flow cytometry was used for detection with an excitation light of 495 nm and an emission light of 530 nm, and the ROS fluorescence intensity was statistically analyzed.

[0113] 1.2 Experimental results

[0114] As Figure 1 shown in A of Figure 1 the Antarctic fish NPAFP protein is obtained by tandem repeats of different numbers of AFPIII domains. NPAFP1 contains one AFPIII domain, NPAFP2 contains two AFPIII domains, NPAFP3 contains three AFPIII domains, NPAFP4 contains four AFPIII domains, and NPAFP12 contains twelve AFPIII domains. The NPAFP protein expression plasmid was transfected into 293T cells using a chemical transfection reagent and treated with H2O2, and the cell viability and ROS level were detected. The results showed that the expression of NPAFP protein could significantly increase the cell viability ( Figure 1 shown in B of Figure 1 ), and reduce the ROS level in the cells ( Figure 1 shown in C of

[0115] ), indicating that the NPAFP protein can improve the antioxidant stress ability of 293T cells, and the antioxidant ability is proportional to the number of AFPIII domains. This finding supports the potential application value of NPAFP in antioxidant stress and the prevention of AD. Example 2 Immunofluorescence detection of mitochondrial mass

[0116] 2.1 Experimental method

[0117] SH-SY5Y / WT cells and cells stably expressing the Swedish mutant APP (SH-SY5Y / APP695swe) were selected as AD models;

[0118] The expression plasmid of the NPAFP gene was transfected into WT (SY5Y) and APP (SH-SY5Y / APP695swe) cells. After transfection, the cells were subjected to immunofluorescence staining;

[0119] The cells were fixed to preserve their structure; the cell membrane was treated with Triton X-100 to allow antibodies to enter the cells;

[0120] The primary antibody (anti-TOM20) was added, incubated, and unbound primary antibody was removed by washing;

[0121] The secondary antibody (Alexa Fluor 594-conjugated goat anti-rabbit IgG(H+L)) was added, incubated further, and excess secondary antibody was removed by washing, and the coverslip was sealed with an anti-fluorescence quencher;

[0122] The expression of TOM20 in the cells was observed and recorded.

[0123] 2.2 Test Results

[0124] Wild-type cells (SH-SY5Y / WT) and cells stably expressing human APP695 with the Swedish familial mutation (SHSY5Y / APP695swe) were used as Alzheimer's disease cell models with high endogenous expression of APP.

[0125] The NPAFP protein expression plasmid was transfected into these two types of cells using a chemical transfection reagent ( Figure 2 A in Figure 2 and Figure 2 B in

[0126] ). Immunofluorescence staining of the mitochondrial mass protein TOM20 showed that the NPAFP protein could significantly increase the mitochondrial mass of APP cells, and the effect was positively correlated with the number of AFPIII domains in the npafp gene (

[0127] C in

[0128] ). This finding indicates the potential application value of NPAFP in preventing AD.

[0129] Example 3 Detection of Aβ42 Secretion in APP Cells

[0127] 3.1 Test Method

[0128] The detection of Aβ42 secreted by cells was performed by enzyme-linked immunosorbent assay (ELISA).

[0129] The constructed NPAFP expression plasmid was transfected into APP cells using a chemical transfection reagent, and the cell supernatant was collected. An ELISA kit was used to quantitatively analyze Aβ42 in the supernatant. First, a capture antibody specific for Aβ42 was pre-coated on a microplate. After incubation, the sample was added, and Aβ42 would bind to the capture antibody. Then, a labeled detection antibody was added. After this antibody bound to Aβ42, a color change was produced through a substrate reaction. The absorbance value was measured and compared with the standard curve to quantify the content of Aβ42.

[0130] 3.2 Test Results

[0131] The secretion of Aβ protein into the interstitial fluid of cells and its aggregation to form oligomers, protofibrils, and ultimately plaques in an environment of enhanced neuronal activity is an important cause of Alzheimer's disease.

[0132] The NPAFP protein expression plasmid was transfected into cells of APP695 (SHSY5Y / APP695swe) using a chemical transfection reagent, and the content of Aβ42 in the cytoplasm was detected.

[0133] As Figure 3 shown, the NPAFP protein can significantly reduce the secretion of Aβ42 in APP695 cells, and the inhibitory effect is positively correlated with the number of AFPIII domains in the npafp gene. These results indicate that the NPAFP protein has the potential to reduce Aβ accumulation and can provide new ideas for the treatment of AD.

[0134] Example 4 Detection of pTau Pathology in Mice Transgenic for Antarctic Fish npafp Gene

[0135] 4.1 Test Methods

[0136] Twelve-week-old mice, 7 wild-type (WT) mice and 10 experimental mice, were selected for transgenic treatment.

[0137] In vitro-expressed tau PFF was injected into the hippocampi of WT (C57BL6) and heterozygous mice transgenic for npafp, npafp4, and npafp12 via stereotaxic injection. The hippocampal region was inoculated with tau PFF, and pathological examinations were performed on WT and transgenic mice (N = 7 and 10) at 3 months post-inoculation (3 mpi).

[0138] To confirm the pathological transformation, hyperphosphorylated Tau (pTau) deposition was detected at 3 mpi after a single inoculation with PFF.

[0139] The whole brain of the mouse was fixed after perfusion and then dehydrated with a sucrose solution. Immunohistochemistry (IHC) was performed on cryosections of the brain. Primary antibody and working dilution: p-Tau 1:500. The sections labeled with the primary antibody were incubated with a secondary antibody labeled with horseradish peroxidase (Invitrogen). Images were observed, photographed, and recorded.

[0140] 4.2 Test Results

[0141] The abnormal aggregation of Tau protein is an important cause of Alzheimer's disease.

[0142] As Figure 4 shown in A of [], the Tau PFF protein was stereotactically microinjected into the hippocampal region of transgenic mice, and the pathology of the hippocampal region was detected several months later.

[0143] As Figure 4 shown in B of [], it was found that the NPAFP protein could significantly reduce the pathology of Alzheimer's disease caused by Tau PFF, and the inhibitory effect was positively correlated with the number of AFPIII domains in the npafp gene. These results indicate that the NPAFP protein has the potential to reduce the pathology caused by Tau PFF and can provide new ideas for the treatment of AD. The multi-target effect of the NPAFP protein makes it a potential new candidate drug for the treatment of AD, with broad clinical application prospects.

Claims

1. Use of NPAFP protein in the preparation of a medicament for treating Alzheimer's disease, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are respectively shown as SEQ ID NO: 1-5; and their amino acid sequences are respectively shown as SEQ ID NO: 6-10.

2. Use of the NPAFP protein as claimed in claim 1 in the preparation of a medicament for treating Alzheimer's disease, wherein the functions of the NPAFP protein include at least one of the following: Improving antioxidant stress capacity; Increasing cell viability; Reducing ROS levels; Reducing Aβ42 secretion in a subject; Reducing the pathological conditions of Alzheimer's disease caused by Tau PFF.

3. Use of the NPAFP protein as claimed in claim 1 in the preparation of a medicament for treating Alzheimer's disease, wherein the dosage form of the medicament is selected from tablets, powders, granules, capsules, oral liquids, injectable preparations, or aerosols.

4. Use of NPAFP protein in the preparation of a product for improving antioxidant stress capacity, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are respectively shown as SEQ ID NO: 1-5; and their amino acid sequences are respectively shown as SEQ ID NO: 6-10; and the product includes health products.

5. Use of NPAFP protein in the construction of a biological model for anti-Alzheimer's disease, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, and / or NPAFP12; the nucleotide sequences of the NPAFP1, NPAFP2, NPAFP3, NPAFP4, and NPAFP12 proteins are respectively shown as SEQ ID NO: 1-5; and their amino acid sequences are respectively shown as SEQ ID NO: 6-10; and the biological model includes mammalian models and aquatic biological models.

6. Use of the NPAFP protein as claimed in claim 5 in the construction of a biological model for anti-Alzheimer's disease, wherein the functions of the NPAFP protein include at least one of the following: Improving antioxidant stress capacity; Increasing cell viability; Reducing ROS levels; Reducing Aβ42 secretion in a subject; Reducing the pathological conditions of Alzheimer's disease caused by Tau PFF.

Images