Pharmaceutical composition for preventing / treating Alzheimer's disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a pharmaceutical composition for preventing / treating Alzheimer's disease. According to the pharmaceutical composition based on the NPAFP protein provided by the invention, the synergistic effect of natural and artificially synthesized compounds is utilized, so that the pharmaceutical composition can play a role in a plurality of pathological levels, and the AD symptom is remarkably improved. The composition has the remarkable effects of resisting oxidation, resisting inflammation, promoting A beta removal, improving mitochondrial quality, reducing A beta 42 secretion and pathological states caused by Tau PFF and the like, and can protect neurons by adjusting the metabolic process. By screening and optimizing the compound with synergistic effect, the action mechanism of the compound is further defined, a novel safe and efficient potential solution is provided for AD treatment, and the compound has wide clinical application prospect. The medicine is high in safety, strong in specificity, simple and convenient in preparation method and suitable for mass production.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a pharmaceutical composition for preventing and / or 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. It is one of the most common types of dementia, and is mainly manifested by memory loss, cognitive decline, and behavioral changes. The etiology of AD is complex, involving the combined effects of genetic and environmental factors. Its pathogenesis is not yet fully understood, but it is generally believed to be closely related to multiple pathological processes such as β-amyloid protein (Aβ) deposition, neurofibrillary tangles (NFTs), neuronal synaptic dysfunction, metabolic abnormalities, neuroinflammation, and oxidative stress. Among them, excessive production and clearance of β-amyloid protein (Aβ) lead to its deposition in the cerebral cortex and hippocampus to form plaques, leading to 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 proteins. In the brains of AD patients, tau proteins are overphosphorylated, causing them to lose their ability to bind to microtubules, thereby destroying the microtubule structure and causing neuronal dysfunction (Wang Jianzhi, Tian Qing. Mechanism of Tau protein overphosphorylation 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 increased year by year, causing great life troubles for patients and also causing huge economic pressure on the global medical system.

[0003] Currently, the treatments for AD mainly include two categories: drug therapy and non-drug therapy. 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 patients' cognitive function 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).); 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 inLow- and Middle-Income Countries." PLoS Medicine(2009).. Martin J. Prince, Daisy Acosta.); Drugs targeting Aβ pathology include monoclonal antibodies (such as aducanumab, solazumab, 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, diet conditioning 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-drug interventions have been shown 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] The latest research shows that some natural and synthetic compounds can act on the pathological process of AD through multiple biological pathways, exerting anti-inflammatory, antioxidant, and Aβ clearance-promoting effects. For example, natural compounds such as curcumin and resveratrol can reduce neuroinflammation and improve neurological 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 neuroregeneration-promoting 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 generation. In addition, studies on the gut-brain axis have also revealed that gut microbial metabolites (such as short-chain fatty acids) may further delay the course of AD by regulating neuroimmune responses and brain metabolism.

[0005] However, the pathological mechanism of AD is complex, and there are significant differences between different patients, resulting in different treatment effects. Existing treatment options generally have defects such as limited efficacy, single target, and obvious side effects, which make it difficult to fundamentally improve the symptoms of AD and delay the course of the disease (AE Abdallah. "Review on anti-alzheimer drug development: approaches, challenges and perspectives." RSC Advances (2024).). Therefore, there is an urgent need to develop innovative treatments with multi-target effects, higher efficacy and better safety.

[0006] In the previous study (“Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict”, Deng et al. PNAS, 2010.), it was first discovered and proved that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein AFPIII through neofunctionalization in the process of escaping adaptive conflict, and named it LdAFPII. This protein evolved from SAS-B and has multiple repeated functional domains (such as LdAFPIII-1 with one repeated functional domain, LdAFPIII-4 with four repeated functional domains, LdAFPIII-12 with twelve repeated functional domains, etc.), and each AFPIII domain contains 62 amino acids. The sequence of LdAFPII protein was published in the previous study (“Cloning and evolutionary analysis of the gene of the multimeric type III antifreeze protein 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. Alphafold3 was used to predict and optimize the protein structure. 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. In order 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 abilities. NPAFP1 contains one AFPIII domain, NPAFP4 contains four AFPIII domains, and NPAFP12 contains twelve AFPIII domains.

[0007] There are currently no studies on the use of NPAFP protein to treat Alzheimer's disease. Summary of the invention

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

[0009] In a first aspect, the present invention provides a pharmaceutical composition for preventing and / or treating Alzheimer's disease, the pharmaceutical composition containing NPAFP protein, 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.

[0010] 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 in SEQ ID NOs: 1-5; and their amino acid sequences are respectively shown in SEQ ID NOs: 6-10.

[0011] Furthermore, the NPAFP protein can enhance the ability to resist oxidative stress.

[0012] Furthermore, the NPAFP protein can improve cell survival rate.

[0013] Furthermore, the NPAFP protein can reduce ROS levels.

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

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

[0016] Furthermore, the pharmaceutical composition may also contain other active ingredients for preventing / treating Alzheimer's disease.

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

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

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

[0020] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

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

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

[0024] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

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

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

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

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

[0029] Furthermore, 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-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0030] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0031] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0032] Furthermore, the pharmaceutical composition can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0033] In a second aspect, the present invention provides the use of NPAFP protein in the preparation of a drug for preventing and / or treating 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 the NPAFP1, NPAFP2, NPAFP3, NPAFP4 and NPAFP12 proteins are respectively shown in SEQ ID NOs: 1-5; and their amino acid sequences are respectively shown in SEQ ID NOs: 6-10.

[0035] Furthermore, the NPAFP protein improves the ability to resist oxidative stress.

[0036] Furthermore, the NPAFP protein increases cell survival and / or reduces ROS levels.

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

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

[0039] Furthermore, the pharmaceutical composition may also contain other active ingredients for preventing / treating Alzheimer's disease.

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

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

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

[0043] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0044] 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.

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

[0046] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, 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, tristearin, cocoa butter and / or hydrogenated oil.

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

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

[0050] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

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

[0052] Furthermore, 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-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

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

[0054] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0055] Furthermore, the pharmaceutical composition can be introduced into the body by physical or chemical methods, such as intramuscularly, intradermally, subcutaneously or intravenously.

[0056] In a third aspect, the present invention provides a product for preventing and / or treating Alzheimer's disease, wherein the product contains 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 in SEQ ID NOs: 1-5; and their amino acid sequences are respectively shown in SEQ ID NOs: 6-10.

[0058] Furthermore, the NPAFP protein can enhance the ability to resist oxidative stress.

[0059] Furthermore, the NPAFP protein can improve cell survival rate.

[0060] Furthermore, the NPAFP protein can reduce ROS levels.

[0061] Furthermore, the NPAFP protein can reduce the secretion of Aβ42 in a subject.

[0062] Furthermore, the NPAFP protein can reduce the pathological conditions of Alzheimer's disease caused by Tau PFF.

[0063] Furthermore, the products include but are not limited to health products, functional foods or experimental reagents.

[0064] In a fourth aspect, the present invention provides a use of NPAFP protein in the preparation of a product for preventing and / or treating Alzheimer's disease, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0065] 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 in SEQ ID NOs: 1-5; and their amino acid sequences are respectively shown in SEQ ID NOs: 6-10.

[0066] Furthermore, the NPAFP protein can enhance the ability to resist oxidative stress.

[0067] Furthermore, the NPAFP protein can improve cell survival rate.

[0068] Furthermore, the NPAFP protein can reduce ROS levels.

[0069] Furthermore, the NPAFP protein can reduce the secretion of Aβ42 in a subject.

[0070] Furthermore, the NPAFP protein can reduce the pathological conditions of Alzheimer's disease caused by Tau PFF.

[0071] Furthermore, the products include but are not limited to health products, functional foods or experimental reagents.

[0072] In a fifth aspect, the present invention provides an application of NPAFP protein in constructing an anti-Alzheimer's disease biological model, wherein the NPAFP protein is selected from one or more of NPAFP1, NPAFP2, NPAFP3, NPAFP4, NPAFP5, NPAFP6, NPAFP7, NPAFP8, NPAFP9, NPAFP10, NPAFP11 and / or NPAFP12.

[0073] 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 in SEQ ID NOs: 1-5; and their amino acid sequences are respectively shown in SEQ ID NOs: 6-10.

[0074] Furthermore, the biological model includes but is not limited to a mammalian model and an aquatic biological model.

[0075] Furthermore, the NPAFP protein can enhance the ability to resist oxidative stress.

[0076] Furthermore, the NPAFP protein can improve cell survival rate.

[0077] Furthermore, the NPAFP protein can reduce ROS levels.

[0078] Furthermore, the NPAFP protein can reduce the secretion of Aβ42 in a subject.

[0079] Furthermore, the NPAFP protein can reduce the pathological conditions of Alzheimer's disease caused by Tau PFF.

[0080] Beneficial Effects The present invention proposes a pharmaceutical composition based on NPAFP protein, which utilizes the synergistic effect of natural and synthetic compounds to play a role at multiple pathological levels and significantly improve AD symptoms. The composition has significant antioxidant, anti-inflammatory, Aβ clearance promotion, mitochondrial quality improvement, reduction of Aβ42 secretion and pathological states caused by Tau PFF, and can protect neurons by regulating metabolic processes. By screening and optimizing compounds with synergistic effects and further clarifying their mechanism of action, a new safe and efficient potential solution is provided for the treatment of AD, which has broad clinical application prospects. The drug has high safety, strong specificity, simple preparation method, and can be mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 NPAFP protein improves the anti-oxidative stress ability of 293T cells.

[0082] Note: (A) Plasmid structure diagram of NPAFP gene; (B) CCK8 detection of cell survival rate; (C) Relative ROS content detection.

[0083] Figure 2 NPAFP protein improves mitochondrial mass in APP cells.

[0084] Note: (A) Mitochondrial TOM20 staining of wild-type SY5Y cells; (B) Mitochondrial TOM20 staining of cells transfected with NPAFP gene and empty vector plasmid; (C) Relative fluorescence intensity statistics after TOM20 staining.

[0085] Figure 3 NPAFP protein reduces Aβ secretion in APP cells.

[0086] Figure 4 NPAFP protein reduces the pathological state of Alzheimer's disease caused by Tau PFF. DETAILED DESCRIPTION

[0087] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0088] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0089] Explanation of terms SY5Y cells: A human neuroblastoma cell line derived from malignant neuroblastoma in the human brain. It has high proliferation and differentiation potential. It expresses a variety of receptors and proteins on the cell surface, such as TrkA, BDNF receptors, etc. These receptors play an important role in neuronal function and signal transduction. During cell differentiation, it can express neuronal markers (such as neurofilament protein, synaptophysin, etc.) and show certain neuroblastoma-like characteristics. It is widely used in neuroscience research.

[0090] 293T cells: 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 efficient protein expression and easy-to-manipulate genetic background. It is a mammalian cell line widely used in biomedical research, which is derived from human embryonic kidney cells and transformed by adenovirus vectors. This cell line is widely used because of its high transfection efficiency, easy culture, and flexibility in genetic manipulation.

[0091] DNA sequence of NPAFP DNA sequence of NPAFP1: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGA TGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA DNA sequence of NPAFP2: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA DNA sequence of NPAFP3: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAA DNA sequence of NPAFP4: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA DNA sequence of NPAFP12: Amino acid sequence of NPAFP Amino acid sequence of NPAFP1: MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE Amino acid sequence of NPAFP2: MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE Amino acid sequence of NPAFP3: MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVM MKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE Amino acid sequence of NPAFP4: MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLM PDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE Amino acid sequence of NPAFP12: MKSVVLTGLLFVLLCVDHMSSANKASVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAV QTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQARAVYLEQTLM PDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMV KNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYEDGTTCPGIKSVVANQLIPINTALTLVMMKAEEVSPKGIPAEEIPRIVGMQANRAVYLEQTLMPDMVKNYE Example 1 H2O2 treatment test NPAFP protein anti-oxidation ability 1.1 test method (1) Processing The constructed NPAFP expression plasmid was transferred into 293T cells using a chemical transfection reagent; after transfection, the cells were treated with hydrogen peroxide (H2O2), and fresh culture medium was replaced and culture was continued.

[0092] (2) CCK8 detection of cell survival rate The cells treated with H2O2 were replaced with fresh culture medium, and after continued culture, CCK8 reagent was added and the cells were placed in an incubator for further incubation; then, the OD value was read; the OD value treated with H2O2 was compared with the OD value not treated, and the survival rate was calculated.

[0093] (3) Detection of intracellular ROS levels 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 and washed to fully remove the CM-H2DCFDA that did not enter the cells; flow cytometry was used to detect with 495 nm excitation light and 530 nm emission light, and the ROS fluorescence intensity was counted.

[0094] 1.2 Test results like Figure 1 As shown in A, the Antarctic fish NPAFP protein is obtained by tandem duplication 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 to detect cell survival rate and ROS level. The results showed that the expression of NPAFP protein can significantly increase cell survival rate ( Figure 1 B) and reduce the level of ROS in cells ( Figure 1 C), indicating that NPAFP protein can improve the anti-oxidative stress ability of 293T cells, and the antioxidant capacity is proportional to the number of AFPIII domains. This finding supports the potential application value of NPAFP in anti-oxidative stress and prevention of AD.

[0095] Example 2 Immunofluorescence detection of mitochondrial mass 2.1 Test methods 1) SH-SY5Y / WT and cells stably expressing Swedish mutant APP (SH-SY5Y / APP695swe) were selected as AD models; 2) 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; 3) Fix the cells to preserve their structure; treat the cell membrane with Triton X-100 to allow antibodies to enter the cells; 4) Add primary antibody (anti-TOM20), incubate, and remove unbound primary antibody after washing; 5) Add secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L)), continue incubation, remove excess secondary antibody after washing, and seal the slide with anti-fluorescence attenuation agent; 6) Observe and record the expression of TOM20 in cells.

[0096] 2.2 Test results Wild-type cells (SH-SY5Y / WT) and cells stably expressing Swedish familial mutation of human APP695 (SHSY5Y / APP695swe) were used as cell models of Alzheimer's disease with endogenous high expression of APP.

[0097] The NPAFP protein expression plasmid was transfected into these two cells using chemical transfection reagents ( Figure 2 A and Figure 2 B), immunofluorescence staining of mitochondrial mass protein TOM20, the results showed that NPAFP protein can significantly improve the mitochondrial mass of APP cells, and the effect is similar to npafp The number of AFPIII domains in the gene was positively correlated ( Figure 2 This finding suggests the potential application value of NPAFP in the prevention of AD.

[0098] Example 3 Detection of Aβ42 secretion from APP cells 3.1 Test methods The secreted Aβ42 was detected by enzyme-linked immunosorbent assay (ELISA).

[0099] The constructed NPAFP expression plasmid was transferred into APP cells using a chemical transfection reagent, and the cell supernatant was collected. Aβ42 in the supernatant was quantitatively analyzed using an ELISA kit. The specific anti-Aβ42 capture antibody was first pre-coated on a microplate, and the sample was added after incubation, and Aβ42 will bind to the capture antibody. Next, the labeled detection antibody was added, and after the antibody binds to Aβ42, a color change is produced through a substrate reaction, and the absorbance value is measured and compared with the standard curve to quantify the content of Aβ42.

[0100] 3.2 Test results Aβ protein is secreted and released into the interstitial fluid under the environment of enhanced neuronal activity, aggregates to form oligomers and fibrils, and finally forms plaques, which is an important cause of Alzheimer's disease.

[0101] The NPAFP protein expression plasmid was transfected into APP695 cells (SHSY5Y / APP695swe) using a chemical transfection reagent, and the content of Aβ42 in the cytoplasm was detected.

[0102] like Figure 3 As shown in the results, 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 NPAFP protein has the potential to reduce Aβ accumulation and can provide new ideas for the treatment of AD.

[0103] Example 4: Transgenic Antarctic fish npafp Detection of pTau pathology in genetic mice 4.1 Test methods Twelve-week-old mice, 7 WT mice, and 10 experimental mice were selected for transgenic treatment.

[0104] The in vitro expressed tau PFFs were injected into WT (C57BL6) and transfected npafp, npafp4 and npafp12 Hippocampus of heterozygous mice. WT and transgenic mice (N = 7 and 10) were inoculated with tau PFFs in the hippocampus and pathological examination was performed at 3 months (3 mpi).

[0105] To confirm pathological transformation, hyperphosphorylated Tau (pTau) deposition was examined after a single inoculation of PFFs 3 mpi.

[0106] The whole brain of the mouse was fixed after perfusion and then dehydrated with sucrose solution. Immunohistochemistry (IHC) was performed on frozen brain sections. Primary antibody and working dilution: p-Tau 1:500, sections labeled with primary antibody were incubated with secondary antibody labeled with horseradish peroxidase (Invitrogen). Images were observed, photographed and recorded.

[0107] 4.2 Test results Abnormal aggregation of Tau protein is an important cause of Alzheimer's disease.

[0108] like Figure 4 As shown in A, Tau PFF protein was stereomicroinjected into the hippocampus of transgenic mice, and the pathology of the hippocampus was detected several months later.

[0109] like Figure 4As shown in Figure B, it was found that NPAFP protein can significantly reduce the pathological conditions of Alzheimer's disease caused by Tau PFF, and the inhibitory effect is similar to npafp The number of AFPIII domains in the gene was positively correlated. These results indicate that NPAFP protein has the potential to reduce the pathology caused by Tau PFFs and provide new ideas for AD treatment. The multi-target effect of NPAFP protein makes it a potential new candidate drug for AD treatment with broad clinical application prospects.

Claims

1. Use of NPAFP protein in the preparation of a drug for preventing and / or 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 in SEQ ID NOs: 1-5; and their amino acid sequences are respectively shown in SEQ ID NOs: 6-10.

2. The use of the NPAFP protein according to claim 1 in the preparation of a drug for preventing and / or treating Alzheimer's disease, wherein the functions of the NPAFP protein include at least one of the following: Improve the ability to resist oxidative stress; Improve cell survival rate; Reduce ROS levels; Reduce Aβ42 secretion in subjects; Reduces Alzheimer's disease pathology caused by Tau PFFs.

3. Use of the NPAFP protein as claimed in claim 1 in the preparation of a drug for preventing and / or treating Alzheimer's disease, wherein the dosage form of the pharmaceutical composition is selected from tablets, powders, granules, capsules, oral solutions, injection preparations or aerosols.

4. Use of NPAFP protein in the preparation of a product for preventing and / or 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 in SEQ ID NOs: 1-5; their amino acid sequences are respectively shown in SEQ ID NOs: 6-10; the product includes a health product, a functional food or an experimental reagent.

5. Use of the NPAFP protein according to claim 4 in the preparation of a product for preventing and / or treating Alzheimer's disease, wherein the functions of the NPAFP protein include at least one of the following: Improve the ability to resist oxidative stress; Improve cell survival rate; Reduce ROS levels; Reduce Aβ42 secretion in subjects; Reduces Alzheimer's disease pathology caused by Tau PFFs.

6. Application of NPAFP protein in constructing an anti-Alzheimer's disease biological model, 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 in SEQ ID NOs: 1-5; their amino acid sequences are respectively shown in SEQ ID NOs: 6-10; the biological model includes a mammalian model and an aquatic biological model.

7. The use of the NPAFP protein in constructing an anti-Alzheimer's disease biological model according to claim 6, wherein the functions of the NPAFP protein include at least one of the following: Improve the ability to resist oxidative stress; Improve cell survival rate; Reduce ROS levels; Reduce Aβ42 secretion in subjects; Reduces Alzheimer's disease pathology caused by Tau PFFs.

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