WO2023186062A1 - Use of peptide in treating neurodegenerative disease or ameliorating cognitive function - Google Patents

Abstract

The present invention relates to use of a peptide in treating a neurodegenerative disease or ameliorating a cognitive function. The present invention relates to the treatment of neurodegenerative diseases and cognitive disorders, and the amelioration and improvement of cognitive functions. In particular, the present invention relates to use of a peptide in preventing or treating a neurodegenerative disease or a cognitive disorder, preferably Alzheimer's disease and Parkinson's disease. The present invention further relates to use of a peptide in ameliorating or improving a cognitive function in a subject.

Description

Use of peptides to treat neurodegenerative diseases or improve cognitive function Field of invention

The present invention relates to the treatment of neurodegenerative diseases and cognitive impairment; and the improvement and enhancement of cognitive function. In particular, the present invention relates to the use of peptides for the prevention or treatment of neurodegenerative diseases and cognitive disorders, preferably Alzheimer's disease and Parkinson's disease. The invention also relates to the use of peptides for improving or enhancing cognitive function in a subject.

Background technique

Neurodegenerative disease or neurodegenerative disease (Neurodegenerative Disorder or Neurodegenerative Disease) is a disease directly caused by the gradual degeneration of neurons. Degenerative processes may involve progressive loss of neuronal structure, progressive loss of neuronal function, or progressive neuronal cell death. This progressive neurodegeneration often leads to physical disability and mental deterioration. Many neurodegenerative diseases are severe, progressive and ongoing diseases with few treatments. Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most important neurodegenerative diseases.

Neurocognitive Disorder, also known as Dementia, is a type of brain disease that causes long-term and gradual deterioration of thinking ability and memory, and affects a person's daily life activities. The most common type of dementia is Alzheimer's disease, which accounts for 50 to 70 percent of all dementia patients. Dementia affects 36 million people worldwide. About 10% of the population will develop the disease during their lifetime. Dementia is closely related to age (aging), with approximately 3% of the population developing dementia between the ages of 65 and 74, another 19% between the ages of 75 and 84, and nearly half of the population over the age of 85 developing dementia. .

Alzheimer's disease (AD) is a chronic neurodegenerative disease that develops slowly and worsens over time. The most common early symptom is loss of short-term memory. As the disease progresses, symptoms may gradually appear, including language difficulties, disorientation, mood swings, loss of motivation, inability to care for oneself, and many behavioral problems. When the condition worsens, patients often become disconnected from family or society, gradually lose physical function, and eventually die. Although the course of the disease varies from person to person, the average life expectancy after diagnosis is about three to nine years. In AD, the hippocampus is the first area to be damaged, with symptoms such as memory decline and loss of directional awareness (see Yangling Mu et al., Adult hippocampal neurogenesis and its role in Alzheimer's disease, Mol Neurodegener, 2011; 6 :85). The progression of AD is related to the deposition of beta-amyloid (Aβ) plaques and tau protein in the brain (see Clive Ballard et al., Alzheimer's disease, The Lancet. 2011 March, 377(9770):1019–1031). Recent studies have found that neuroinflammation or microglial activation is involved in the spread of tau tangles in the neocortex in Alzheimer's disease, which in turn leads to the occurrence of cognitive dysfunction in Alzheimer's disease patients (see Tharick A .Pascoal et al., Microglial activation and tau propagate jointly across Braak stages, Nature Medicine, 27, pages 1592–1599 (2021 August)). Microglial activation is part of the immune response in the human brain and is a key factor associated with the development of Alzheimer's disease. Excessive activation of microglia is not only an epiphenomenon of inflammation, but also a key upstream mechanism that is crucial to the development of AD. Research has also found that neuroinflammation and certain inflammatory cytokines are involved in the pathology of AD (see Rishika Dhapola et al., Recent advances in molecular pathways and therapeutic implications targeting neuroinflammation for Alzheimer's disease, Inflammopharmacology volume 29, pages1669–1681 (2021November)) . Therefore, reducing Aβ plaque deposition, alleviating neuroinflammation, and inhibiting microglia activation can be regarded as effective means to treat or alleviate AD.

Parkinson's disease (PD) is a chronic neurodegenerative disease affecting the central nervous system, mainly affecting the motor nervous system. Its symptoms usually appear slowly over time. The most obvious early symptoms are tremor, limb stiffness, reduced motor function and abnormal gait. Cognitive and behavioral problems may also be present. Dementia is quite common in severely ill patients. The main pathological changes of Parkinson's disease occur in the ventral substantia nigra pars compacta of the midbrain, and most of the main symptoms are due to the degeneration of dopamine neurons in the substantia nigra pars compacta (Jose A Obeso et al., Functional organization of the basal ganglia:therapeutic implications for Parkinson's disease, Movement Disorders, Volume 23, Issue S3 p.S548-S559, Sept. 2008). Selegiline is a monoamine oxidase (MAO)-B inhibitor (MAOI). In 2006, the US FDA approved selegiline for the treatment of PD patients.

There is a need in the art for drugs that are effective in treating neurodegenerative diseases or cognitive impairment caused by neurodegenerative diseases.

WO2013/173941 and CN104321337A disclose an analgesic peptide with 11 to 14 amino acid residues, which matches the fragment of rabbit α1-antiprotease through sequence alignment. WO2016/165101 discloses that the peptide can effectively inhibit HCV replication. In addition, WO2016/165102 and CN107847550A disclosed that the peptide is used to treat stroke (an acute cerebrovascular disease), verified that the peptide can pass through the brain-blood barrier, and verified the effect of the peptide on PC12 cells through in vitro cell experiments. Protection from glutamate- and hydrogen peroxide-induced cytotoxicity. These documents are incorporated by reference into this article. The PC12 cells tested in WO2016/165102 are a type of pheochromocytoma derived from rat adrenal medulla that exhibit certain characteristics of ganglion cells and are particularly useful in establishing a model of cellular hypoxic injury, thereby simulating acute cerebrovascular disease. rapid hypoxic and toxic states. However, Alzheimer's disease and Parkinson's disease are chronic diseases that are not caused by acute cerebral hypoxia and toxicity caused by cerebral blood vessel embolism or rupture. The prior art does not mention the possibility of using said peptides to treat AD or PD.

Contents of the invention

The object of the present invention includes providing drugs for treating neurodegenerative diseases and cognitive impairment, as well as providing active ingredients or compositions for improving or enhancing the cognitive function of subjects. It is also an object of the present invention to provide medicaments for the treatment of Alzheimer's disease and Parkinson's disease.

One of the technical problems of the present invention is solved by providing the peptides of the present invention and compositions comprising said peptides.

The present invention relates to a peptide having the amino acid sequence of SEQ ID NO: 1 or 2 or a variant or fragment thereof. "Variant" and "fragment" mean a peptide that has certain amino acid residue changes compared to the peptide of the invention and substantially retains the same or similar biological function or activity as the peptide. A variant of an amino acid sequence may be a variant of an amino acid sequence that has at least a certain percent identity with the amino acid sequence, or a variant of an amino acid sequence that has at least one or more amino acid mutations compared with the amino acid sequence. For example, the variant may be at least 70% identical to the amino acid sequence of SEQ ID NO: 1 or 2. Alternatively, the variant may have 0 to 4 amino acid mutations in the amino acid sequence of SEQ ID NO: 1 or 2. A fragment of an amino acid sequence may be an amino acid sequence having one or more deletions at the C-terminus and/or N-terminus compared to the amino acid sequence. The deletions may be 1-5, such as 1, 2, 3, 4 or 5. Fragments of the amino acid sequence may be 8 to 20 amino acids in length, such as 10 to 15 amino acids, such as 10, 11, 12, 13, 14 or 15 amino acids.

In one aspect, the peptide of the invention is selected from:

a) A peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSH (SEQ ID NO: 1);

b) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1);

c) A peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or

d) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).

In one aspect, the peptides of the invention comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 sexual amino acid sequence.

In one aspect, the peptide of the invention is comprised in or has 0 to 4 amino acid mutations relative to SEQ ID NO: 1, such as 0 to 3, such as 1 to 3, such as 0, 1, 2, Amino acid sequence with 3 or 4 amino acid mutations.

In one aspect, the peptides of the invention comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2 sexual amino acid sequence.

In one aspect, the peptide of the invention is comprised in or has 0 to 4 amino acid mutations relative to SEQ ID NO: 2, such as 0 to 3, such as 1 to 3, such as 0, 1, 2, Amino acid sequence with 3 or 4 amino acid mutations.

Amino acid mutations can be selected from additions, deletions or substitutions of amino acids. The addition can be the insertion of an amino acid between two amino acid residues in the amino acid sequence, or the addition of an amino acid at the C-terminus or N-terminus of the amino acid sequence. The deletion can be the deletion of an amino acid between two amino acid residues in the amino acid sequence, or the deletion of the C-terminus or N-terminus of the amino acid sequence. The substitution may be of any amino acid residue in the amino acid sequence. Preferably, the addition, deletion or substitution of amino acids occurs at the C-terminus or N-terminus of the sequence. Substitutions may be conservative substitutions. Conservative substitution refers to the substitution between amino acid molecules with similar properties, including the ionicity, hydrophobicity and molecular weight of the molecule. Amino acids with similar properties can be classified as follows: aliphatic amino acids (such as glycine, alanine, valine, leucine, isoleucine); hydroxyl-containing or sulfur-containing amino acids (such as serine, cysteine, threonine). amino acids, methionine); cyclic amino acids (such as proline); aromatic amino acids (such as phenylalanine, tyrosine, tryptophan); basic amino acids (such as histidine, lysine) , arginine) and acidic or amide amino acids (such as aspartic acid, glutamic acid, asparagine, glutamine).

Those skilled in the art understand that the twenty common amino acids that constitute peptides, polypeptides or proteins and their abbreviations are shown in Table 1 below. The skilled person can determine the amino acid residue sequence and composition of the peptide of the present invention, for example, based on Table 1 below or common knowledge.

Table 1. Various amino acids and their abbreviations

Amino acid mutations may be located at the C-terminus and/or N-terminus of the amino acid sequence. For example, the peptide may have 1, 2 or 3 amino acid additions at the C-terminus or N-terminus of SEQ ID NO: 1. For example, the peptide may have 1, 2 or 3 amino acid deletions at the C-terminus or N-terminus of SEQ ID NO:2.

In one aspect, the peptides of the invention may be from 8 to 20 amino acids in length, preferably from 10 to 18 amino acids in length, more preferably from 11 to 14 amino acids in length. For example, the peptide may be 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acids in length.

In one aspect, the peptides of the invention may be selected from

1) A peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 1; or

2) A peptide that contains, consists essentially of, or is represented by the amino acid sequence of SEQ ID NO:2.

In one aspect, a peptide of the invention consists of the amino acid sequence of SEQ ID NO: 1. Alternatively, the peptide of the invention consists of the amino acid sequence of SEQ ID NO:2.

In one aspect, the invention relates to the use of a peptide described herein in the preparation of a composition for preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof. In one aspect, the invention relates to peptides for use in preventing, treating or ameliorating neurodegenerative diseases in a subject in need thereof. In one aspect, the invention relates to a method of preventing, treating or ameliorating a neurodegenerative disease in a subject in need thereof, comprising administering an effective amount of a peptide to the subject in need thereof. Neurodegenerative diseases can include cognitive impairment.

In one aspect, the neurodegenerative disease is a chronic neurodegenerative disease.

In one aspect, the invention relates to the use of a peptide described herein for the preparation of a composition for preventing, treating or ameliorating dementia in a subject in need thereof. In one aspect, the invention relates to peptides for use in preventing, treating or ameliorating dementia in a subject in need thereof. In one aspect, the invention relates to a method of preventing, treating or ameliorating dementia in a subject in need thereof, comprising administering an effective amount of a peptide to the subject in need thereof. Dementia can be caused by neurodegenerative diseases. Alternatively, dementia can accompany neurodegenerative diseases.

In one aspect, the neurodegenerative disease or cognitive disorder is selected from Alzheimer's disease or Parkinson's disease. Therefore, the peptides of the present invention can be used to prevent, treat or improve Alzheimer's disease. In addition, the peptide of the present invention can be used to prevent, treat or improve Parkinson's disease.

Cognition can be the intelligent processing process by which the body recognizes and acquires knowledge, involving a series of random, psychological and social behaviors such as learning, memory, language, thinking, spirit, and emotion. Cognitive impairment can include abnormalities in the brain's high-level intelligent processing related to the above-mentioned learning, memory, and thinking judgment, causing severe learning and memory impairments, accompanied by pathological processes such as aphasia, apraxia, agnosia, or aphasia.

In one aspect, the invention relates to the use of a peptide described herein in the preparation of a composition for improving, enhancing or restoring cognitive function or cognitive ability in a subject in need thereof. In one aspect, the invention relates to peptides for use in improving, enhancing or restoring cognitive function or cognitive ability in a subject in need thereof. In one aspect, the invention relates to a method of improving, enhancing or restoring cognitive function or cognitive ability in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of a peptide. In this aspect, cognitive function or cognitive ability includes perceptual ability, logical thinking ability, memory ability, language ability, learning ability, emotional control ability, social ability or attention. In this aspect, the subject has a cognitive function or cognitive ability decline, such as a cognitive function or cognitive ability decline caused by or accompanying aging. Alternatively, the subject may suffer from cognitive impairment or a neurodegenerative disease, preferably a cognitive impairment caused by a neurodegenerative disease, more preferably Alzheimer's disease or Parkinson's disease.

In one aspect, dementia is characterized or manifested by communication difficulties, poor judgment, difficulty performing simple tasks, missing or misplacing objects, language impairment, personality changes, reduced behavioral abilities, confused sense of time and space, and reduced comprehension. , decreased problem-solving ability, decreased concentration, decreased social skills, sensory impairment, logical thinking disorder or memory impairment.

In one aspect, the neurodegenerative disease or cognitive disorder is Alzheimer's disease. Alzheimer's disease is characterized by beta-amyloid (Aβ) deposition, neuroinflammation, and/or glial cell abnormalities or activation in the brain, particularly the hippocampus. Prevention, treatment or improvement of Alzheimer's disease is achieved by reducing or alleviating beta-amyloid (Aβ) deposition, neuroinflammation and/or glial cell abnormalities or activation in the brain.

In one aspect, the invention relates to the use of the peptides described herein in the preparation of a method for reducing or alleviating beta-amyloid (Aβ) deposition, neuroinflammation and/or glial abnormality or activation in the brain of a subject, particularly in the hippocampus. Use in compositions. The subject may be a neurodegenerative disease patient or a cognitive impairment patient, preferably an Alzheimer's disease patient.

The peptides described herein can be effective in reducing or alleviating Aβ or Aβ plaque deposition in a subject's brain, such as the hippocampus. In addition, the peptide can reduce or alleviate the expression or levels of inflammatory factors in the subject's brain, such as the hippocampus or cortex. The inflammatory factor can be selected from IL-1β, IL-6 or TNF-α. The peptides may also reduce or alleviate glial cell abnormalities or activation in the brain's hippocampus. Glial cells can also be called glial cells and can be selected from astrocytes or microglia, with microglia being preferred.

In one aspect, the neurodegenerative disease or cognitive disorder is Parkinson's disease. Parkinson's disease is characterized by abnormality or damage to neurons in the substantia nigra of the brain. The prevention, treatment or improvement of Parkinson's disease is achieved by reducing or alleviating neuronal abnormalities or damage in the substantia nigra of the brain.

In one aspect, the present invention relates to the use of a peptide described herein in the preparation of a composition for reducing or alleviating abnormality or damage to neurons in the substantia nigra of the brain of a subject. The subject may be a neurodegenerative disease patient or a cognitive impairment patient, preferably a Parkinson's disease patient.

In one aspect, the subject or patient is a mammal. In one aspect, the subject or patient is a human, such as a middle-aged or elderly person, preferably an elderly person. Middle-aged people can be people between 40 and 59 years old or people between 45 and 59 years old. An elderly person can be a person over 60, over 65, over 70, over 75 or over 80.

In one aspect, the subject may be a subject with cognitive function or cognitive ability decline, such as cognitive function or cognitive ability decline caused by or accompanying aging. The decline generally refers to chronic decline.

Compositions of the invention comprise peptides described herein. The composition of the present invention may also contain pharmaceutical excipients, solvents or carriers; or pharmaceutical excipients, solvents or carriers acceptable to the human body. The composition may be a pharmaceutical composition or a nutritional composition. The pharmaceutical composition may be a drug. The nutritional composition may be in the form of a nutritional supplement, nutritional supplement, dietary supplement, dietary supplement, nutraceutical, nutraceutical, nutraceutical or health food.

"Compositions" as described herein may generally include substances or formulations that improve or enhance the health condition of a subject (eg, a human) or treat a disease in the subject.

"Nutritional composition" as described herein may refer to meeting at least part of the nutrient requirements of a subject (such as a human), regulating the body functions of the subject, regulating the physiological functions of the subject, and/or improving the health status of the subject Substances or formulations that may be taken. For example, the nutritional composition may be a nutraceutical, functional food, dietary supplement, nutraceutical or health food that activates, modulates or improves cognitive function or cognitive ability.

As used herein, a "pharmaceutical composition" may refer to an ingestible substance or formulation for preventing, treating, alleviating, ameliorating, conditioning, or modulating a disease, disorder, symptom, or condition in a subject (eg, a human). For example, pharmaceutical compositions may be in the form of pharmaceuticals, including oral medications, such as tablets, pills, capsules, and the like.

Administration of the polypeptides or compositions of the invention may be in oral form. Compositions for oral administration may be formulated in dosages suitable for oral administration using carriers, vehicles or excipients known in the art. Such carriers enable the compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, suitable for ingestion by a subject.

In addition, the polypeptide or composition of the present invention can be prepared as an injection, such as intramuscular injection, intraperitoneal injection, intraperitoneal injection, subcutaneous injection or intravenous injection. Injections may be in the form of solutions, emulsions, or suspensions, as well as powders or concentrated solutions for solution or suspension before use. Those skilled in the art understand that injections may also contain solvents such as water, isotonic agents, buffers, preservatives, co-solvents, solubilizers, suspending agents and emulsifiers.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a peptide or composition of the invention that is effective in treating a disease or disorder or ameliorating or enhancing the health condition of a subject. Improving or improving one's health includes improving or improving cognitive functions or abilities. The peptide of the present invention can be used at 0.01mg/kg to 10mg/kg, 0.02mg/kg to 8mg/kg, 0.04mg/kg to 5mg/kg, 0.05mg/kg to 1mg/kg, 0.1mg/kg to 0.8mg/kg. kg or 0.2 mg/kg to 0.5 mg/kg is administered to the subject. For example, the peptide of the invention can be administered to a subject in an amount of 0.04 mg/kg, 0.16 mg/kg or 0.64 mg/kg. A therapeutically effective amount may be these doses. The peptide or composition of the invention may be administered at once daily intervals for at least 1 week, for example 1 to 4 weeks.

The peptides of the present invention may be administered to a subject as a combination of peptides or prepared into a composition. Compositions of the invention may comprise such combinations of peptides. For example, the peptide combination includes a first peptide having the amino acid sequence of SEQ ID NO: 1 or a variant thereof and a second peptide having the amino acid sequence of SEQ ID NO: 2 or a variant thereof.

Brief description of the drawings

Figure 1. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on short-term working memory, conditioned fear memory and social ability of APP/PS1 mice. (A) Statistical chart showing the percentage of time mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups entered the novel arm in the Y maze experiment. (B) In the new object recognition experiment, the statistical chart of the new object recognition cognitive index of the four groups of mice. (C) Statistical chart of freezing time of mice in four groups during the conditioned fear memory experiment. (D) In the social preference and social memory experiment, (left) statistical graph of the percentage of time mice entered the Empty chamber and Stranger-1 chamber; (right) statistical graph of the percentage of time mice entered the Stranger-1 chamber and Stranger-2 chamber . Results are expressed as mean ± standard error, n = 13-15 per group. The data are in Figure D. The comparison of the percentage of time that the four groups of mice entered the Empty chamber and Stranger-1 and the comparison of the percentage of time that the four groups of mice entered the Stranger-1 chamber and Stranger-2 chamber were compared using T test. Other statistical graphs One-way analysis of variance was used. Enter number: number of entries; Recognition index: cognitive index; Time spent: time spent; % of freezing: percentage of freezing time.

Figure 2. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on Aβ plaque deposition in the hippocampus and cortex of APP/PS1 mice. (A) Fluorescence images of thioflavin S staining of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. Scale bar is 200 μm. (B) Immunofluorescence experiment shows fluorescence images of 6E10 expression in the cortex and hippocampus of four groups of mice. Scale bar is 200 μm. (C) Quantitative statistical diagram of Thioflavin S-positive Aβ plaques in the cortex and hippocampus of four groups of mice. (D) Statistical chart of the percentage of 6E10 positive expression areas in the cortex and hippocampus of four groups of mice. The results are expressed as mean ± standard error, n = 4-5 per group, and the statistical graph uses one-way analysis of variance. Cortext: cortex; Hippocampus: hippocampus; Relative Density: relative density; Positive area of 6E10+: the positive area of 6E10+.

Figure 3. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on glial cell activation around hippocampal Aβ plaques in APP/PS1 mice. (A) Immunofluorescence experiment shows fluorescence images of Iba1, GFAP and 6E10 expression in the hippocampus of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. Scale bar is 50 μm. (B) Statistical chart of the percentage of positive expression areas of Iba1 and GFAP in the hippocampus of four groups of mice. The results are expressed as mean ± standard error, n = 4-5 per group, and the statistical graph uses one-way analysis of variance. Positive area of Iba1+: the positive area of Iba1+; Positive area of GFAP+: the positive area of GFAP+.

Figure 4. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on glial cell activation around cortical Aβ plaques in APP/PS1 mice. (A) Immunofluorescence experiment shows fluorescence images of Iba1, GFAP and 6E10 expression in the cortex of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. Scale bar is 50 μm. (B) Statistical chart of the percentage of positive expression areas of Iba1 and GFAP in the cortex of four groups of mice. The results are expressed as mean ± standard error, n = 5 per group, and the statistical graph uses one-way analysis of variance. Positive area of Iba1+: the positive area of Iba1+; Positive area of GFAP+: the positive area of GFAP+.

Figure 5. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on the contents of IL-1β, IL-6 and TNF-α in the hippocampus and cortex of APP/PS1 mice. (A) ELISA detects the expression of inflammatory factors IL-1β, IL-6 and TNF-α in the hippocampus of mice in the control group (Con), PKT101, PKT002 and PKT101/PKT002 combined treatment groups. (B) ELISA detects the expression of inflammatory factors IL-1β, IL-6 and TNF-α in the cortex of four groups of mice. The results are expressed as mean ± standard error, n = 5 per group, and the statistical graph uses one-way analysis of variance. Cortext: cortex; Hippocampus: hippocampus; Relative expression of IL-1β: relative expression of IL-1β;; Relative expression of IL-6: relative expression of IL-6;; Relative expression of TNF-α: TNF-α Relative expression.

Figure 6. Effects of PKT101 and PKT002 on body weight changes after subacute model of MPTP in mice. When 0 to 7 days old, n=20 or 28; when 14 days old, n=10 or 14. Sham: normal control group; Selegiline: Selegiline treatment group.

Figure 7. Effects of PKT101 and PKT002 on the total movement distance of mice in the mine field experiment after the MPTP subacute model (1, 3, 7 and 14 days). ^*** p<0.001, ^** p<0.01vs.Sham;^###p<0.001,^##p<0.01,^#p<0.05vs.MPTP.

Figure 8. Effects of PKT101 and PKT002 on T-Turn in the pole climbing experiment of mice after the MPTP subacute model (1, 3, 7 and 14 days). ***p<0.001,**p<0.01vs.Sham; ###p<0.001,##p<0.01,#p<0.05vs.MPTP.

Figure 9. Effects of PKT101 and PKT002 on T-TLA in the pole climbing experiment of mice after the MPTP subacute model (1, 3, 7 and 14 days). ***p<0.001vs.Sham; ###p<0.001,##p<0.01,#p<0.05vs.MPTP.

Figure 10. Effects of PKT101 and PKT002 on the latency period (1, 3, 7 and 14 days) of mice in the rotarod experiment after the subacute model of MPTP. ***p<0.001vs.Sham; ###p<0.001vs.MPTP.

Figure 11. Effects of PKT101 and PKT002 on the number of Nissl-positive neurons in the substantia nigra pars compacta of mice after the subacute model of MPTP (7 and 14 days). ***p<0.001vs.Sham; ##p<0.01,#p<0.05vs.MPTP; Scale bar is 250μm.

Figure 12. Effects of PKT101 and PKT002 on the number of TH-positive neurons in the substantia nigra pars compacta of mice after the subacute model of MPTP (7 and 14 days). *p<0.05vs.Sham; ###p<0.001,##p<0.01vs.MPTP; Scale bar is 250μm.

Figure 13. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of GL261 cells.

Figure 14. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of LN229.

Figure 15. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of WERI-RB-1 cells.

Figure 16. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of N2A cells.

Figure 17. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of HT22 cells.

Figure 18. Effects of PKT001 and PKT002 pretreatment on LPS-induced apoptosis of SK-N-SH cells.

Detailed ways

Unless otherwise defined, all scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Example methods and materials are described below, equivalents thereof may be used. All publications and other references mentioned herein are incorporated by reference in their entirety.

The following examples are provided to further illustrate the invention. The following examples are not intended to limit the scope of the invention for any reason.

Example

Example 1 - Study on the neuroprotective effect of the peptide of the present invention on the Alzheimer's disease model animal APP/PS1 mice

PKT101 peptide: DEAQETAVSSHEQD(SEQ ID NO:2)

PKT002 peptide: DEAQETAVSSH (SEQ ID NO:1)

The above peptides can be obtained by the methods described in WO2013/173941 and WO2016/165102.

1. Materials and methods

Sixty 5-month-old APP/PS1 male mice were randomly divided into 4 groups, 15 in each group. They were intraperitoneally injected with normal saline, PKT101 (8mg/kg), PKT002 (8mg/kg) and PKT101/PKT002 ( 8 mg/kg) for 4 weeks. The final volume of intraperitoneal injection in each group was 80 μL. After weekly administration, 20-35 mice were randomly taken out and measured for weight measurement. The average weight was used as an important basis for the next week's dosage.

The mice were tested for short-term learning and memory (Y maze and new object recognition), conditioned fear memory and social behavior, and the plaque deposition in the cerebral cortex and hippocampus was detected through thioflavin staining and 6E10 immunofluorescence staining; through immunofluorescence staining Observe the activation of microglia and astrocytes in the cortex and hippocampus; detect the inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumors using enzyme-linked immunosorbent assay (ELISA) Necrosis factor (TNF-α) content.

1.1. Behavioral testing

1.1.1. Short-term learning and memory

1.1.1.1.Y maze

The Y maze device used in this experiment has an angle of 120 degrees between the three arms, which are connected to each other. The three arms are the same size, 29cm long, wide and high 29cm x 8cm x 15cm. Different patterns are affixed to the inside of the arms to represent mice. As visual markers, name the starting arm, novelty arm and other arms respectively. The experimental process is divided into two parts, the adaptation phase and the testing phase, with an interval of 1-2 hours. Adaptation is to separate the novel arms with baffles. Mice can only move freely in the starting arm and other arms. The adaptation time of each mouse is 5 minutes. The test phase is to open the novel arms after 1-2 hours. The mice can move freely within the three arms, and the test time for each mouse is 5 minutes. During the experiment, Topscan software was used to record the time and number of times the mice entered the novel arm in real time. After each experiment, the experimental equipment must be cleaned with 75% alcohol to remove the mouse odor.

1.1.1.2. New object recognition

Before testing this device, the mice must be eliminated from any sense of strangeness and the mice must be stroked every day to avoid irritation to the mice during operation. In the first stage (familiarization period), put two identical objects (AB, make sure the objects have no smell and are not pushed) into the device. The objects are 10cm away from both sides of the walls. Put the mouse into the device from the middle of the two objects. Use a camera and software to record the mouse's exploration time on each object (touching the object with the mouth or nose and approaching the object within about 2-3cm are considered exploration of the object), within 5 minutes (many experiments have confirmed that the familiarization period At 2 minutes, the animal already has a preference for novel objects, and after 3 minutes of familiarity, the preference becomes more obvious.) The number, time and distance of the animal exploring each object were measured. In the second stage (test period), 1 hour after the completion of the first stage is used as the time interval to detect memory. One of the two identical objects is replaced with a different object and placed in the device (AC or BC). The small object is also placed in the device (AC or BC). The mouse is put into the device from the middle of the two objects, and the number, time and distance of the mouse's exploration of the old and new objects within 5 minutes are recorded, that is, the number, time and distance of the mouse's activities around the old and new objects, and the cognitive status of the mouse is detected. After each experiment, the experimental equipment needs to be cleaned with 75% alcohol. If the mouse has poor cognitive ability, there will be no difference in the exploration of new and old objects; if the mouse has normal cognitive ability, the exploration time of new objects will be longer than that of old objects. The calculation formula of recognition index (RI) is: RI=new object/(new object+old object)×100%.

1.1.2. Conditioned fear memory

On the first day of this experiment, the mice were put into the box (the bottom of the box is a copper fence that can be powered). After adapting for 3 minutes, the mice were allowed to stay in the box for another 6 minutes. During this period, a single frequency sound was given at the same time every 2 minutes. Stimulation (1.0 KHZ, 70 db, 30 s) and inescapable foot shock (0.8 mA, 2 s) were performed three times in total. The total time of sound and electric shock-induced freezing behavior of the mice within 6 min was recorded, and then placed back in the cage. After each experiment, wipe the bottom of the box with 75% alcohol. After 24 hours, the mice with established fear were placed in the original box, and the same intensity of sound stimulation (1.0KHZ, 70db, 30s) was immediately given, and the sound-induced freezing behavior of the mice was recorded within 3 minutes. Catalepsy was defined as no movement other than breathing. Percentage of environmentally and sound-induced freezing time was recorded.

1.1.3.Social behavior

1.1.3.1. Social preference stage

Before the start of the experiment, the mice were placed in the behavioral testing room to adapt for 0.5 h, and the tested rectangular box was equally divided into three areas: Empty, Center and Stranger-1. Mice of the same sex, background, and age were placed into the metal cage in the Stranger-1 area, and the metal cage on the other side of the box was empty. Put the test mouse into the center box so that the test mouse can move freely in the three boxes for 5 minutes. Photograph and record relevant parameters: the duration of entering each box. When the mouse's head and four paws enter a box, it is considered to be in that box.

1.1.3.2. Social memory stage

During the social memory phase of the experiment, mice of the same sex and background and different ages (Stranger-2) were placed in an empty metal cage, and then recorded for 5 minutes to observe the time the test mice spent in the Stranger-1 and Stranger-2 areas.

1.2. Brain tissue collection and section preparation

1.2.1. Collection of materials

Collect the materials immediately after the mouse behavioral test is completed. After anesthesia, blood was collected through the eyeball. Perfuse with physiological saline from the left ventricle of the heart until the liver turns khaki, replace with 4% paraformaldehyde solution and perfuse for 5 minutes. Remove the brain, place it in 4% paraformaldehyde, and fix it overnight at 4°C. Brain tissues from the hippocampus and cortex were excised and subjected to sucrose gradient dehydration.

1.2.2. Frozen section preparation

The OCT resin-embedded brain tissue was sliced coronally using a Leica freezing microtome, with a thickness of 10 μm, and collected in PBS for immunofluorescence staining and Aβ histopathological analysis.

1.3. Immunofluorescence staining

The sections were blocked with 10% fetal bovine serum PBS solution for 1 h at room temperature. Discard the blocking solution, add mouse-derived 6E10 antibody, chicken-derived glial fibrillary acidic protein antibody, and rabbit-derived calcium ion binding receptor molecule 1 (inized calcium binding adapter molecule 1, Iba1) antibody, and incubate overnight at 4°C. The next day, the primary antibody was washed away and washed 3 times with PBS for 5 minutes each time. Add secondary antibodies of corresponding species (goat anti-chicken IgG-488; donkey anti-rabbit IgG-488; donkey anti-mouse IgG-647) and incubate at room temperature in the dark for 1 hour. Discard the secondary antibody, wash 3 times with PBS, 5 minutes each time, stain with DAPI for 10 minutes at room temperature, discard DAPI, and wash 3 times with PBS. Anti-fluorescence quenching agent sealing, Take pictures under fluorescence microscope.

1.4. Thioflavin S staining

Thioflavin S can mark Aβ core plaques on brain slices. Dewaxed and hydrated tissue sections can be stained with 1% thioflavin for 5 minutes, washed with running water for 1 minute, separated with 70% alcohol for 30 seconds, and then quenched with anti-fluorescence. The slides were sealed with agent and photographed under a fluorescence microscope.

1.5.Image analysis

Immunofluorescence-stained tissue sections were photographed using a Leica microscope, and ImageJ software was used to use grayscale threshold analysis to count the percentage of positive areas for GFAP, Iba1, 6E10, and thioflavin. There were 5 mice in each group, and 3 sections were selected for each mouse. , take its average value as the statistical result of this group.

1.6. Enzyme-linked immunosorbent assay (ELISA)

1.6.1. Collection of materials

After the mice were anesthetized, they were killed by decapitation, the skin on the neck and head was peeled off, the skull was exposed, the skull was cut along the sagittal suture, and the brain tissue was removed after the skull was separated. Put the cortical tissue and hippocampal tissue into marked centrifuge tubes, quickly freeze them in liquid nitrogen, and transfer them to a -80°C refrigerator for storage.

1.6.2.Sample preparation

Take about 10-20 mg of cortex and hippocampus tissue from the ice box, weigh it into a 1.5 mL centrifuge tube, and add RIPA lysis solution at a mass-to-volume ratio of 1:10. The remaining tissue is returned to the original tube and quickly frozen in liquid nitrogen. At the same time, add two steel balls to the centrifuge tube, put it into the homogenizer to balance, homogenize and lyse, and then shake on ice for 30 minutes. Remove the steel beads, centrifuge at 12000 rpm, 4°C for 15 min, fully absorb the supernatant, and transfer to a new 1.5 mL centrifuge tube. Protein concentration was quantified using protein quantification method.

1.6.3.ELISA

Add diluent to the sample to dilute it to an appropriate concentration. Add the diluted sample to the enzyme-labeled reaction well for testing interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). ) 3 pro-inflammatory indicators, add at least two wells to each sample, 45 μL of standard and sample in each well, then add 50 μL of biotinylated antibody working solution to the sample and standard wells, seal the reaction well plate with sealing paper, and place Incubate at room temperature for 2 hours (use micro shaker, frequency, 300 rpm). Prepare the enzyme-binding substrate 30 minutes in advance and store it at room temperature away from light. Discard the liquid in the wells and pat dry on absorbent paper. Wash 5 times. Washing method: absorb the reaction solution in the wells, add the washing solution to the well plate, and leave it for 2 minutes with slight shaking. Add 100 μL of enzyme-bound substrate working solution to each well except the blank well, and incubate at room temperature for 1 hour (use a micro shaker, frequency, 300 rpm). Discard the liquid in the wells and pat dry on absorbent paper. Wash 5 times. Add 100 μL of chromogenic substrate working solution to each well except the blank wells, and place at room temperature in the dark for 15 minutes. Add 100 μL of stop solution to each well except the blank well to stop the reaction, and measure the experimental results within 20 minutes. Detection at 450nm wavelength, absorption values of standard holes and test holes. A standard curve is drawn based on the standard wells and known concentrations, and then the actual concentration of the test wells is converted from the standard curve. Finally, the target protein amount per milligram of brain tissue is obtained based on the protein concentration.

2. Effects of PKT101, PKT002 and PKT101/PKT002 combined administration on cognitive behavior of APP/PS1 mice

The Y maze test was used to evaluate the short-term memory of mice in each group. The results showed that compared with the control group APP/PS1 mice, the time for APP/PS1 mice in the PKT101 treatment group to enter the novel arm increased, while PKT002 and PKT101/PKT002 The time for APP/PS1 mice in the combination treatment group to enter the novel arm was significantly increased (Figure 1A). The new object recognition experiment was used to evaluate the short-term memory of mice in each group. The results showed that compared with the control group, the index of APP/PS1 mice in the above three treatment groups to recognize new objects increased significantly (Figure 1B). Conditioned fear memory was used to evaluate the fear memory of mice in each group. The results showed that the percentage of fear time of APP/PS1 mice in the PKT101, PKT002 and PKT101/PKT002 combined treatment groups did not show significant changes, but it was reduced compared to the control group ( Figure 1C); the three-box experiment was used to evaluate the social ability of mice in each group. The results showed that during the social preference stage, PKT101 showed a preference for same-sex mice (Stranger1) (Figure 1D left), suggesting that social learning ability has been improved. ; In the social memory stage, there is still a certain preference for Stranger1 in the PKT101 group (Figure 1D right). The experimental results are also shown in Table 2 below.

3. Effects of PKT101, PKT002 and PKT101/PKT002 combined administration on Aβ deposition in APP/PS1 mice

Thioflavin staining and 6E10 immunofluorescence staining were used to evaluate the fibrous and diffuse Aβ plaque deposition in the cortex and hippocampus of mice in each group. The results of thioflavin staining showed that compared with the control group, PKT101, PKT002 and PKT101/PKT002 combined The Aβ plaques in the hippocampus of APP/PS1 mice in the treatment group were significantly reduced, and there was no effect on the deposition of Aβ plaques in the cortex (Figure 2A, C). The 6E10 staining results showed that the combined administration of PKT101, PKT002 and PKT101/PKT002 significantly reduced Aβ deposition in the hippocampus of APP/PS1 mice, but had no effect on Aβ deposition in the cortex (Figure 2B, D). The experimental results are also shown in Table 3.

Table 3. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on Aβ plaque deposition in the hippocampus and cortex of APP/PS1 mice

*p<0.05, vs. control group.

4. Combined administration of PKT101, PKT002 and PKT101/PKT002 improves glial cell activation around hippocampal Aβ plaques in APP/PS1 mice

The activation of microglia and astrocytes around Aβ plaques in the hippocampus and cortex of APP/PS1 mice were evaluated by co-labeling staining of 6E10, GFAP and Iba1. The results showed that compared with the control group, PKT101, PKT002 and PKT101 The activation degree of hippocampal microglia in mice in the /PKT002 combination treatment group was significantly reduced (Figure 3A-B). The combined administration of PKT002 and PKT101/PKT002 significantly reduced astrocyte activation in the mouse hippocampus (Figure 3A, C). Compared with the control group, the activation of glial cells in the cerebral cortex of mice in the PKT101, PKT002 and PKT101/PKT002 combined treatment groups showed no significant improvement (Figure 4A-B). The experimental results are also shown in Table 4.

Table 4. Effects of combined administration of PKT101, PKT002 and PKT101/PKT002 on glial cell activation around Aβ plaques in the cortex and hippocampus of APP/PS1 mice

*p<0.05, vs. control group.

5. Effects of PKT101, PKT002 and PKT101/PKT002 combined administration on IL-1β, IL-6 and TNF-α contents in hippocampus and cerebral cortex of APP/PS1 mice

ELISA was used to detect the contents of inflammatory factors IL-1β, IL-6 and TNF-α in the hippocampus and cerebral cortex of mice in each group. The results showed that compared with the control group, PKT101, PKT002 and PKT101/PKT002 combined treatment of hippocampal inflammatory factors in mice The levels of IL-6 and TNF-α were significantly reduced. The levels of IL-1β in the PKT002 and PKT101/PKT002 combined treatment groups were significantly reduced (Figure 5A). The expression of IL-6 in the cerebral cortex of mice in the above three groups was significantly reduced. , there was no significant difference in the expression of other inflammatory factors IL-1β and TNF-α (Figure 5B). The experimental results are also shown in Table 5.

6 Conclusion

PKT101 and PKT002 treatment alone and PKT101/PKT002 combined treatment can improve short-term cognitive impairment, reduce hippocampal Aβ load deposition, glial cell activation and inflammatory factors in APP/PS1 mice to varying degrees.

Example 2 - Experimental study on the pharmacodynamics of the peptide of the present invention on MPTP subacute Parkinson's disease mouse model

PKT101 peptide: DEAQETAVSSHEQD(SEQ ID NO:2)

PKT002 peptide: DEAQETAVSSH (SEQ ID NO:1)

The above peptides can be obtained by the methods described in WO2013/173941 and WO2016/165102.

1. Materials and methods

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was used to establish a subacute Parkinson's disease mouse model. After the mice were weighed and recorded, 20 mg/kg MPTP was injected subcutaneously into the back of the neck once a day for 5 consecutive days. Follow-up experiments were conducted on days 1, 3, 7 and 14 after the last administration.

168 healthy adult C57BL/6J male mice were randomly divided into eight groups according to body weight: normal control (Sham) group (n=20), MPTP model group (n=28), PKT101 low-dose (0.5mg/ kg/d) treatment group (n=20), PKT101 medium-dose (2mg/kg/d) treatment group (n=20), PKT101 high-dose (8mg/kg/d) treatment group (n=20), PKT002 ( 8mg/kg/d) treatment group (n=20), PKT101 (8mg/kg/d)+PKT002 (8mg/kg/d) combined treatment group (n=20) and the positive control drug Selegiline (Selegiline, Imidopyr) (0.5mg/kg/d) treatment group (n=20). Treatment began with intraperitoneal injection of the corresponding drug 1 hour after the first MPTP administration and continued once a day for 7 or 14 days. Mice in the MPTP model group were given an equal volume of physiological saline (0.1mL/10g). The mice in the Sham group were only given an equal volume of physiological saline (0.1mL/10g).

Open field, rod climbing and rotarod experiments were conducted on days 1, 3, 7 and 14 after the last administration of MPTP to evaluate the behavioral changes of mice. On days 7 and 14 after the last administration of MPTP, Nissl and TH staining was performed on the midbrain, the number of Nissl and TH positive cells in the substantia nigra pars compacta was counted, and the degree of damage to dopaminergic neurons in the corresponding brain areas was evaluated.

2. Behavioral evaluation

Behavioral evaluations were conducted on days 1, 3, 7, and 14 after the last administration of MPTP.

2.1. Open field test

The open field test was used to evaluate the voluntary movement ability of mice. The open field device is made of opaque blue plastic and measures 60cm × 60cm × 45cm. During the test, each mouse was placed into the box from the central area, and at the same time, the movement trajectory of each mouse within 5 minutes was recorded, and the total movement distance was calculated using software (Clever Sys Inc., VA, USA). After each mouse test, use 75% ethanol to wipe the open area to prevent the smell of the previous mouse from affecting the next mouse.

2.2. Pole climbing test

The pole climbing test was used to evaluate the motor coordination function of mice. The pole used has a diameter of 1cm and a height of 50cm. A wooden ball with a diameter of 1.2cm is fixed on the top of the pole. The pole is wrapped with anti-slip tape to prevent mice from slipping. Place the mouse's head upward on the top of the pole. When the mouse starts to climb up, start the timer. Record the time it takes for the mouse to move over the top of the pole (T-Turn) and the time it takes for the mouse to climb to the bottom of the four paws. Time to Landing (T-TLA). Before formal testing, each mouse needs to be trained three times consecutively. During the test, three consecutive measurements are required, and the shortest time is chosen.

2.3. Rotarod test

The rotarod test was used to evaluate the motor balance ability and limb coordination of mice. Place the mouse on the drum of the rotarod tester, and set the rotation speed to increase from 5rpm/min to 25rpm/min at a constant speed within 2 minutes. When the mouse falls from the drum to the sensing area below, an infrared sensor will receive the signal and record it. Time for mice to move on the instrument roller. Before formal testing, each mouse needs to be trained three times consecutively. During the test, three consecutive measurements are required to calculate the average drop time (latency period).

3. Immunohistochemical study

On days 7 and 14 after the last administration of MPTP, half of the mice from each group were taken, anesthetized with 4% chloral hydrate, and perfused with physiological saline + 4% PFA to obtain brain tissue. After dehydration through 20% (v/v) and 30% (v/v) sucrose solution gradient for 3 days each, the mouse brain tissue was embedded in O.C.T. glue and frozen, and serial tissue sections (20 μm) were made using a freezing microtome (Leica). ), collect brain slices from relevant areas, place them in cryopreservation solution (glycerol: 0.01M PBS=1:1), and store them at -80°C for later use.

3.1. Nissl staining

Nissl bodies are basophilic substances in the cytoplasm that are widely found in various neurons and are used to count the number of brain neurons. Stain according to Nissl staining kit.

3.2.TH (Tyrosine hydroxylase, tyrosine hydroxylase) staining

TH mainly labels dopaminergic neurons in the substantia nigra pars compacta, and TH-positive neurons in this brain area will be significantly reduced after MPTP modeling. Remove the midbrain brain slices, wash them once with 0.01M PBS for 10 minutes, incubate with 3% H ₂ O ₂ for 15 minutes to remove endogenous peroxidase activity, wash away H ₂ O ₂ with 0.01 M PBS for 10 minutes × 3, and then use PBS containing 5% BSA and 0.3% Triton After three times, diaminobenzidin (DAB) was used to develop color in the dark.

4. Cell counting and data processing and analysis

Immunohistochemistry-positive cells were counted using a Stereo Investigator (MBF bioscience) counting system to count the number of Nissl body-positive cells and TH ⁺ cells in the substantia nigra pars compacta. Cell counting was performed under a ×20 objective lens.

All data are expressed as Mean±S.E.M, and GraphPad Prism 8.0 statistical analysis software was used for statistical processing. The measurement data were analyzed using One-way ANOVA combined with Dunnett’s test to analyze the differences between each group and the modeling group. p<0.05 indicated that the difference was statistically significant.

5.Experimental results

5.1. Effects of PKT101 and PKT002 on survival rate after subacute MPTP model in mice

Subacute administration of MPTP caused the mice to become listless, anorexic, lose weight, and develop a tail-erector reaction after modeling. However, no mice died in this experiment, and mice in each group of drug treatments also did not die.

5.2. Effects of PKT101 and PKT002 on body weight changes after subacute MPTP model in mice

Subacute administration of MPTP caused the mice to eat less after modeling. The weight of the mice decreased slightly within 7 days. After 14 days, the mice ate gradually increased and their weight gradually recovered. The treatments in each group had no significant effect on the body weight of mice. The results are shown in Figure 6.

5.3. Effects of PKT101 and PKT002 on voluntary movement ability in mice after subacute MPTP model in the mine experiment

As shown in Table 6 and Figure 7, in the MPTP subacute model, mice showed a decrease in the total movement distance in the open field test at 1, 3, 7, and 14 days after modeling, indicating a decrease in autonomous activity ability. The positive control drug Selegiline can significantly improve MPTP-induced decrease in total exercise distance on days 1, 3, 7 and 14. PKT101 and PKT002 also had significant improvement within 14 days. At 14 days, the low, medium and high doses of PKT101 alone increased to 221.1%, 253.9% and 255.3% of the model group; PKT002 alone increased to 213.0% of the model group; the combined administration of PKT101 and PKT002 increased to the model group 214.8%.

Table 6. Effects of PKT101 and PKT002 on total movement distance of mice in the mine experiment after MPTP subacute model

^*** p<0.001, ^** p<0.01vs.Sham;^###p<0.001,^##p<0.01,^#p<0.05vs.MPTP

5.4. Effects of PKT101 and PKT002 on the motor coordination ability in the pole climbing experiment after the subacute model of MPTP in mice

As shown in Tables 7-8 and Figures 8-9, in the MPTP subacute model, mice showed prolonged T-Turn and T-TLA times in the pole climbing test at 1, 3, 7 and 14 days after modeling, indicating movement coordination. Decline in ability. The positive control drug Selegiline can significantly improve MPTP-induced T-Turn and T-TLA time extension on days 1, 3, 7 and 14. PKT101 and PKT002 also had significant improvement within 14 days. For T-Turn, at 14 days, single PKT101 administration at low, medium and high doses was reduced to 26.1%, 24.6% and 26.0% of the model group respectively; single PKT002 administration was reduced to 24.4% of the model group; PKT101 and PKT002 combined The drug administration was reduced to 21.6% of the model group. For T-TLA, at 14 days, single administration of low, medium and high doses of PKT101 was reduced to 68.8%, 65.1% and 38.6% of the model group respectively; single administration of PKT002 was reduced to 46.0% of the model group; PKT101 and PKT002 were combined The drug administration was reduced to 46.3% of the model group.

Table 7. Effects of PKT101 and PKT002 on T-Turn of mice in the pole climbing experiment after the subacute model of MPTP

^*** p<0.001, ^** p<0.01vs.Sham;^###p<0.001,^##p<0.01,^#p<0.05vs.MPTP

Table 8. Effects of PKT101 and PKT002 on T-TLA in the pole-climbing experiment in mice following the MPTP subacute model.

^*** p<0.001vs.Sham;^###p<0.001,^##p<0.01,^#p<0.05vs.MPTP

5.5. Effects of PKT101 and PKT002 on the motor balance ability of mice in the rotarod test after subacute MPTP model

As shown in Table 9 and Figure 10, in the MPTP subacute model, mice showed a shortening of the falling latency in the rotarod test at 1, 3, 7, and 14 days after modeling, indicating a decrease in motor balance ability. The positive control drug Selegiline can significantly improve the shortening of MPTP-induced latency time on days 1, 3, 7 and 14. Both PKT101 and PKT00 had significant improvement within 14 days. At 14 days, the incubation period of low, medium and high doses of PKT101 alone was prolonged to 121.7, 119.9% and 118.3% of the model group respectively; the latency of PKT002 alone was prolonged to 120.3% of the model group; the combined administration of PKT101 and PKT002 was prolonged to 121.7%, 119.9% and 118.3% of the model group. 120.9% of the model group.

Table 9. Effects of PKT101 and PKT002 on the latency period of mice in the rotarod experiment after the subacute model of MPTP

^*** p<0.001vs.Sham;^###p<0.001vs.MPTP

5.6. Effect of Lizazhi on the number of Nissl-positive neurons in the substantia nigra pars compacta in the subacute model of mouse MPTP

As shown in Table 10 and Figure 11, in the MPTP subacute model, mice showed a decrease in the number of Nissl stain-positive neurons in the substantia nigra pars compacta 7 and 14 days after modeling, indicating the death of neurons in this brain area. The positive control drug Selegiline could significantly improve the MPTP-induced decrease in the number of Nissl stain-positive neurons in the substantia nigra pars compacta on both 7 and 14 days. PKT101 and PKT002 also had significant improvement effects at 7 and 14 days. At 7 days, the number of Nissl-positive cells treated with low, medium and high doses of PKT101 alone increased to 120.9%, 124.0% and 130.9% of the model group, respectively; 124.5%; the number of Nissl-positive cells after combined administration of PKT101 and PKT002 increased to 128.0% of the model group. At 14 days, the number of Nissl-positive cells treated with low, medium and high doses of PKT101 alone increased to 124.9%, 130.9% and 141.1% of the model group, respectively; 128.5%; the number of Nissl-positive cells after combined administration of PKT101 and PKT002 increased to 141.5% of the model group.

Table 10. Effects of PKT101 and PKT002 on the number of Nissl-positive neurons in the substantia nigra pars compacta of mice after subacute model of MPTP

^*** p<0.001vs.Sham;^##p<0.01,^#p<0.05vs.MPTP

5.7. Effects of PKT101 and PKT002 on the number of TH-positive neurons in the substantia nigra pars compacta after subacute model of mouse MPTP.

As shown in Table 11 and Figure 12, in the MPTP subacute model, mice showed a decrease in the number of TH-positive neurons in the substantia nigra pars compacta 7 and 14 days after modeling, indicating the death of dopaminergic neurons in this brain area. The positive control drug Selegiline could significantly improve the MPTP-induced decrease in the number of TH-stained positive neurons in the substantia nigra pars compacta on both 7 and 14 days. PKT101 and PKT002 also had significant improvement effects at 7 and 14 days. At 7 days, the number of TH-positive cells after single administration of low, medium and high doses of PKT101 increased to 114.5%, 124.9% and 129.7% of the model group respectively; the number of TH-positive cells after single administration of PKT002 increased to 129.9% of the model group. ; The number of TH-positive cells treated with PKT101 and PKT002 combined increased to 130.3% of that in the model group. At 14 days, the number of TH-positive cells after single administration of PKT101 at low, medium and high doses increased to 125.6%, 136.0% and 137.5% of the model group respectively; the number of TH-positive cells after single administration of PKT002 increased to 128.1% of the model group. ; The number of TH-positive cells after combined administration of PKT101 and PKT002 increased to 137.7% of that in the model group.

Table 11. Effects of PKT101 and PKT002 on the number of TH-positive neurons in the substantia nigra pars compacta of mice after subacute model of MPTP

^* p<0.05vs.Sham;^###p<0.001,^##p<0.01,^#p<0.05vs.MPTP.

6 Conclusion

Continuous treatment of peptides PKT101 and PKT002 for 7 and 14 days can significantly improve the behavioral disorders of mice after the MPTP subacute model, increase the total movement distance in the open field test, shorten the T-Turn time and T-TLA time in the pole climbing test, The incubation period of the rotarod experiment was prolonged, but the dose-dependent relationship between the groups was not obvious. At the same time, both PKT101 and PKT002 can alleviate the MPTP-induced decrease in the number of Nissl-positive and TH-positive neurons in the substantia nigra pars compacta. These results indicate that both PKT101 and PKT002 have neuroprotective effects on the subacute model of MPTP. There is no significant difference between combined administration of PKT101 and PKT002 compared with administration alone.

Example 3 - Preliminary study on the protective effect of the peptide of the present invention on damage to glial cells and neurons in neurodegenerative disease models

1. Experimental purpose

At the cellular level, we will initially explore the protective effects of PKT001 and PKT002 on damage to glial cells and neurons, and discuss the general mechanism for the treatment of neurodegenerative diseases.

2. Experimental plan

Mouse or human glioma cells and neuronal cells were pretreated with 1, 3, 9ug/ml PKT001, PKT002 experimental groups and control groups respectively for 24 hours, and then lipopolysaccharide (LPS, 1μg/ml) was added for induction. At 48 hours, Annexin V/propidium iodide (PI) double staining was performed to analyze the proportion of apoptotic cells by flow cytometry, and the effects of PKT001 and PKT002 administration on the apoptosis of various nervous system cells were detected.

3. Experimental materials

cell lines

GL261 mouse glioblastoma cells, culture medium: DMEM+10% FBS+1% P/S;

LN229 human glioma cells, culture medium: DMEM+10% FBS+1% P/S;

WERI-RB-1 human retinal glioma cells, culture medium RPMI-1640+10% FBS+1% P/S;

SK-N-SH human neuroblastoma cells, culture medium: MEM (containing NEAA) + 10% FBS + 1% P/S;

HT22 mouse hippocampal neuron cells, culture medium: DMEM+10% FBS+1% P/S;

neuro-2a (N2A) mouse brain neuroma cells, culture medium: MEM (containing NEAA) + 10% FBS + 1% P/S;

Culture conditions: 37°C, 5% CO ₂ .

The cells used in the experiment were all in logarithmic growth phase.

4. Experimental steps

Effects of PKT001 and PKT002 on apoptosis of nervous system cells

(1) Cell passage and plating: Take the normally cultured cells, aspirate the original culture medium, add PBS to wash, add trypsin for digestion for 1-3 minutes, then terminate the digestion, pipette into single cells with a pipette, and centrifuge the cell suspension at 200g for 5 minutes. , resuspend with culture medium, add 20 μl of cell suspension to 20 μl of trypan blue for counting, plate at 3*10 ⁵ cells/well (6-well plate), 3 duplicate wells, 5% CO ₂ , and culture at 37°C overnight.

(2)Dosing treatment:

①Cell;

②Cell+LPS(1μg/ml);

③Cell+PKT001(1μg/ml)+LPS(1μg/ml);

④Cell+PKT001(3μg/ml)+LPS(1μg/ml);

⑤Cell+PKT001(9μg/ml)+LPS(1μg/ml);

⑥Cell+PKT002(1μg/ml)+LPS(1μg/ml);

⑦Cell+PKT002(3μg/ml)+LPS(1μg/ml);

⑧Cell+PKT002(9μg/ml)+LPS(1μg/ml);

There were 8 groups in total. The drugs were first administered for 24 hours, followed by LPS induction treatment for 48 hours, and then related tests were conducted.

(3)Apoptosis detection:

①Collect cells: centrifuge at 200g for 5 minutes at 4°C to collect cells.

② Wash the cells twice with pre-cooled PBS, 200g each time, and centrifuge at 4°C for 5 minutes.

③Aspirate the PBS and add 100μL 1×Binding Buffer to resuspend the cells.

④Add 5μL Annexin FITC and 2μL PI and mix gently.

⑤ Protect from light and react at room temperature for 15 minutes.

⑥Add 300μL 1×Binding Buffer, mix well and place on ice. The sample will be detected by flow cytometry within 1 hour.

5.Experimental results

5.1. Effects of PKT001 and PKT002 on LPS-induced apoptosis of glioblastoma cells in GL261 mice

Using Annexin V-FITC/PI double-staining flow cytometry, the experimental results showed that compared with the LPS control group, after pretreatment with 1-9 μg/ml PKT001, the proportion of LPS-induced apoptosis in GL261 cells at 48 h was significantly reduced; 1-9 μg/ml After pretreatment with ml PKT002, the proportion of LPS-induced apoptosis in GL261 cells at 48 h was significantly reduced. The results are shown in Figure 13.

5.2. Effects of PKT001 and PKT002 on LPS-induced apoptosis of LN229 human glioma cells

Compared with the LPS control group, after pretreatment with 1 μg/ml PKT001, the proportion of LN229 cell apoptosis induced by LPS at 48 h did not change significantly. After pretreatment with 3 μg/ml and 9 μg/ml PKT001, the proportion of LN229 cell apoptosis induced by LPS at 48 h was significantly reduced. . After pretreatment with 1-9 μg/ml PKT002, the proportion of LN229 cell apoptosis induced by LPS at 48 h was significantly reduced. The results are shown in Figure 14.

5.3. Effects of PKT001 and PKT002 on LPS-induced apoptosis of WERI-RB-1 human retinal glioma cells

Compared with the LPS control group, after pretreatment with 1-9 μg/ml PKT001, the proportion of WERI-RB-1 cells induced by LPS at 48 h was significantly reduced; after pretreatment with 1-9 μg/ml PKT002, the proportion of LPS-induced WERI-RB-1 cells at 48 h was significantly reduced. The proportion of cell apoptosis was significantly reduced. The results are shown in Figure 15.

5.4. Effects of PKT001 and PKT002 on LPS-induced apoptosis of neuro-2a (N2A) mouse brain neuroma cells

Compared with the LPS control group, after pretreatment with 1μg/ml and 3μg/ml PKT001, the proportion of N2A cell apoptosis induced by LPS at 48h did not change significantly. After pretreatment with 9μg/ml PKT001, the proportion of LPS-induced apoptosis at 48h N2A cells decreased. There is a significant difference. After pretreatment with 1-9 μg/ml PKT002, the proportion of N2A cell apoptosis induced by LPS at 48 h was significantly reduced. The results are shown in Figure 16.

5.5. Effects of PKT001 and PKT002 on LPS-induced apoptosis of hippocampal neurons in HT22 mice

The experimental results showed that compared with the LPS control group, after pretreatment with 1-9 μg/ml PKT001, the proportion of LPS-induced apoptosis in HT22 cells at 48h was significantly reduced; after pretreatment with 1-9 μg/ml PKT002, the proportion of LPS-induced apoptosis in HT22 cells at 48h was significantly reduced. significantly reduced. The results are shown in Figure 17.

5.6. Effects of PKT001 and PKT002 on LPS-induced apoptosis of SK-N-SH human neuroblastoma cells

Compared with the LPS control group, after pretreatment with 1 μg/ml PKT001, the proportion of LPS-induced apoptosis in SK-N-SH cells did not change significantly at 48 h. After pretreatment with 3 μg/ml and 9 μg/ml PKT001, LPS induced SK-N cells at 48 h. -SH cell apoptosis ratio was significantly reduced. After pretreatment with PKT002 at 1-9 μg/ml, the apoptosis rate of SK-N-SH cells induced by LPS at 48 h was significantly reduced. The results are shown in Figure 18.

Claims (15)

1. Use of a peptide selected from the group consisting of:

   a) A peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSH (SEQ ID NO: 1);

   b) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1);

   c) A peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or

   d) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).
2. The use of claim 1, wherein said cognitive impairment is caused by a neurodegenerative disease.
3. The use of claim 1 or 2, wherein the neurodegenerative disease or cognitive disorder is selected from Alzheimer's disease or Parkinson's disease.
4. The peptide is prepared to improve, enhance or restore cognitive function or cognitive ability in a subject in need, preferably including perceptual ability, thinking logical ability, memory ability, language ability, learning ability, emotional control ability, social ability or For use in a composition of attention, the peptide is selected from:

   a) A peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSH (SEQ ID NO: 1);

   b) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1);

   c) A peptide comprising an amino acid sequence that is at least 70% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or

   d) A peptide comprising an amino acid sequence having 0 to 4 amino acid mutations in DEAQETAVSSHEQD (SEQ ID NO: 2).
5. The use of claim 4, wherein

   The subject has cognitive function or cognitive ability decline, such as cognitive function or cognitive ability decline caused by or accompanying aging; or

   The subject suffers from cognitive impairment or neurodegenerative disease, preferably cognitive impairment caused by neurodegenerative disease, more preferably Alzheimer's disease or Parkinson's disease.
6. The use of any one of claims 1-3 and 5, wherein the dementia is characterized by communication difficulties, poor judgment, difficulty in performing simple tasks, missing or misplacing objects, language impairment, personality mutations, behavioral decline, time and confusion in spatial sense, decreased understanding, decreased problem-solving ability, decreased concentration, decreased social skills, perceptual impairment, logical thinking impairment, or memory impairment.
7. The use in any one of the preceding claims, wherein the neurodegenerative disease or cognitive impairment is Alzheimer's disease, wherein

   a) the Alzheimer's disease is characterized by beta-amyloid (Aβ) deposition, neuroinflammation, and/or glial cell abnormalities or activation in the brain; or

   b) Prevention, treatment or improvement of Alzheimer's disease is achieved by reducing or alleviating beta-amyloid (Aβ) deposition, neuroinflammation and/or glial cell abnormalities or activation in the brain.
8. The use in any one of the preceding claims, wherein the neurodegenerative disease or cognitive impairment is Parkinson's disease, wherein

   a) The Parkinson's disease is characterized by abnormality or damage to neurons in the substantia nigra of the brain; or

   b) The prevention, treatment or improvement of Parkinson's disease is achieved by reducing or alleviating neuronal abnormalities or damage in the substantia nigra of the brain.
9. Use in any one of the preceding claims, wherein the subject is a human, preferably an elderly person.
10. The use in any one of the preceding claims, the composition is a pharmaceutical composition or a nutritional composition, preferably the pharmaceutical composition or nutritional composition contains the peptide and pharmaceutical excipients, solvents or carriers.
11. Use in any one of the preceding claims, said peptide being selected from:

    a) A peptide comprising an amino acid sequence that is at least 80% identical to DEAQETAVSSH (SEQ ID NO: 1);

    b) A peptide comprising an amino acid sequence having 0 to 3 amino acid mutations in DEAQETAVSSH (SEQ ID NO: 1);

    c) A peptide comprising an amino acid sequence that is at least 80% identical to DEAQETAVSSHEQD (SEQ ID NO: 2); or

    d) Contained in DEAQETAVSSHEQD (SEQ ID NO:2) with 0 to 3 amino acid mutations The amino acid sequence of the peptide.
12. Use in any one of the preceding claims, said amino acid mutation being selected from the group consisting of addition, deletion or substitution of amino acids.
13. The use in any one of the preceding claims, the amino acid mutation is located at the C-terminus and/or N-terminus of the amino acid sequence, preferably an addition or deletion at the C-terminus and/or N-terminus of the amino acid sequence.
14. Use in any one of the preceding claims, said peptide having a length of 8 to 20 amino acids, preferably 10 to 18 amino acids in length, more preferably 11 to 14 amino acids in length.
15. Use in any one of the preceding claims, said peptide being selected from the group consisting of

    1) A peptide comprising, consisting essentially of, or represented by the amino acid sequence of SEQ ID NO: 1; or

    2) A peptide that contains, consists essentially of, or is represented by the amino acid sequence of SEQ ID NO:2.