KR100973641B1 - A novel peptide for increasing the protein expression of Brain-derived neurotrophic factor in the hippocampal neuronal cell, the hippocampal tissue and the cerebral cortex tissue - Google Patents

Abstract

The present invention relates to novel peptides that increase protein expression of brain-derived neurotrophic factor (BDNF). Specifically, the present invention increases the expression of BDNF mRNA and protein in hippocampus and cerebral cortex, such as Alzheimer's disease, Parkinson's disease, depression, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative diseases or diabetic neuropathy. Provided are novel peptides that can be used as a prophylactic or therapeutic agent for a disease. MVG (Methionine-Valine-Glycine) peptide consisting of three amino acids of the present invention is capable of expressing mRNA and protein of BDNF, which are important for functions such as learning and memory, protection of dopamine neurons, and antidepression, such as hippocampal neurons, hippocampus tissues and Increased in cerebral cortex.

BDNF, MVG, Peptides, Hippocampus, Cortex.

Description

A novel peptide for increasing the protein expression of Brain-derived neurotrophic factor in the hippocampal neuronal cell, the hippocampal tissue and the cerebral cortex tissue}

The present invention relates to novel peptides that increase protein expression of BDNF with various effects such as neuronal growth and differentiation and learning, memory, antidepression and the like.

Neurotrophic factor regulates the survival and differentiation of neurons during development (Davies, 1994), and maintains neural structure and activity of ion channels, neurotransmitter release, and axon pathways during the lifetime of the individual axon path-finding) (Schnell et al., 1994; Song and Poo, 1999; Schinder and Poo, 2000). Examples of such neurotrophic factors include neuronal growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophs. Pin-4 / 5 (Neurotrophin-4 / 5, NT-4 / 5) is present.

Among the neurotrophic factors, BDNF is expressed in the hippocampus, cortex and basal brain which are responsible for learning, memory, and higher thinking in the central nervous system (CNS). BDNF expressed in this position is a major regulator of synaptic plasticity and recognition processes, which is the neurobiochemical basis for synaptic transmission and learning and memory (Lu B, 2003). Not only does it promote long-term potentiation (LTP), a cellular phenomenon in learning and memory processes, but it also inhibits long-term depression, LTD (opposite) (Ikegaya, Y. et al., 2002; Huber, K. et al., 1998). Among neurotrophic factors, this unique role is consistent with the fact that BDNF and its receptor, TrkB (tropomyosin-related kinase B), are widely distributed in glutamate synapses.

BDNF, an important regulator of synaptic transmission and synaptic plasticity, is associated with a variety of diseases. Typical examples include degenerative brain diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), depression from stress, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative diseases or diabetic neuropathy Etc. can be mentioned.

Alzheimer's disease causes deterioration of cognitive ability by the degeneration of cholinergic neurons in the whole brain and a decrease in the release of the neurotransmitter acetylcholine (Murer MG et al., 2001). BDNF is known to promote the survival and differentiation of basal cholinergic neurons as well as to stimulate the release of acetylcholine in these neurons (Knipper M et al., 1994). Therefore, it is suggested that deterioration of homeostasis of cells due to lack of synthesis of BDNF will cause Alzheimer's disease. It is also known that neuroplastic deficiency, ie reduction in synaptic contact, presents neuropathological and clinical signs (Mesulam MM., 1999). Clinical results showed that post-mortem brain necropsy in Alzheimer's disease significantly reduced the expression of BDNF and its receptor, TrkB, in the hippocampus and cortex, which are responsible for learning and memory (Phillips HS et al., 1991; Holsinger RM). et al., 2000; Allen SJ et al., 1999). BDNF is known to regulate long-term potentiation (LTP), a phenomenon in cellular processes of learning and memory through synaptic plasticity at these sites (Figurov, A. et al., 1996). Therefore, it is also thought that a decrease in the function of the recognition and memory process by reducing the expression of BDNF will cause Alzheimer's disease.

Parkinson's disease results in a decrease in the number of substantia nigral dopaminergic neurons, leading to depletion of striatal dopamine in the striatum, leading to symptoms such as motor dysfunction. Another characteristic of Parkinson's disease patients is that the cognitive function is deteriorated. As a protein associated with the cause of Parkinson's disease, neurotrophic factors are expected to play an important role in the protection of dopaminergic neurons (Siegel GJ and Chauhan NB., 2000). Among neurotrophic factors, the interaction of BDNF with dopaminergic neurons is well known. Dopaminergic neurons in the ventral midbrain, melanoma and ventral tegmental areas express BDNF (Seroogy KB et al., 1994), resulting in reduced BDNF expression in the melanoma region, resulting in melanoma dopaminergic neurons. The number of is significantly reduced (Porritt MJ et al., 2005). In addition, we report that BDNF is necessary to maintain the appropriate number of dopamine neurons in the vaginal cells (Baquet ZC et al., 2005), and in fact, in Parkinson's patients, the BDNF expression of the stratified dopamine neurons was significantly reduced (MogiM et. al., 1999; Howells DW et al., 2000) reported that the reduction of dopaminergic neurons in Parkinson's disease is associated with a deficiency of BDNF biosynthesis.

The number of depressed patients is increasing rapidly in the modern society, which is heavily stressed. Stress is known to reduce the volume of various brain regions, including nervous breakdown and hippocampus (Duman, RS and Monteggia, LM 2006), as well as reducing BDNF mRNA expression in the hippocampus (Duman, RS and Monteggia, LM 2006 Smith, MA et al., 1995). In postmortem brain necropsy of depressed patients, the expression of BDNF was significantly reduced compared to normal subjects, and the volume of hippocampus was significantly reduced in brain imaging (Sheline, Y.I et al., 2003). Various chemical antidepressants have been used to treat these depressions. Commonly, the expression of BDNF mRNA is significantly increased in the hippocampus, anterior cerebral cortex or both regions (Duman, R.S. and Monteggia, L.M. 2006). These results suggest the hypothesis of 'neurotrophin hypothesis of depression'. In other words, the treatment of mental disorders such as depression by restoring neuronal breakdown and cell number reduction by re-increasing the expression of reduced BDNF in the hippocampus and the anterior cerebral cortex by stress (Duman, R.S. and Monteggia, L.M. 2006).

As described above, BDNF exhibits various functions such as neuronal cell growth and differentiation, learning and memory, and antidepressant as well as maintenance of neural structure, activation of ion channels, and release of neurotransmitters. Along with these diverse functions, BDNF can be used for Alzheimer's disease, Parkinson's disease, depression, stroke, a chronic stress disorder (Schabitz et al., Stroke, 38: 2165-2172, 2007), and Huntington's chorea (Zuccato C et al., Science 293, 20, July 2001), cerebral ischemic disease (Han BH et al., The Journal of neuroscience, 2000, 20 (15): 5775-5781, August 1, 2000), neurodegenerative disease (Tsuzaka K et al., Muscle Nerve 24 (4): 474-80, April 2001) or diabetic neuropathy (Nitta A et al., Neurotoxicology and Teratology, 24: 695-701, 2002). However, BDNF itself is a 14kDa macromolecular protein that does not cross the blood-brain barrier (BBB) and has problems such as administration method. Therefore, it is easy to cross the blood brain barrier and has very low cytotoxicity, and by discovering a substance that increases the expression of BDNF by administering a small amount, it is possible to detect neurological diseases such as Alzheimer's disease, Parkinson's disease, depression, stroke, which are chronic stress diseases. It can be used for prevention and treatment.

The present inventors prepared an MVG peptide consisting of three amino acids by selecting the amino acids with the highest expression of BDNF at each position through a peptide library technology called PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library). The present invention was completed by confirming that BDNF mRNA and protein expression is increased in hippocampal neurons, hippocampal tissues and cerebral cortical tissues by MVG peptide.

It is an object of the present invention to provide peptides that increase mRNA expression of Brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissues and cerebral cortical tissues.

Another object of the present invention is to provide a peptide for increasing protein expression of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissues and cerebral cortical tissues.

Another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of neurological diseases comprising a therapeutically effective amount of the peptide.

In order to achieve the above object, the present invention is composed of the amino acid (MVG; Methionine-Valine-Glycine) described in SEQ ID NO: 1, BDNF (Brain-derived neurotrophic factor) in hippocampal neurons, hippocampal tissue and cerebral cortical tissue Peptides that increase the expression of mRNA (hereinafter referred to as 'MVG peptide').

The present invention also provides a peptide consisting of the amino acids set forth in SEQ ID NO: 1, which increases the protein expression of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissues and cerebral cortical tissues.

The present invention also provides a polynucleotide consisting of a DNA sequence encoding the peptide. The polynucleotide comprises an equivalent nucleic acid sequence, that is, a codon degeneracy sequence that encodes MVG peptides of different but identical sequences.

Specifically, the present inventors selected the amino acid with the highest expression of BDNF at each position through a peptide library technology called PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library) (MVG; Methionine-Valine-Glycine) Afterwards, it was again confirmed by RT-PCR and Western blotting that expression increased substantially at the cellular level. In addition, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) test was performed to determine whether MVG peptide is toxic at the cellular level. To confirm the expression of BDNF at the actual animal level, 0.1 μg / kg of peptide was injected through the tail vein, and the hippocampus and cerebral cortex were removed and Western blotting was performed.

As a result, the present inventors confirmed that the MVG peptide consisting of three amino acids induced an increase in BDNF expression not only in hippocampal neuronal cell line but also in hippocampal tissue and cerebral cortical tissue, and showed no cytotoxicity up to 1 mM at the cellular level. It was. Hereinafter, the present invention will be described in more detail.

In one embodiment, the present inventors incubated HT22 cells, a mouse hippocampal neuronal cell line, in a 24 well plate at 5 × 10 ⁴ per well for 12 hours, and then prepared PS-SPCL stock (stock). Was treated to each well. After 12 hours, the protein was obtained using RIPA (Radio-immunoprecipitation Assay) buffer to which the protease inhibitor was added, and the amount of BDNF was measured through Enzyme-Linked ImmunoSorbent Assay (ELISA). As a result, as shown in FIG. 1, M (Methionine) at the first position, V (Valine) at the second position, and G (Glycine) at the third position expressed the most BDNF.

In another embodiment, the results show that the peptide composed of the amino acid (MVG) having the highest expression of BDNF at each position is resynthesized and treated to H19-7 cells of rat hippocampal neuronal cell line to increase the expression of BDNF mRNA. It was confirmed by RT-PCR. As a result, BDNF mRNA expression was increased in a dose-dependent manner in the experimental group treated with 0.001, 0.01, 0.1 μM MVG peptide as shown in FIG.

In another embodiment, the BDNF protein expression was confirmed by Western blotting by treating the hippocampal neuronal H19-7 cells with MVG peptide. As a result, as shown in FIG. 3, BDNF protein expression was increased in a dose-dependent manner in the experimental group treated with 0.001, 0.01, and 0.1 μM MVG peptide as compared to the control group.

In another embodiment, male SD (Sprague-Dawely) rats (4 weeks of age) are purchased and subjected to stabilization for 1 week, after which 1X PBS (control) and the MVG peptides determined above are 0.1 μg / Kg injections and hippocampal and cerebral cortical tissues were performed and Western blotting was performed. As a result, it was confirmed that the expression of BDNF and proBDNF protein in the experimental group increased as shown in Figure 4, 5 compared with the control group.

In another embodiment, H19-7 cells treated with MVG peptide from 10nM to 1mM to confirm the cytotoxicity of the MVG peptide, and then confirmed the survival rate of the cell through the MTT solution did not show a significant difference from the control group It was confirmed (FIG. 6).

The present invention provides a pharmaceutical composition for the prevention and treatment of neurological diseases comprising a therapeutically effective amount of the MVG peptide of the present invention. Examples of the neurological diseases include Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative disease or diabetic neuropathy, and the like. In general, the pharmaceutical composition is prepared in various forms by mixing a pharmaceutically acceptable non-toxic carrier that is commercially available according to the method of administration with the MVG peptide of the present invention, the prepared pharmaceutical composition is a neurological disease described above It can be administered as a prophylactic and therapeutic agent of.

Pharmaceutically acceptable carriers compatible with various formulations include all forms of diluents or solvents, fillers, fillers, binders, dispersants, disintegrants, surfactants, lubricants, excipients, and wetting agents. Furthermore, if necessary, commercially available dissolution aids, buffers, preservatives, colorants, flavors, sweeteners, and the like may also be added to the drug.

The dosage unit form of the pharmaceutical composition according to the present invention is not particularly limited and may be widely selected according to various therapeutic purposes, for example, tablets, capsules, granules, pills, syrups, solutions, emulsions, suspensions. Oral dosages such as and the like, injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, etc.) and parenteral administration such as suppositories. Among these formulations, oral dosage formulations are preferred.

The various types of medicaments described above can be prepared by conventional methods, for example in the preparation of oral dosage forms such as tablets, capsules, granules and pills, they are known as sucrose, lactose, glucose, starch, mannitol Excipients; Binders such as syrup, gum arabic, sorbitol, trigacanth rubber, methylcellulose, polyvinylpyrrolidone and the like; Disintegrants such as starch, carboxymethyl cellulose and its calcium salts, microcrystalline cellulose, polyethylene glycol and the like; Lubricants such as talc, magnesium stearate, calcium stearate, silica and the like; It may be prepared by conventional methods using wetting agents such as sodium laurate, glycerol and the like.

In the preparation of injectables, solutions, emulsions, suspensions and syrups, they are prepared by conventional methods, such as ethyl alcohol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol, castor oil and the like. Solvent for component dissolution; Surfactants such as sorbitol fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene ester, polyoxyethylene ether of hydrogenated castor oil, lecithin and the like; Manufactured using appropriate preservatives such as cellulose derivatives such as sodium carboxymethyl cellulose, methyl cellulose, suspending agents such as natural rubbers such as trigacanth rubber, gum arabic, esters of paraoxybenzoic acid, benzalkonium chloride, sorbitan fatty acid salt Can be.

In preparing suppositories, they can be prepared using excipients such as polyethylene glycol, lanolin, coconut oil and the like using conventional methods.

The dose of the pharmaceutical composition for the prevention and treatment of neurological diseases, including the therapeutically effective amount of the MVG peptide according to the present invention, depends on the method of administration, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease. It may be appropriately selected and is not specifically limited. The therapeutically effective amount of the MVG peptide included in the pharmaceutical composition of the present invention may be administered at 0.1 μg / kg / day to 10 μg / kg / day. Of course, the therapeutically effective amount may be in an amount outside the ranges described above.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration can be envisaged, for example, by oral, subcutaneous, intravenous, intramuscular, nasal, intraperitoneal, rectal, intrauterine dural or intracerebroventricular injection. The MVG peptide of the present invention has little toxicity and side effects, and therefore can be used with confidence even for prolonged administration for prophylactic purposes.

MVG peptide consisting of three amino acids according to the present invention not only increases the expression of BDNF without cytotoxicity in the hippocampal neuronal cell line but also increases the expression of BDNF in hippocampal and cortical tissues. Therefore, MVG peptides, such as the present invention, are small in molecular weight, easily cross the Blood Brain Barrier (BBB), and have no cytotoxicity. It is very effective as a preventive and therapeutic agent for neurological diseases such as cerebral ischemic disease, neurodegenerative disease and diabetic neuropathy.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these embodiments, it will be apparent to those skilled in the art and those skilled in the art. will be.

Example  1: PS- SPCL Initial screening

PS-SPCL trimmer package stock was supported by the Peptide library support facility at Pohang University of Science and Technology for the discovery of peptides that increase BDNF expression. PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library), a peptide library technology used in the present invention, is a peptide consisting of three amino acids, the first position of which is substituted with tyrosine in alanine except cysteine. And the remaining second and third positions are fixed in such a way as to identify the most effective amino acid sequence at each position, thereby finding the amino acid with the highest BNDF expression at each position.

First, 57 mouse hippocampal neuronal cell lines, HT22 cells, were placed in a 24-well plate (SPL) at a total of 57 by ⁵ × 10 ⁴ per well for 12 hours at 37 ° C. in a 5% CO ₂ incubator (Incubator, VISION). Incubated in the. After spin down of the PS-SPCL trimmer package stock, the tube was opened, tapped with 15 μl of distilled water (DW) for cell culture, and then spun down again. 15 μl of PS-SPCL trimmer package stock prepared on a 24-well plate in which HT22 cells are incubated is placed in each well and incubated in an incubator for 12 hours. After 12 hours, the medium in the well was discarded and washed twice with 1 ml of 1X PBS (Potassium persulfate). 10 μl of protease inhibitor (Amersham) per 1 mL of RIPA was prepared, placed in each well, and the cell contents were placed in a 1.5 mL tube using a cell lifter (Cell Lifter, Corning). Pelletize the cell membrane by centrifugation (Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) for 5 minutes at 4 ° C., 9000 rpm, and transfer the supernatant containing protein to a new 1.5 ml tube. The concentration of was quantified using the Bradford assay.

In order to measure the amount of BDNF expression, ELISA (Enzyme-linked immunosorbent assay, BDNF ELISA Kit, Promega) was performed according to the protocol. As shown in FIG. 1, M (Methionine) in the first position, V (Valine) in the second position, and G (Glycine) in the third position showed the highest BDNF expression.

Example  2: Hippocampal nerve cell line H19 From -7 MVG On peptides  by BDNF mRNA  Increase in expression

As a result of Example 1, an MVG synthetic peptide consisting of three amino acids was purchased from Peptron Co., Ltd. H19-7 cells, the hippocampal neuronal cell lines of rats purchased from the American Type Culture Collection (ATCC), were placed in 6 well plates (NUNC) at 10 ⁶ per well and incubated in a 34 ° C., 5% CO ₂ incubator for 12 hours. , MVG synthetic peptides were treated with 0.001, 0.01, 0.1, 1μM and further incubated for 12 hours. RNA was isolated from each sample according to the protocol of the Easy-blue Total RNA extraction kit (Intron), and then 1 ug total RNA, 1 ol oligo (dT) ₁₈ and the rest were processed using DW treated with DEPC (Diethylpyrocarbonate). The volume was added to the tube for PCR to 20 μl and heated at 70 ° C. for 5 minutes. Complementary DNA (cDNA) was prepared by transferring 20 μl of the tube heated with RT premix (Reverse transcriptase premix, Bioneer) tube and performing reverse transcription at 42 ° C. for 1 hour. 5 μl of the prepared cDNA, 2 μl of BDNF primer and 13 μl of DDW (Double Distilled Water) PCR were placed in a PCR premix (Bioneer) tube for 5 minutes at 94 ° C., 30 seconds at 94 ° C., 30 seconds at 60 ° C., 45 at 72 ° C. The cycle was turned 5 minutes and 29 cycles at 72 ° C. As a result, as shown in Figure 2 it can be seen that the amount of BDNF mRNA expression increases by concentration at 0.001, 0.01, 0.1μM. This demonstrates that MVG synthetic peptides increase BDNF mRNA expression in hippocampal neuronal cell lines of rats, and that peptide selection through Example 1 was achieved.

Example  3: Hippocampal nerve cell line H19 From -7 MVG On peptides  by BDNF  Increase in protein expression

As a result of Example 1, an MVG synthetic peptide consisting of three amino acids was purchased from Peptron Co., Ltd. H19-7 cells, rat hippocampal neuronal cell lines purchased from the American Type Culture Collection (ATCC), were placed in 6 well plates (NUNC) at 10 ⁶ per well and incubated in a 34 ° C., 5% CO ₂ incubator for 12 hours. , MVG synthetic peptides were treated with 0.001, 0.01, 0.1, 1μM and further incubated for 12 hours. Obtain protein in the same manner as in Example 1, make 15% acrylamide gel through 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and load 30 μg sample ) Was run for 2 hours and 30 minutes at 100V with an electrophoresis device (Electrophoresis Power supply, EPS 601, Amersham). Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and a rabbit anti BDNF polyclonal antibody (AB1534, chemicon) in a ratio of 1: 1000 React overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, as shown in Figure 3 it can be seen that the amount of BDNF protein expression increased by concentration at 0.001, 0.01, 0.1μM. In addition, this shows the same increase pattern as the BDNF mRNA expression results shown in FIG.

Example  4: in the hippocampus MVG On peptides  by BDNF  Increase in protein expression

Four-week-old male SD (Sprague-Dawley) rats were purchased from Orient Co., Ltd. and separated into three groups of experimental groups (MVG-administered group) and control group (1X PBS-administered group). Adaptation and stabilization were made. After one week, the experimental group was tail vein injection of 0.1 μg / kg of MVG and the control group of 0.1 μg / kg of 1 × PBS. After 12 hours, anesthesia was temporarily used in ether, and blood was collected from the heart after laparotomy. The brains of the rats were quickly extracted, rapidly cooled in liquid nitrogen, and stored at -80 ° C. After separating only the hippocampus from the brain, the RIPA buffer and protease inhibitor were prepared in the same ratio as in Example 1, and the hippocampal tissue was completely ground using a Homogenizer (RZR2020M, Heidolph). Centrifuge at 4 ° C and 2,000rpm for 30 minutes (Centrifuge, UNION 32R, Hanil Science Industrial Co., Ltd), transfer only the supernatant to 1.5ml tube, and then centrifuge for 30 minutes at 4 ° C, 9,000rpm ( Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) was used to quantify the protein by Bradford assay once again transfer only the supernatant to 1.5ml tube.

A 15% acrylamide gel was made through a 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and 30 μg of sample was loaded to obtain an electrophoresis power supply. EPS 601, Amersham) for 2 hours 30 minutes at 100V. Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and a rabbit anti BDNF polyclonal antibody (AB1534, chemicon) in a ratio of 1: 1000 React overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, as shown in FIG. 4, the amount of BDNF and its precursor (precursor) form proBDNF in the experimental group compared to the control group was confirmed to increase compared to β-actin.

Example  5: in cortical tissue MVG On peptides  by BDNF  Increase in protein expression

Four-week-old male SD (Sprague-Dawley) rats were purchased from Orient Co., Ltd. and separated into three groups of experimental groups (MVG-administered group) and control group (1X PBS-administered group). Adaptation and stabilization were made. After one week, the experimental group was tail vein injection of 0.1 μg / kg of MVG and the control group of 0.1 μg / kg of 1 × PBS. After 12 hours, anesthesia was temporarily used in ether, and blood was collected from the heart after laparotomy. The brains of the rats were quickly extracted, and then rapidly cooled in liquid nitrogen and stored at -80 ° C. After only the cerebral cortex was separated from the extracted brain, RIPA buffer and protease inhibitor were prepared in the same ratio as in Example 1, and the cortical tissue was completely ground using a Homogenizer (RZR2020M, Heidolph). Centrifuge at 4 ° C and 2,000rpm for 30 minutes (Centrifuge, UNION 32R, Hanil Science Industrial Co., Ltd), transfer only the supernatant to 1.5ml tube, and then centrifuge for 30 minutes at 4 ° C, 9,000rpm ( Centrifuge, MICRO 17TR, Hanil Science Industrial Co., Ltd) was used to quantify the protein by Bradford assay once again transfer only the supernatant to 1.5ml tube.

A 15% acrylamide gel was made through a 1.5 mm Western blotting gel caster (Gel caster, Bio-Rad) and 30 μg of sample was loaded to obtain an electrophoresis power supply. EPS 601, Amersham) for 2 hours 30 minutes at 100V. Polyvinylidene Difluoride (PVDF) was used to transfer the gel (Mighty small transphor, Amersham) using an electrophoresis power supply (EP 301, GE healthcare) at 400 mA for 2 hours and 30 minutes. Blocking PVDF for 1 hour with 7% skimmed milk (Skim milk, Difco ^™ Skim milk, BD) and a rabbit anti BDNF polyclonal antibody (AB1534, chemicon) in a ratio of 1: 1000 React overnight. After washing for 1 hour with 0.05% TBST (Tris-Buffered Saline Tween 20) (3 times for 20 minutes), Goat anti-rabbit IgG: Horseradish Peroxidase Conjugate, SAB-300, Bioreagents Incubate for 50 minutes at a ratio of 1: 3000. After washing for 1 hour with 0.05% TBST (3 times for 20 minutes), detection was performed with a chemiluminescent HRP substrate kit (Millipore). As a result, as shown in FIG. 5, the amount of BDNF and its precursor (precursor) form proBDNF in the experimental group was higher than that of the control group, compared to β-actin.

Example  6: MVG Peptide  Cytotoxic effect measurement

H19-7 cells were incubated in 96 well plates (FALCON) at 2 × ¹⁰ 3/200 μl and incubated in 34 ° C., 5% CO ₂ incubator for 12 hours, and then treated with MVG synthetic peptide from 10 nM to 1 mM and 3 days Incubated for After 3 days, 20 μl of MTT (50 mg / ml) solution was treated, wrapped in aluminum foil, and incubated again in an incubator. After 4 hours, the medium and the MTT solution were removed, treated with 200 μl of DMSO (Dimetyl sulfoxide) solution, wrapped in foil, and placed at room temperature for 4 hours. The absorbance was measured at 570 nm using a microplate reader (Molecular device) to convert the control to 100% to calculate the value. As a result, as shown in Figure 6 it did not show cytotoxicity at a concentration from 10nM to 1mM compared to the control. Therefore, MVG synthetic peptide according to the present invention increases the protein expression of BDNF in hippocampal neuronal cell line, hippocampal tissue and cerebral cortex, and there is no cytotoxicity, Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea It can be effectively used as a pharmaceutical composition for the prevention and treatment of cerebral ischemic diseases, neurodegenerative diseases, diabetic neuropathy and the like.

While the invention has been described above with reference to specific embodiments, various modifications, changes or modifications are possible in the art within the spirit and scope of the appended claims, and thus the foregoing description and drawings illustrate the invention. It should not be construed as limiting the technical spirit of the present invention but as illustrating the present invention. All documents cited in the present invention are incorporated herein by reference.

Figure 1 shows the results of finding the amino acid with the highest BDNF expression at each position of the peptide consisting of three amino acids (Methionine-Valine-Glycine) through the PS-SPCL (Positional Scanning-Synthetic Peptide Combinatorial Library) .

Figure 2 shows the results of mRNA expression of BDNF after treatment of MVG synthetic peptide of the present invention in rats (Hat), a hippocampal nerve cell line of rat (Rat) by concentration.

Figure 3 shows the results of protein expression of BDNF after treatment of the MVG synthetic peptide of the present invention to the rat hippocampal neuronal cell line H19-7 by concentration.

Figure 4 shows the results of BDNF protein expression by extracting the hippocampal tissue site after the rat intravenous injection of 0.1g / kg MVG synthetic peptide of the present invention.

Figure 5 shows the results of BDNF protein expression by extracting the cerebral cortical tissue site after the tail vein injection of 0.1V / kg MVG synthetic peptide of the present invention.

Figure 6 shows the cytotoxicity results of MVG synthetic peptides by MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) test.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> A novel peptide for increasing the protein expression of          Brain-derived neurotropic factor in the hippocampal neuronal          cell, the hippocampal tissue and the cerebral cortex tissue <130> IPDC-35086 <160> 1 <170> KopatentIn 1.7 <210> 1 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Methionine-Valine-Glycine (MVG) peptide <400> 1 Met val gly   One  

Claims (10)

A peptide for increasing mRNA expression of a brain-derived neurotrophic factor (BDNF), consisting of the amino acids set forth in SEQ ID NO: 1. The peptide of claim 1, wherein the peptide increases mRNA expression of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampus, and cerebral cortex. A peptide that increases the protein expression of a brain-derived neurotrophic factor (BDNF), consisting of the amino acids set forth in SEQ ID NO: 1. 4. The peptide according to claim 3, which increases protein expression of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, hippocampal tissue and cerebral cortex. The peptide according to claim 2 or 4, wherein the hippocampal neurons are mouse HT22 cell lines or rat H19-7 cell lines. A polynucleotide consisting of a DNA sequence encoding the peptide of claim 1. Prevention and treatment of Alzheimer's disease, Parkinson's disease, chronic stress depression, stroke, Huntington's chorea, cerebral ischemic disease, neurodegenerative disease, or diabetic neuropathy comprising a therapeutically effective amount of the peptide of claim 1 or 3 Pharmaceutical composition for. 8. The pharmaceutical composition of claim 7, wherein the therapeutically effective amount of the peptide is from 0.1 μg / kg / day to 10 μg / kg / day. 8. A pharmaceutical composition according to claim 7, which is administered orally, subcutaneously, intravenously, intramuscularly, intranasally, intraperitoneally, rectally, intrauterine or intraventricularly. 8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is prepared in the form of tablets, capsules, granules, pills, syrups, solutions, emulsions, suspensions, injections or suppositories.

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