WO2015108372A1 - Composition for preventing or treating neurological disorders caused by excitotoxicity or synaptic dysfunction, containing osmotin, and method for preventing or treating neurological disorders by using same - Google Patents

Abstract

The present invention relates to: a composition for preventing, alleviating or treating neurological disorders caused by excitotoxicity or synaptic dysfunction; and a method for preventing or treating neurological disorders by using the same. Specifically, the present invention relates to: a pharmaceutical composition or a food composition for preventing, alleviating or treating neurological disorders caused by excitotoxicity or synaptic dysfunction, containing osmotin as an active ingredient; and a method for preventing or treating neurological disorders caused by excitotoxicity or synaptic dysfunction, by using the composition.

Description

A composition for preventing or treating neurological diseases caused by excitatory toxicity or synaptic dysfunction, including osmotin, and a method for preventing or treating neurological diseases using the same

The present invention relates to a composition for preventing, ameliorating or treating neurological diseases caused by excitatory toxicity or synaptic dysfunction, and a method for preventing or treating neurological diseases using the same.

Specifically, the present invention relates to a pharmaceutical composition or a food composition for preventing, ameliorating or treating a neurological disease caused by excitatory toxicity or synaptic dysfunction, including osmotin as an active ingredient, and using the composition, excitatory toxicity or synapse A method for preventing or treating a neurological disorder caused by a dysfunction.

Excitotoxicity refers to a pathological process in which nerve cells are damaged or killed by nerve cells being overstimulated by neurotransmitters such as glutamate or the like. Excitatory toxicity-mediated damage or death of nerve cells has been reported in various central nervous system diseases including ischemia and neurodegenerative diseases.

Previous studies conducted in vitro and in vivo on excitatory toxicity report that excess stimulation of NMDA or AMPA glutamate receptors induces neuronal apoptosis, and glutamate and glutamate activation excess in some neurological diseases It is reported to appear. During the excessive stimulation of glutamate receptors, conventional studies on NMDA glutamate receptors suggest that intracellular calcium increase, activation of catabolic enzymes and caspases, and free radicals and nitrogen free by the continuous activation of NMDA glutamate receptors. It is reported that by inducing the production of radicals, the resulting cell apoptosis.

However, the exact mechanism of glutamate-induced excitatory toxicity has not been elucidated yet.

Osmotin is a multifunctional plant protein derived from Nicotiana tabacum and the like and has a molecular weight of about 24 kDa. Osmotin is a protein belonging to the family of pathogenesis related-5 (PR-5) that provides plants with osmotolerance and exhibits antimicrobial activity. Osmotin is believed to be a structural and functional homologue with the mammalian hormone adiponectin and can act biologically as an adiponectin counterpart. The anti-inflammatory, anti-diabetic, and anti-atherogenic effects have been reported in relation to the efficacy of adiponectin, and the effects of osmotin as an adiponectin counterpart on the anti-inflammatory effect of adiponectin have been shown in murine colitis animal model Reduction has been reported. In addition, only the effects related to obesity and diabetes have been studied in relation to osmotin, and studies on the efficacy related to neurological diseases of osmotin are insufficient.

Regarding the efficacy of osmotin against neurodegenerative diseases, Korean Patent No. 10-1308232 discloses a composition for treating neurodegenerative diseases by alcohol containing osmotin, but the registered patent discloses an alcohol of osmotin. Efficacy of the neurodegenerative diseases induced by the present invention was only identified, and no effect was suggested for the neurological diseases caused by excitatory toxicity or synaptic dysfunction.

The present inventors have diligently tried to determine the effect of osmotin in neurological diseases caused by excitatory toxicity or synaptic dysfunction. As a result, the osmotin is an AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptor. By reducing the expression of, such as, it was confirmed that there is an effect of preventing or treating excitatory toxicity or synaptic dysfunction due to excessive stimulation by glutamate, and came to complete the present invention utilizing the same.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurological diseases caused by excitatory toxicity or synaptic dysfunction comprising osmotin as an active ingredient.

Another object of the present invention to provide a food composition for the prevention or improvement of neurological diseases caused by excitatory toxicity or synaptic dysfunction comprising osmotin as an active ingredient.

Still another object of the present invention is to provide a method for preventing or treating neurological diseases caused by excitatory toxicity or synaptic dysfunction using the composition.

The composition containing the osmotin of the present invention as an active ingredient may be usefully used as a substance for preventing or treating excitatory toxicity or synaptic dysfunction induced by excessive stimulation of a neurotransmitter, and the neurological diseases caused thereby. . In addition, the effect of inhibiting the expression of the AMPA glutamate receptor is excellent, particularly the effect of preventing or treating excitatory or synaptic dysfunction induced by glutamate, and the neurological diseases caused thereby.

1A and 1B show changes in expression of AMPA glutamine receptor (AMPAR), p-AMPAR and CaMKII in the cortex and hippocampus of osmotin treatment.

1C and 1D show changes in expression of p-CREB and synaptopycin in the cortex and hippocampus of the brain following osmotin treatment.

2A and 2B show changes in expression of p53, Bax and Bcl-2 in the cortex and hippocampus of the brain following osmotin treatment.

2C and 2D show changes in the expression of cytochrome-C, caspase-3 and PARP-1 in the cortex and hippocampus of the brain following osmotin treatment.

3A and 3B show changes in the degree of DNA fragmentation in three regions of the hippocampus (CA1, CA3 and DG) following osmotin treatment.

4A and 4B show changes in the expression of p-JNK, p-PI3K and p-Akt in cortex and hippocampus following osmotin treatment.

4C and 4C show changes in expression of p-JNK and p-PI3K in CA1 region of hippocampus following osmotin treatment.

5A and 5B show changes in distribution and colocation of p53 and p-Akt in CA1 region of hippocampus following osmotin treatment.

5C and 5D show changes in the distribution and colocation of p53 and caspase-3 in the CA1 region of the hippocampus following osmotin treatment.

6 is a simplified schematic of the mechanism of action of osmotin on biomechanisms induced by glutamate.

1 to 6, "C" is a control group, "Glu" is a glutamate treatment group, "Os" is an osmotin treatment group, and "Glu + Os" means a glutamate and osmotin treatment group.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases caused by excitatory toxicity or synaptic dysfunction comprising osmotin as an active ingredient as one embodiment.

As used herein, the term "osmotin" is a multifunctional plant protein derived from Nicotiana tabacum and the like, and is composed of about 150 to 250 amino acids depending on the individual, and has a molecular weight of about 24 kDa. It is known. Osmotin is a member of the PR-5 (Pathogenesis related-5) family that provides plants with osmotolerance and exhibits antimicrobial activity. It is believed that it can act as an adiponectin counterpart. The anti-inflammatory, anti-diabetic, and anti-atherogenic effects have been reported in relation to the efficacy of adiponectin, and the effects of osmotin as an adiponectin counterpart on the anti-inflammatory effect of adiponectin have been shown in murine colitis animal model Reduction has been reported. Osmotin is also known to be effective in treating obesity, diabetes and cancer.

In the prior art related to the neuropathy of osmotin, Korean Patent No. 10-1308232 discloses that osmotin exhibits an effect of inhibiting the death of alcohol-induced nerve cells, and by using the neurodegenerative disease caused by alcohol A therapeutic composition is provided. However, the registered patent is limited to alcohol-induced neurodegenerative diseases, and there is no disclosure about neurological diseases induced by excitatory toxicity or synaptic dysfunction among neurological diseases, and the efficacy of osmotin on such neurological diseases. There was no suggestion about.

Accordingly, the present inventors first identified that osmotin has an effect of treating neurological diseases caused by excitatory toxicity or synaptic dysfunction, and particularly, has a turbulent effect in the treatment of excitatory toxicity or synaptic dysfunction caused by glutamate overstimulation. It was confirmed.

Neurodegenerative diseases caused by alcohol are neurological diseases in which neuronal cell death is induced by alcohol. In the prior patent, only the osmotin inhibits neuronal cell death, but the excitatory toxicity or synaptic function of the present invention. Neurological diseases induced by the disorder are induced by excessive stimulation by neurotransmitters (particularly, glutamate). In the present invention, the first clarification of glutathione receptor expression inhibition of osmotin and the like is performed. There is a difference in that it was first disclosed the therapeutic use of neurological diseases caused by excessive stimulation.

In a specific embodiment of the present invention, when treated with glutamate alone in Sqrague-Dawley rats at 7 days of age, the expression of AMPA glutamate receptors is increased, the expression of synaptopycin is decreased, and the expression of CaMKII is increased. Synaptic dysfunction symptoms were induced, but when osmotin was treated after glutamate treatment, all of the above symptoms were reversed, confirming that osmotin is effective in preventing or treating excitatory toxicity or synaptic dysfunction (Experimental Example). 1 and 2).

In the present invention, the osmotin can be used without limitation in its origin or type. The osmotin may be separated from various plants or artificially synthesized according to genetic engineering methods, or may be purchased from domestic and foreign companies.

In the present invention, the neurological disease caused by the excitatory toxicity or synaptic dysfunction may be included without limitation to the type or condition of the disease, as long as it is induced by the excitatory toxicity or synaptic dysfunction.

For example, the neurological disorder may be a mental neurological disorder, and examples of the mental neurological disorder include, but are not limited to, depression, schizophrenia, OCD, anxiety neurosis, and the like.

In addition, the neurological diseases include neurological diseases induced by nerve damage caused by excitotoxicity or synaptic dysfunction.

As used herein, the term "prevention" means any action that inhibits or delays neurological symptoms caused by excitatory or synaptic dysfunction by administering the pharmaceutical composition of the present invention to a subject.

As used herein, the term "treatment" refers to any action by which the pharmaceutical composition of the present invention is administered to a subject to improve or benefit neurological symptoms caused by excitatory toxicity or synaptic dysfunction.

The prevention or treatment of neurological diseases caused by the excitatory toxicity or synaptic dysfunction using the pharmaceutical composition comprising the osmotin of the present invention as an active ingredient may be performed by reducing expression of AMPA glutamate receptor or decreasing phosphorylation of AMPA glutamate receptor. Can be.

As used herein, the term “decreased expression” or “increased expression” means that the expression of the protein is reduced or increased at the level of transcription or the activity of the protein as compared to the condition prior to treatment of the composition comprising the osmotin of the invention. It means all cases that are reduced or increased. Said "reduced expression" or "increased expression" is for the purpose of the present invention, more preferably the expression of the protein of interest increased or decreased compared to the normal state induces excitatory or synaptic dysfunction, administration of the composition of the present invention It means all cases where the expression of the protein is reduced or increased than the condition before the administration of the composition of the present invention.

As used herein, the term "AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptor" refers to a type of channel glutamate receptor.

In a specific embodiment of the present invention, when treated with glutamate in rats of 7 days of age, it was confirmed that the expression of AMPA glutamate receptor and p-AMPA glutamate receptor is increased, but the osmotin of the present invention after the glutamate treatment In this case, it was confirmed that the expression of the AMPA glutamate receptor and the p-AMPA glutamate receptor was significantly reduced (Experimental Example 1).

Prevention or treatment of neurological diseases caused by the excitatory toxicity or synaptic dysfunction using the pharmaceutical composition comprising the osmotin of the present invention as an active ingredient, may be performed by increasing the expression of synaptopycin.

As used herein, the term "synaptophysin" is a glycoprotein of about 30 kDa present in the synaptic vesicle of nerve cells or secretory cells in the nerve, also known as synaptic vesicle protein p38. Mean protein.

In a specific embodiment of the present invention, the expression of synaptopycin was significantly reduced when glutamate was treated in the rat at 7 days of age, but the osmotin treatment of the present invention after the glutamate treatment resulted in the reduction of synaptopycin. It was confirmed that the expression level is increased (Experimental Example 2).

The prevention or treatment of neurological diseases caused by the excitatory toxicity or synaptic dysfunction using the pharmaceutical composition comprising the osmotin of the present invention as an active ingredient may be performed by reducing the expression of CaMKII.

As used herein, the term “CaMKII (Ca ²⁺ / calmodulin-depandent protein kinase II)” is a kind of serine / threonine-specific protein kinase, and means a kinase regulated by a Ca ²⁺ / calmodulin complex.

In a specific embodiment of the present invention, the expression of CaMKII was significantly increased when glutamate was treated in the rat at 7 days of age, but as a result of treating the osmotin of the present invention after the glutamate treatment, the expression level of CaMKII was increased. It was confirmed that the decrease by (experimental example 1).

Prevention or treatment of neurological diseases caused by the excitatory toxicity or synaptic dysfunction using the pharmaceutical composition comprising the osmotin of the present invention, the expression of p-JNK in the JNK / PI3K / Akt pathway, p-PI3K It can be carried out by increasing the expression of or by increasing the expression of p-Akt.

As used herein, the term "JNK / PI3K / Akt pathway" is a kind of intracellular signal transduction pathway, and a factor involved in the pathway "JNK" means "c-Jun N-terminal kinase" and " PI3K "means" Phosphoinositide 3-kinase "and" Akt "means" Protein kinase B (PKB) ".

In a specific embodiment of the present invention, p-JNK expression was increased and p-PI3K and p-Akt expression was reduced when glutamate was treated in rats of 7 days of age, but the osmotin of the present invention after the glutamate treatment As a result, it was confirmed that the increased expression level of p-JNK was significantly decreased, and the expression levels of p-PI3K and p-Akt were decreased significantly (Experimental Example 4).

The pharmaceutical composition comprising the osmotin of the present invention as an active ingredient also exhibits the effect of inhibiting apoptosis of nerve cells.

As used herein, the term "apoptosis" is a form of a cell controlled by genes and kills itself, and is a concept distinguished from necrosis, which is necrosis or pathological death of cells. Also called.

In a specific embodiment of the present invention, the expression changes of p53, Bcl-2, Bax, which are known to be involved in apoptosis, the delivery of mitochondrial cytochromes to the cell substrate and the activation of caspase-3 and PARP-1 were observed. It was.

The term "p53" used in the present invention is one of tumor suppressor factors, and is known to play a role in inhibiting abnormal proliferation of cells and inducing cancer cells to die.

As used herein, the term "Bcl-2" is a type of Bcl-2 family protein having a molecular weight of about 26 kDa and is known as an apoptosis inhibitor. Bcl-2 blocks apoptosis mediated by Fas antigen and TNF receptors, and overexpression of Bcl-2 inhibits apoptosis and is known to prolong cell life. Bcl-2 is also known to play a role in inhibiting the action of Bax and inhibiting the release of cytochrome C.

As used herein, the term "Bax" is known as an apoptosis inducing factor and belongs to the Bcl-2 family because Bcl-2 and its gene sequence are similar. When Bax is activated, apoptosis is promoted and cell death is increased. On the contrary, when Bcl-2 is activated, apoptosis is suppressed and cell death is reduced.

In a specific embodiment of the present invention, treatment of glutamate in rats at 7 days of age increased the expression level of p53 protein and the Bax / Bcl-2 ratio, the delivery of mitochondrial cytochrome C and the caspase-3 and PARP-1. Activation was promoted, but as a result of treating the osmotin of the present invention after the glutamate treatment, all of the above aspects were found to be reversed, and it was confirmed that the apoptosis of nerve cells was suppressed (Experimental Example 3).

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, in addition to including osmotin as an active ingredient.

In the present invention, the "pharmaceutically acceptable" means that it is commonly used in the pharmaceutical field that does not impede the biological activity and properties of the compound to be administered without stimulating the organism upon administration thereof.

In the present invention, the type of the carrier is not particularly limited and any carrier can be used as long as it is commonly used in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, ethanol, and the like. Can be. These may be used alone or in combination of two or more thereof.

In addition, the pharmaceutical composition of the present invention may be used by adding other pharmaceutically acceptable additives, such as excipients, diluents, antioxidants, buffers or bacteriostatic agents, if necessary, fillers, extenders, wetting agents, disintegrants, dispersants, surfactants , Binders or lubricants may be additionally added and used.

In the pharmaceutical composition of the present invention, osmotin may be included in 0.00001% to 99.99% by weight based on the total weight of the pharmaceutical composition, preferably 1% to 99.99% by weight, more preferably 10 It may be included in the weight% to 99.99% by weight, more preferably 50% to 99.99% by weight, and may also be included in the total content of the pharmaceutical composition.

The pharmaceutical compositions of the present invention can be formulated and used in a variety of formulations suitable for oral or parenteral administration.

Non-limiting examples of the formulation for oral administration include troches, lozenges, tablets, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, and the like. Can be mentioned.

In order to formulate the pharmaceutical composition of the present invention for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin and the like; Excipients such as dicalcium phosphate and the like; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax and the like can be used, and sweeteners, fragrances, syrups and the like can also be used.

Furthermore, in the case of a capsule, a liquid carrier such as fatty oil may be additionally used in addition to the above-mentioned materials.

Non-limiting examples of the parenteral preparations include injection liquids, suppositories, respiratory inhalation powders, spray aerosols, ointments, application powders, oils, creams, and the like.

In order to formulate the pharmaceutical composition for parenteral administration, a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a freeze-dried preparation, an external preparation, and the like may be used. As the non-aqueous solvent and the suspension, propylene glycol, polyethylene Glycols, vegetable oils such as olive oil, injectable esters such as ethyloleate and the like can be used.

Further, more specifically, when the pharmaceutical composition of the present invention is formulated as an injection solution, the composition of the present invention is mixed with water with a stabilizer or buffer to prepare a solution or suspension, which may be used as an ampoule or vial. It may be formulated for unit administration. In addition, when the pharmaceutical composition of the present invention is formulated with an aerosol, a propellant or the like may be combined with the additive to disperse the dispersed concentrate or the wet powder.

In addition, when the pharmaceutical composition of the present invention is formulated into an ointment, a cream, or the like, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide It may be formulated using such as carrier.

A pharmaceutically effective amount, effective dosage of the pharmaceutical composition of the present invention may vary depending on the method of formulating the pharmaceutical composition, the mode of administration, the time of administration and / or the route of administration, and the likeness of the reaction to be achieved by the administration of the pharmaceutical composition. The type and extent of the subject, the type, age, weight, general state of health, condition or extent of the disease, sex, diet, excretion, components of the drug or other composition used concurrently or simultaneously with the subject, Various factors and similar factors well known in the medical arts can be varied, and one of ordinary skill in the art can easily determine and prescribe a dosage effective for the desired treatment.

Administration of the pharmaceutical composition of the present invention may be administered once a day, may be divided into several times. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

In the case of injection administration of the pharmaceutical composition of the present invention, for a more preferable effect, it may be administered in an amount of 0.01 mg / kg / day to 100 mg / kg / day, more preferably from 0.1 mg / kg / day to It may be administered in an amount of 70 mg / kg / day, more preferably 1 mg / kg / day to 50 mg / kg / day. The administration may be administered one time, divided into several times, and the administration cycle may be appropriately selected.

The route of administration and mode of administration of the pharmaceutical composition of the present invention may be independent of each other, and are not particularly limited in the way, and may be any route of administration and mode of administration as long as the pharmaceutical composition can reach the desired site. Can follow. The pharmaceutical composition may be administered by oral or parenteral administration.

As the parenteral administration method, for example, intravenous administration, intraperitoneal administration, intramuscular administration, transdermal administration or subcutaneous administration may be used, and the method of applying, spraying or inhaling the composition to a diseased site may also be used. May be, but is not limited to these.

According to another embodiment of the present invention, there is provided a food composition for the prevention or improvement of neurological diseases caused by excitatory toxicity or synaptic dysfunction comprising osmotin as an active ingredient.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with alleviation or treatment of a condition, such as the degree of symptoms.

The food composition of the present invention is not particularly limited and includes a health functional food composition.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.

The kind of the food is not particularly limited, and includes all foods in a general sense. Non-limiting examples of foods that can be added to the material include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products, including other noodles, gums, ice cream, various soups, drinks, tea , A drink, an alcoholic beverage, and a vitamin complex.

When the health functional food composition of the present invention is a beverage composition, it may contain various flavors or natural carbohydrates and the like as additional ingredients, as in the usual beverage. Non-limiting examples of the natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin, cyclodextrin; Synthetic sweeteners such as saccharin and aspartame; and the like. The proportion of the additional components added above may be appropriately determined by the choice of those skilled in the art.

In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the health functional food composition of the present invention may contain a flesh for preparing natural fruit juice, fruit drink or vegetable drink. These components can be used independently or can be used in combination of 2 or more. The proportion of such additives may also be appropriately selected by those skilled in the art.

According to another embodiment of the present invention, there is provided a method for preventing or treating neurological diseases caused by excitotoxicity or synaptic dysfunction, comprising administering to a subject the pharmaceutical composition comprising osmotin as an active ingredient.

According to another embodiment of the present invention, there is provided a method for preventing or ameliorating neurological diseases caused by excitatory toxicity or synaptic dysfunction, comprising administering to a subject the food composition comprising osmotin as an active ingredient.

As used herein, the term "individual" means all animals, including mammals, including rats, domestic animals, humans, and the like.

In the present invention, the description of the dosage, route of administration, mode of administration and the like of the composition is replaced with the contents described in connection with the pharmaceutical composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention by way of example, and the scope of the present invention should not be construed as being limited by these examples.

Example 1: Animal and Drug Treatment

Twenty Sprague-Dawley rats (average weight 18 g) after 7 days of birth were divided into four groups of five animals per group, each of which was added to each group (0.9%) [control], glutamate (10 mg / kg). , Osmotin (15 μg / g), and glutamate (10 mg / kg) + osmotin (15 μg / g) were treated by subcutaneous injection method. In the drug treatment, glutamate treatment was performed 1 hour or 2 hours before osmotin. Rats from each group were sacrificed 4 to 12 hours after the drug treatment. All experimental and ethical procedures of this example were made in accordance with the Division of Applied Life Sciences Local Animal Ethics Committee at the Department of Biology, Gyeongsang National University, Korea.

In the above glutamate is Sigma-Aldrich Co. It was purchased from LLC and osmotin was purified from salt-adapted cultured Nicotiana tabacum cells. The endotoxin content of osmotin was <0.03 EU / mg protein.

Example 2: Tissue Collection and Sample Preparation

Brain sections of pups treated with saline, glutamate or glutamate + osmotin in Example 1 were analyzed 12 hours after drug treatment. Transcardial perfusion was performed by treatment with 1 × phosphate buffered saline (PBS) and 4% ice-cold treated paraformaldehyde. The brain tissue was then post-fixed in 4% paraformaldehyde and transferred to 20% Sukurose until the brain tissue sank to the bottom of the tube. The brain was frozen in OCT (AO, USA) and then the brain was cut into 14 μm sections in the coronal plane using a CM 3050S cryostat (Leica, Germany). The brain sections were thawed-fixed to probes on positively charged slides (Fisher, USA).

Example 3: Western Blot Analysis

Western blot analysis was used to measure the expression levels of various proteins. Rats of each group of Example 1 were sacrificed 4 hours after the drug treatment, and the cortex and hippocampus were excised and frozen with dry ice. The cortex and hippocampus were then homogenized in 0.2 M PBS with a protease inhibitory factor cocktail. Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, CA, USA) was used to measure the protein concentration of the homogenate. An amount of total protein (30 μg per sample) was used for 10% to 15% SDS-PAGE electrophoresis.

In order to determine the molecular weight of the detected protein, a pre-stained protein ladder (GangNam-STAIN ^™ , iNtRon Biotechnology, Inc. Republic of Korea) containing a wide range of molecular weights from 10 kDa to 245 kDa was operated in parallel. Was used. In order to minimize membrane blocking and non-specificity, blocking was performed using 5% (w / v) skim milk.

Antibodies used in immunoblotting to detect proteins include rabbit-derived anti-actin, anti-Bcl-2, anti-Bax and anti-caspase-3; Chlorine-derived anti-cytochrome c and anti-p53; And mouse-derived polyclonal antibodies anti-PARP-1 and anti-JNK (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In addition, rabbit-derived anti-p-CREB (Ser133), anti-CaMKII, anti-p-Akt (Ser473), anti-p-PI3K (Y458 / Y199), anti-p-JNK (Thr183 / Tyr185), anti -AMPA, anti-p-AMPA (Ser845) and anti-synaptopycin (Cell Signaling Technology, Inc) were used.

Membrane-bound secondary antibodies were visualized using ECL detection reagents according to the manufacturer's instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). The optical density of the band was then analyzed by densitometry using a computer-based Sigma Gel program version 1.0 (SPSS, Chicago, IL, USA).

Example 4: Immunofluorescence Assay

The tissue-containing slides according to Example 2 were washed twice for 15 minutes in 0.01 M PBS and proteolytic enzyme K solution was added to the tissues and incubated at 37 ° C. for 5 minutes. The tissues were then incubated for 90 minutes in blocking solution containing 0.3% Triton X-100 and normal pig serum in PBS. The primary antibodies (rabbit polyclonal p-Akt, p-PI3K and caspase-3 (Cell Signaling Technology, Inc), and goat polyclonal IgG p53 and p-JNK (Santa cruz); All present at 1: 100 in PBS) overnight at 4 ° C. Secondary antibodies (pig anti-rabbit TRITC (Dako), and rabbit anti-goat FITC (Santa cruz); all of these antibodies are present at 1:50 in PBS) were applied at room temperature for 90 minutes. The slides were then washed twice with PBS for 5 minutes. Then, the culture was performed in parallel for double staining. Glass cover slides were encapsulated on glass slides with an encapsulant and images were captured using a confocal microscope (FluoView FV 1000 Olympus, Japan).

Example 5: Tunnel Staining

To detect cellular apoptosis induced by glutamate, nuclear DNA was stained with TUNEL (GenScript Corporation, USA) and contrast staining was performed using PI. TUNEL staining was performed according to the recommendations of In Situ Cell Death Detection Kit Fluorescein (GenScript, NJ, USA). Glass cover slides were encapsulated on glass slides with an encapsulant and FITC filters were used for TUNEL staining (green color) detection. TUNEL-positive (green) staining patterns were obtained with a confocal laser scanning microscope (FluoView FV 1000, Olympus, Japan), and TUNEL-positive cells in different regions of each section were counted with a computer based program.

Example 6: Analysis and Statistics of Data

The band of Western blot was scanned and the optical density of the band was analyzed by densitometer using Sigma Gel System (SPSS Inc., Chicago, IL). Density values are expressed as mean ± SEM. One-way analysis (ANOVA) of variables was performed to identify significant differences, followed by Student's t-test to identify the significance of differences among the treatment groups. P values less than 0.05 were considered significant (p <0.05).

Experimental Example 1: Verification of the efficacy of osmotin to attenuate excitatory toxicity induced by glutamate

(1) Reduction of expression level of AMPA glutamate receptor

AMPA glutamate receptors are ligand-gated ion channels consisting of a combination of four different subunits, GluR1-4, wherein overexpression of glutamate receptors causes excitatory toxicity and may result in neuronal damage.

The rat cortex and hippocampus were observed 4 hours after the first subcutaneous injection of glutamate in rats after 7 days of birth, whereas the AMPA glutamate receptor protein (Glu2 / 3/4) was overexpressed, whereas the glutamate treatment was performed. Later, it was observed that protein expression levels of AMPA receptors were significantly reduced in the animal group treated with osmotin (FIGS. 1A and 1B).

(2) decreased expression level of p-AMPA glutamate receptor

Experiments on phosphorylation of the serine 845 position of AMPA glutamate receptor protein (Glu1), known to be associated with excitatory toxicity and long-term potentiation (LTP), showed that brains in glutamate-treated animals It was observed that the expression level of p-AMPA glutamate receptor is increased in the cortex and hippocampus of, whereas the expression level of p-AMPA glutamate receptor is decreased in the cortex and hippocampus of brains of osmotin treated animals after glutamate treatment. Was observed.

(3) decreased expression level of CaMKII

Accumulation of Ca ⁺² in mitochondria is a significant step in acute glutamate excitatory toxicity, which is associated with a significant increase in free calcium levels in cells. Calmodulin-dependent kinase-II (CaMKII) is an important regulatory factor in the biochemical mechanism leading to neuronal cell death due to acute excitatory toxicity and excitatory toxicity when antagonists inhibit the action of CaMKII. By the death of nerve cells is reduced.

After 4 hours of subcutaneous injection of glutamate in rats after 7 days of birth, the cortex and hippocampus of the rats were observed 4 hours later, whereas the expression level of CaMKII was observed to increase, whereas osmotin was treated after glutamate treatment. The treated animals were found to significantly reduce the expression level of CaMKII. This means that osmotin is effective in protecting the nerve against acute excitatory toxicity induced by glutamate (FIGS. 1A and 1B).

Experimental Example 2: Validation of Osmotin Efficacy in Restoring Synaptic Dysfunction Induced by Glutamate

Increased CaMKII and Ca ⁺² concentrations due to excitatory toxicity induced by glutamate have a negative effect on neuronal synapses.

In order to analyze the inhibitory effect of glutamate and the beneficial effect of osmotin on synaptic plasticity, the expression level of synaptophysin was measured using Western blot. Specifically, 4 hours after the first subcutaneous injection of glutamate in rats after 7 days of birth, the brain cortex and hippocampus of the rats were observed, whereas synaptopycin was significantly reduced, but after the glutamate treatment. Osmotin-treated animals were found to have increased expression levels of synaptophycins (FIGS. 1C and 1D). This means that osmotin is effective in repairing synaptic dysfunction induced by glutamate in the cortex and hippocampus of the brain.

In addition, in order to analyze the effect of glutamate on the phosphorylation of CREB in the cortex and hippocampus of the rat brain, Western blot was used to measure the expression level of p-CREB protein. Compared to the saline-treated animals, the glutamate-treated animals were found to significantly reduce the expression level of p-CREB protein in the brain cortex and hippocampus, whereas in the animals treated with osmotin after the glutamate treatment, p A significant increase in the expression of CREB protein was observed (FIGS. 1C and 1D). This means that osmotin has the effect of inhibiting long-term synergy in the cortex and hippocampus of the brain.

Experimental Example 3: Verification of the efficacy of osmotin to suppress neuronal damage and DNA fragmentation induced by glutamate

(1) Effect of Osmotin on Nerve Damage

Excitotoxic damage induced by glutamate can lead to a wide range of p53-mediated neuronal damage. Therefore, the levels of different cellular apoptosis markers were measured to determine whether neuronal damage was induced 4 hours after glutamate (10 mg / kg) in rats 7 days after birth.

As a result of this measurement, glutamate is expressed in the level of proapoptotic p53, the ratio of proapoptotic Bax and antiapoptotic Bcl-2, the delivery of mitochondrial cytochrome to cell substrate and caspa in both cortex and hippocampus of brain. Toxicity was observed to be induced by increasing the activation of aze-3 and PARP-1.

On the other hand, when osmotin was treated after glutamate treatment, the changes induced by glutamate were reversed. That is, the expression level of p53 protein and Bax / Bcl-2 ratio were decreased in the cortex and hippocampus of the brain, and reduced the delivery of mitochondrial cytochrome C and activation of caspase-3 and PARP-1 (FIGS. 2A-2D).

(2) Effect of Osmotin on DNA Fragmentation

DNA damage is one of the typical features of cellular apoptosis. Thus, TUNEL staining (DNA cleavage analysis based on enzyme labeling at the free 3 'DNA end) was performed to visualize damaged DNA.

Compared with the control group, glutamate treatment resulted in nuclear fragmentation and cellular apoptosis in CA1, CA3, and DG of the hippocampus, whereas osmotin treatment after glutamate treatment resulted in three regions of the hippocampus. Nuclear fragmentation and cellular apoptosis (almost no TUNEL-positive cells) were found to be markedly reduced (FIGS. 3A and 3B).

Experimental Example 4 Verification of the Efficacy of Osmotin to Protect the Brain through Signal Transduction Pathway in JNK / PI3K / Akt Cells from Glutamate-induced Neuronal Injury

(1) decreased expression level of p-JNK

P-JNK (c-Jun N-) in the cerebral cortex and hippocampus of animals treated with glutamate alone and rats treated with osmotin after glutamate treatment in rats after 7 days of birth using Western blot Terminal kinase) expression levels were measured.

As a result of the measurement, the expression level of p-JNK was significantly increased in the animal group treated with glutamate alone, whereas the expression level of p-JNK was significantly decreased in the animal group treated with osmotin after glutamate treatment (FIG. 4a and 4b).

In addition, as a result of measuring the immunoblot of p-JNK expression in the CA1 region of the hippocampus by immunofluorescence staining, a result consistent with that of the western blot was obtained. That is, the expression level of p-JNK in the CA1 region of the hippocampus was increased by glutamate treatment, whereas the osmotin treatment after glutamate treatment decreased the expression level of p-JNK in the CA1 region of the hippocampus. (FIGS. 4C and 4D).

(2) Relevance of JNK / PI3K / Akt Pathway

Phosphoinositide 3-kinase PI3-K / Akt pathway is one of the important pro-survival pathways in neurons. Therefore, it was examined whether the pathway was relevant when glutamate was treated in the cortex and hippocampus of the brain.

As expected, glutamate treatment significantly reduced the expression levels of p-PI3K and p-Akt in the cortex and hippocampus of the brain, whereas p-PI3K and p-Akt were treated with osmotin after glutamate treatment. The expression level of was found to be increased (FIGS. 4C and 4D).

In addition, the expression of p-PI3K in the hippocampus of the brain was observed using immunofluorescence staining. When osmotin was treated in the image observed by confocal microscopy, glutamate was induced in the CA1 region of the hippocampus of the brain. Reductions in p-PI3K and p-Akt expression levels were found to recover (FIGS. 4C and 4D).

Experimental Example 5: Validation of Osmotin's Inhibitory Effect on Distribution Changes and Colocalization of p53, p-Akt and Caspase-3 Induced by Glutamate

Co-location of p-Akt and p53 to confirm Western blot results according to the experimental results and to elucidate the mechanism of the osmotin neuroprotective effect on neuronal damage induced by glutamate via p-Akt and p53 Measurements and changes in their distribution in the hippocampus (CA1) region of the brain were observed.

As a result of the measurement, p-Akt was found to co-locate with p53 mostly. Specifically, when compared to the control group, the treatment of glutamate alone inhibited the distribution of p-Akt (red) and increased the distribution of p53 (green) in the CA1 region of the hippocampus of the brain, whereas osmotin after glutamate treatment In the case of treatment, the distribution of p-Akt and p53 induced by glutamate in the CA1 region of the hippocampus was reversed (Figs. 5A and 5B).

In addition, the distribution of p53 and caspase-3 in the hippocampus was observed in the same manner as described above. When glutamate was treated alone compared to the control group, p53 (green) and caspase-3 (red) in the hippocampus. The expression level of was observed to be increased, confirming that p53 and caspase-3 were co-located. On the other hand, in the case of osmotin treatment after glutamate treatment, co-location of these proteins was also confirmed in light of the decreased expression levels of p53 and caspase-3 in the hippocampus (FIGS. 5C and 5D).

In the present specification, those skilled in the art of the present invention can fully recognize and infer the details that have been omitted, and the technical spirit or essential configuration of the present invention in addition to the specific examples described in this specification are changed. Many more variations are possible without departing from the scope of the invention. Therefore, the present invention can be implemented in a manner different from that specifically described and illustrated herein, which can be understood by those skilled in the art.

Claims (8)

1. A pharmaceutical composition for preventing or treating neurological diseases caused by excitatory toxicity or synaptic dysfunction, comprising osmotin as an active ingredient.
2. The pharmaceutical composition of claim 1, wherein the excitatory or synaptic dysfunction is induced by glutamate.
3. The method of claim 1, wherein the prevention or treatment of the neurological disease is performed by reducing expression of AMPA (α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptor or decreasing phosphorylation of AMPA glutamate receptor. Will, pharmaceutical composition.
4. The pharmaceutical composition of claim 1, wherein the prevention or treatment of the neurological disease is performed by increasing expression of synaptophycin.
5. The pharmaceutical composition of claim 1, wherein the prevention or treatment of neurological disease is performed by decreasing the expression of CaMKII (Ca ²⁺ / calmodulin-depandent protein kinase II).
6. The method of claim 1, wherein the prevention or treatment of the neurological disease is performed by decreasing the expression of p-JNK, increasing the expression of p-PI3K or increasing the expression of p-Akt in the JNK / PI3K / Akt pathway. Composition.
7. Health functional food composition for the prevention or improvement of neurological diseases caused by excitatory toxicity or synaptic dysfunction comprising osmotin (Osmotin) as an active ingredient.
8. A method of preventing or treating neurological diseases caused by excitatory or synaptic dysfunction, comprising administering to a subject a pharmaceutical composition comprising Osmotin as an active ingredient.

Images