KR102634982B1 - Composition for preventing, ameliorating or treating parkinson's disease comprising osmotin protein as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating Parkinson's disease containing osmotin protein as an active ingredient, wherein the osmotin protein alleviates behavioral and motor deficits induced by MPTP/α-synuclein, Protects dopamine-activated neurons, regulates the expression level of neuroinflammation-related proteins, inhibits cell death induced by MPTP/α-synuclein, alleviates cell damage caused by overexpression of α-synuclein (A53T), and AMPK It has the effect of reducing the accumulation of α-synuclein by activation and subsequent autophagy, modulating dendritic complex structure, increasing spine density in pyramidal neurons, alleviating cognitive deficits, and synaptic synapses. It has the effect of restoring marker expression, so it can be useful as a health functional food or medicine to prevent, improve, or treat Parkinson's disease.

Description

Composition for preventing, ameliorating or treating Parkinson's disease comprising osmotin protein as effective component}

The present invention relates to a composition for preventing, improving or treating Parkinson's disease containing osmotin protein as an active ingredient.

Degenerative neurological disease refers to the gradual structural and functional loss of nerve cells (neurons). Degenerative neurological diseases mainly invade specific parts of the nervous system and are accompanied by symptoms such as dementia, extrapyramidal abnormalities, cerebellar abnormalities, sensory disorders, and motor disorders. Multiple areas may be affected at the same time, resulting in complex symptoms. Diagnosis is made based on the clinical manifestations of the patient, but in this case, the symptoms are diverse and different diseases often have common clinical symptoms, making diagnosis difficult.

Parkinson's disease, one of the neurological diseases, is a disease characterized by the destruction and gradual loss of dopamine neurons distributed in the substantia nigra (SNc) of the brain, and a significant decrease in the dopamine content in the striatum. It is a chronic progressive degenerative disease of the nervous system characterized by resting tremor, rigidity, bradykinesia, and postural instability. Parkinson's disease patients are estimated to be approximately 1% of the population over the age of 60.

Meanwhile, plant-derived osmotin is involved in the regulation of fatty acid oxidation, glucose acquisition, phosphorylation (AMP kinase), and signal transduction pathways. Osmotin (24 kDa) is a stable protein included in the PR-5 family with homology to the sweet-testing protein thaumatin, and is known to induce intracellular signaling in yeast. Osmotin is resistant to heat, acid, and enzymes, so it can circulate through the body without being broken down by digestion. Osmotin is known to be a homolog of adiponectin present in animals, and it has been revealed that adiponectin has the potential to have some anti-inflammatory, anti-diabetic, and anti-atherosclerotic abilities.

In addition, osmotin is widely known for its effects mainly related to obesity and diabetes, and is known as a composition for preventing and treating neurological diseases containing osmotin in Korean Patent No. 1308232, but the osmotin protein of the present invention is used as an active ingredient. A composition for preventing, improving, or treating Parkinson's disease containing the composition has not yet been disclosed.

The present invention has been derived from the above needs, and the present invention provides a composition for preventing, improving or treating Parkinson's disease containing osmotin protein as an active ingredient, and the osmotin protein as an active ingredient is MPTP/α. -Alleviates synuclin-induced behavioral and motor deficits, protects dopamine-activated neurons, regulates the expression level of neuroinflammation-related proteins, and inhibits cell death induced by MPTP/α-synuclein. It has the effect of alleviating cell damage caused by overexpression of α-synuclein (A53T), reducing the accumulation of α-synuclein by AMPK activation and subsequent autophagy, regulating the structure of dendritic complexes, and regulating pyramidal cells. The present invention was completed by confirming that it has the effect of increasing spine density in neurons, alleviating cognitive decline, and restoring synaptic marker expression.

In order to achieve the above object, the present invention provides a health functional food composition for preventing or improving Parkinson's disease, characterized in that it contains osmotin protein selected from the following as an active ingredient: (a) amino acid of SEQ ID NO: 1 Osmotin protein with sequence; or (b) an osmotin protein derived from (a), which has one or more amino acid residue substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 1, and is capable of preventing or improving Parkinson's disease.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of Parkinson's disease, characterized in that it contains as an active ingredient an osmotin protein selected from the following: (a) an osmotin protein having the amino acid sequence of SEQ ID NO: 1; or (b) an osmotin protein derived from (a), which has one or more amino acid residue substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 1, and is capable of preventing or treating Parkinson's disease.

In addition, the present invention provides a method for preventing or treating Parkinson's disease, comprising the step of administering a composition containing an osmotin protein selected from the following to an animal other than a human: (a) of SEQ ID NO: 1 Osmotin protein with amino acid sequence; or (b) an osmotin protein derived from (a), which has one or more amino acid residue substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 1, and is capable of preventing or treating Parkinson's disease.

The present invention relates to a composition for preventing, improving or treating Parkinson's disease containing osmotin protein as an active ingredient, wherein the osmotin protein alleviates behavioral and motor deficits induced by MPTP/α-synuclein, Protects dopamine-activated neurons, regulates the expression level of neuroinflammation-related proteins, inhibits cell death induced by MPTP/α-synuclein, alleviates cell damage caused by overexpression of α-synuclein (A53T), and AMPK It has the effect of reducing the accumulation of α-synuclein by activation and subsequent autophagy, modulating dendritic complex structure, increasing spine density in pyramidal neurons, alleviating cognitive deficits, and synaptic synapses. It has the effect of restoring marker expression.

Figure 1 shows that osmotin protein was administered to MPTP-induced (A) and NSE-hαSyn transgenic (B) mice, and the total distance moved, the distance moved in the center, and the time spent in the open field test (OFT) were confirmed. . #, ##, ### indicate a statistically significant decrease in the total distance moved or the distance moved and time spent in the center of the MPTP or α-syn group compared to the control group, # is p<0.05, # # is p<0.01, and ### is p<0.001. * indicates a statistically significant increase in the total moving distance or moving distance and residence time at the center of the osmotin administration group of the present invention compared to the MPTP or α-syn group, p < 0.05. ns means statistically insignificant change.
Figure 2 shows the results of osmotin protein administered to MPTP-induced (A) and NSE-hαSyn transgenic (B) mice, and the pole test and wire-hang test. #, ##, ### indicate a statistically significant increase in the time to come down from the bar to the floor in the MPTP or α-syn group compared to the control group, and a statistically significant decrease in the time hanging on the rope, # indicates p <0.05, ## is p<0.01, and ### is p<0.001. * means that the time to come down to the floor of the osmotin administered group of the present invention was statistically significantly reduced compared to the MPTP or α-syn group, and the time to hang on the line was significantly increased, p < 0.05.
Figure 3 shows the number of TH-positive neurons in SNpc (A) and striatum TH-positive fiber density (B) in TH fluorescent staining and MPTP/NSE-hαSyn transgenic mice according to administration of osmotin protein. This is the result of confirming the TH expression level (C). ##, ### indicate a statistically significant decrease in the number of TH-positive nerves or TH-positive fiber density in the MPTP-induced group or SE-hαSyn transgenic mouse group compared to the control group (CTL), ## indicates p <0.01, and ### is p<0.001. *, ** indicate a statistically significant increase in the number of TH-positive nerves or TH-positive fiber density in the osmotin-administered group of the present invention compared to the MPTP-induced group or SE-hαSyn transgenic mouse group, * indicates p< is 0.05, and ** is p<0.01.
Figure 4 confirms the immune activity of TH-positive nerves in the SNpc and striatum, and ### shows the statistical statistics of the number of nerves (A) and the density of TH-positive nerve fibers (B) in the striatum in the MPTP-induced group compared to the control group (CTL). This means that it decreased significantly, and ### is p<0.001. ** indicates a statistically significant increase in the number of nerves (A) and TH-positive nerve fiber density (B) in the striatum in the osmotin-administered group of the present invention compared to the MPTP-induced group, ** is p < 0.01.
Figure 5 is a Western blot photograph (A) confirming the expression level of TH in the striatum, SNpc, and cortex according to osmotin administration and a graph (B) quantifying it. ##, ### are This means that the expression level of the MPTP-induced group was decreased compared to the control group (Con), ## is p<0.01, and ### is p<0.001. *, ** indicate a statistically significant increase in the expression level of the osmotin-administered group (MPTP+Os) compared to the MPTP-induced group, * is p < 0.05, and ** is p < 0.01.
Figure 6 is a Western blot photograph (A) confirming the expression levels of Nurr1, DAT, and VMAT2 in SNpc according to osmotin protein administration in MPTP/NSE-hαSyn transgenic mice and a graph (B) quantifying this. #, ## are This means that the expression level of the MPTP-induced group was decreased compared to the control group (Con), # means p<0.05, and ## means p<0.01. *, ** indicate a statistically significant increase in the expression level of the osmotin-administered group (MPTP+Os) compared to the MPTP-induced group, * is p < 0.05, and ** is p < 0.01.
Figure 7 is a Western blot result confirming the expression levels of Nurr1, DAT, VMAT2, and TH according to MPTP induction and osmotin treatment in SH-SY5Y cells induced by MPP ⁺ .
Figure 8 shows the results of confirming the protein expression levels of p-p38/p38, p-ERK/ERK, and p-JNK/JNK in SNpc and striatum, following osmotin administration of the present invention in MPTP/NSE-hαSyn transgenic mice. #, ## indicate a statistically significant increase or decrease in the expression level of the MPTP or αSyn group compared to the control group (CTL, WT), # is p < 0.05, and ## is p < 0.01. *, ** indicate a statistically significant decrease or increase in the expression level of the osmotin administration group of the present invention compared to the MPTP or αSyn group, * is p < 0.05, and ** is p < 0.01.
Figure 9 shows the results of confirming the expression levels of p-ERK/ERK and p-JNK/JNK proteins according to administration of osmotin protein in SH-SY5Y cells induced with MPP ⁺ /α-synuclein (A53T). #, ##, ### indicate a statistically significant increase or decrease in the expression level of the MPP ⁺ or αSyn group compared to the control group (CTL, WT), # is p < 0.05, ## is p < 0.01, ### is p<0.001. *, ** indicate a statistically significant decrease or increase in the expression level of the osmotin administration group of the present invention compared to the MPP ⁺ or αSyn group, * is p < 0.05, and ** is p < 0.01. ns means statistically insignificant change.
Figure 10 confirms changes in GFAP and Iba-1 expression levels following osmotin administration in the striatum and SNpc of MPTP/NSE-hαSyn transgenic mice. #, ## indicate a statistically significant increase in the expression levels of GFAP and Iba-1 in the MPTP group compared to the control group, # is p<0.05, and ## is p<0.01. *, ** indicate a statistically significant decrease in the expression levels of GFAP and Iba-1 in the osmotin administration group of the present invention compared to the MPTP group, * is p < 0.05, and ** is p < 0.01. ns means statistically insignificant change.
Figure 11 shows the results of immunofluorescence analysis of the expression of GFAP and Iba-1 according to osmotin administration in the striatum and SNpc region of MPTP/NSE-hαSyn transgenic mice. ##, ### indicate a statistically significant increase in the expression levels of GFAP and Iba-1 in the MPTP group compared to the control group, ## is p<0.01, and ### is p<0.001. *, ** indicate a statistically significant decrease in the expression levels of GFAP and Iba-1 in the osmotin administration group of the present invention compared to the MPTP group, * is p < 0.05, and ** is p < 0.01.
Figure 12 shows ROS assay results following osmotin administration in the striatum and SNpc. #, ## indicate a statistically significant increase in the relative measured amount of DCF (dye drug for measuring ROS) in the MPTP group compared to the control group, # is p<0.05, and ## is p<0.01. * indicates a statistically significant decrease in the measured relative DCF of the osmotin administered group of the present invention compared to the MPTP group, * is p < 0.05.
Figure 13 is a Western blot result confirming the expression levels of iNOS and COX-2 according to osmotin administration in the SNpc region of MPTP/NSE-hαSyn transgenic mice.
Figure 14 shows the results of analysis of cell survival rate (A), cytotoxicity (B), and caspase-3/7 (C) activity according to osmotin administration in the MPTP/α-synuclein-induced group. #, ### indicate a statistically significant decrease in cell survival rate, increased cytotoxicity, and increased caspase-3/7(C) activity in the MPTP/α-synuclein-induced group compared to the control group. p<0.05, and ### is p<0.001. *, **, *** indicate a statistically significant increase in cell survival rate of the osmotin-administered group of the present invention compared to the MPTP/α-synuclein-induced group, cytotoxicity decreased, and caspase-3/7(C) activity. This means that this has decreased, * means p<0.05, ** means p<0.001, and *** means p<0.001.
Figure 15 is a Western blot result confirming the expression level of Bcl-2, Bcl-xL, Bax, and cytochrome-c (Cyto c) involved in neuronal death induced by MPTP/MPP ⁺ according to osmotin protein treatment.
Figure 16 is a Western blot result confirming the expression level of Bcl-2, Bcl-xL, Bax, and cleaved caspase-3 according to osmotin treatment in SH-SY5Y cells induced by MPP ⁺ .
Figure 17 shows the expression levels of Bcl-2, Bax, PARP-1, cytochrome c, and NeuN involved in apoptosis in the striatum (A) and SNpc (B) of NSE-hαSyn transgenic mice according to osmotin protein administration. Western blot results confirmed and the expression levels of Bcl-2, Bcl-xL, Bax, cytochrome c, and NeuN involved in apoptosis in SH-SY5Y cells (C) induced with α-synuclein (A53T). It is a result.
Figure 18 shows the number of neurons in the striatum and SNpc induced by MPTP/α-synuclein (A, B) and the number of Nissl-stained neurons in PTP/NSE-hαSyn mice (C). ### indicates a statistically significant decrease in the number of nerves in the MPTP group compared to the control group, p<0.001, and *, ** indicate a statistically significant increase in the number of nerves in the osmotin-administered group of the present invention compared to the MPTP group. In other words, * means p<0.05, and ** means p<0.01.
Figure 19 shows Nissl staining results in CA1, CA3 and DG regions of NSE-hαSyn mice following osmotin administration.
Figure 20 shows the structure of the dendritic cell complex (A), the dendrite length of NSE-hαSyn mouse (B), and the number of complexes (C) and spine density (D) in the basal and apical regions of the NSE-hαSyn mouse brain. This is the result of Golgi staining. ## indicates a statistically significant decrease in dendrite length, complex number or spine density of the NSE-hαSyn mouse group compared to the control group, p<0.01, * is the osmotin administered group of the present invention compared to the NSE-hαSyn mouse group There was a statistically significant increase in dendrite length, complex number, or spine density, p<0.05.
Figure 21 shows the results of confirming filopodia spines in the NSE-hαSyn group, showing a photo and number (B) of filopodia spines (A). # indicates a statistically significant decrease in the number of spines in the NSE-hαSyn mouse group compared to the control group, p<0.05, * means a statistically significant decrease in the number of spines in the osmotin-administered group of the present invention compared to the NSE-hαSyn mouse group. It means that it has increased, which means p<0.05. ns means statistically insignificant change.
Figure 22 shows the results of immunofluorescence analysis of SYP (ophysin) (A) and PSD-95 (postsynaptic density protein-95) (B) in the cortex and CA1 according to osmotin administration. ##, ### indicate a statistically significant decrease in the expression levels of SYP and PSD-95 in the NSE-hαSyn mouse group compared to the control group, ## is p<0.01, ### is p<0.001, *, ** indicate that the expression levels of SYP and PSD-95 in the osmotin administered group of the present invention were statistically significantly increased compared to the NSE-hαSyn mouse group, * is p < 0.05, and ** is p < 0.01.
Figure 23 is a Western blot result of SYP, PSD-95, SNAP-25, and pCREB/CREB in the cortex (A) and hippocampus (B) according to osmotin administration. ##, ### indicate a statistically significant decrease in the expression levels of SYP, PSD-95, SNAP-25, and pCREB/CREB in the NSE-hαSyn mouse group compared to the control group, # indicates p<0.05, and ## indicates a statistically significant decrease. p<0.01, *, ** indicate a statistically significant increase in the expression levels of SYP, PSD-95, SNAP-25 and pCREB/CREB in the osmotin administered group of the present invention compared to the NSE-hαSyn mouse group, * indicates p <0.05, and ** is p<0.01.
Figure 24 shows the results confirming the effect of osmotin on behavioral changes induced by α-synuclein.

The present invention relates to a health functional food composition for preventing or improving Parkinson's disease, characterized in that it contains osmotin protein selected from the following as an active ingredient: (a) osmotin protein having the amino acid sequence of SEQ ID NO: 1; or (b) an osmotin protein mutated from (a), which has one or more amino acid residue substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 1, and can prevent or improve Parkinson's disease.

In the present invention, osmotin is a protein contained in large quantities in ripened fruits such as grapes, and is a protein composed of about 150 to 250 amino acids depending on the individual.

The osmotin protein is preferably contained in an amount of 0.1 to 100% by weight based on the total weight of the composition, but is not limited thereto.

The osmotin protein is Nurr1 (Nuclear receptor related 1 protein), VMAT2 (vesicular monoamine transporter 2), DAT (dopamine transporter), TH (tyrosine hydroxylase) p-p38, p-JNK (c-Jun N-terminal kinase) and The expression level of one or more genes or proteins selected from p-ERK (extracellular signal-regulated kinase) proteins can be controlled. The health functional food composition can be manufactured from any one food selected from beverages, pills, tablets, capsules, and powders, or by adding it to other foods or food ingredients, using conventional methods. It can be manufactured appropriately.

Examples of foods to which the composition of the present invention can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, and tea. , it may be in any form selected from drinks, alcoholic beverages, and vitamin complexes, and includes all health foods in the conventional sense.

The health functional food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alkynic acid and its salts, organic acids, and protective colloids. It may contain thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Additionally, it may contain pulp for the production of natural fruit juice and vegetable drinks. These ingredients can be used independently or in combination, and various flavors or natural carbohydrates can be contained as additional ingredients. The natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; and polysaccharides such as dextrins and cyclodextrins; Sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used.

In addition, the present invention relates to a pharmaceutical composition for the prevention or treatment of Parkinson's disease, characterized in that it contains as an active ingredient an osmotin protein selected from the following: (a) an osmotin protein having the amino acid sequence of SEQ ID NO: 1; or (b) an osmotin protein mutated from (a), which has one or more amino acid residue substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 1, and is capable of preventing or treating Parkinson's disease.

In addition to the active ingredients of the pharmaceutical composition, pharmaceutically acceptable saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia Rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals. It may further contain one or more carriers selected from oils, and in addition to the above active ingredients, pharmaceutically acceptable antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, and suspending agents. And it may further contain one or more auxiliaries selected from among preservatives.

The pharmaceutical composition can be administered in an oral or parenteral dosage form according to a conventional method, and when formulated, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. . Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. In addition, non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

In addition, the present invention relates to a method for preventing or treating Parkinson's disease, comprising the step of administering a composition containing an osmotin protein selected from the following to an animal other than a human: (a) of SEQ ID NO: 1 Osmotin protein with amino acid sequence; or (b) an osmotin protein derived from (a), which has one or more amino acid residue substitutions, deletions or insertions in the amino acid sequence of SEQ ID NO: 1, and is capable of preventing or treating Parkinson's disease.

The animals may be animals including or excluding humans.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

[Experimental animals]

As animals to induce Parkinson's disease through MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatment, male wild-type C57BL/6J mice (25-28 g, 8 weeks old) were purchased from the Jackson Laboratory ( Jackson Laboratory; Bar Harbor, ME, USA), and C57BL/6-Tg (NSE-hαSyn) Korl mice were purchased from the National Institute of Food and Drug Safety Evaluation (NIFDS, Cheongju, Korea). Purchased. The animal model was acclimatized for one week under conditions of a 12-hour light/dark cycle, 21 to 23°C, and 60±10% humidity, and had free access to food and water.

MPTP (Sigma-Aldrich, MO, USA) was prepared in sterile distilled water and administered at a dose of 30 mg per kg intraperitoneally for 5 consecutive days according to conventional guidelines.

C57BL/6J mice were randomly divided into three groups. Twelve mice per group were assigned to the control group (control, CTL), MPTP administration group, and MPTP+osmotin administration group (MPTP+Os).

C57BL/6-Tg(NSE-hαSyn) Korl mice were assigned to three groups: wild-type group (WT), control group (NSE-hαSyn; α-syn), and NSE-hαSyn + osmotin group (α-syn + Os ), 12 mice were assigned to each group.

The osmotin was administered in an amount of 15 mg per kg along with purified osmotin and saline solution.

[Cell culture]

Human neuroblastoma SH-SY5Y cells were purchased from the Korea Cell Line Bank (KCLB, South Korea), and were cultured in 1% antibiotic-antibacterial solution and 10% bovine fetus at 37°C in a 5% CO ₂ environment. Cultured in DMEM (Dulbecco's modified Eagle's medium) containing serum. Mouse hippocampal cell line (Cedarlane, Canada) from mHippoE-14 embryos was extracted on embryonic day 14 and cultured in DMEM without sodium pyruvate. SH-SY5Y cells were MPP ⁺ (Sigma-Aldrich, MO, USA). EGFP-alpha-synuclein-A53T plasmid was obtained from David Rubinsztein (Addgene #40823). Adiponectin receptor (AdipoR1) was used as a commercial AdipoR1 CRISPR/Cas9-KO plasmid. (Santa Cruz, CA, USA).

The puromycin gene was inserted as a selection marker gene using the AdipoR1 HDR plasmid (Santa Cruz, CA, USA), the plasmid was transfected into cells using Lipofectamine 3000 (Invitrogen), and the cells were cultured. did. Thereafter, the medium was removed 24 hours before selection, and a pure and stable pool of knockout cell lines was obtained using 2.5 μg/ml of puromycin added as a selection marker to the growth medium.

Example 1. Confirmation of the alleviating effect of behavioral and motor deficits induced by MPTP/α-synuclein following administration of osmotin protein

To confirm the effect of osmotin on the motor deficits seen in MPTP-induced and NSE-hαSyn transgenic mice, behavioral tests for motor impairment were performed.

In the open field test (OFT), the total distance moved was decreased in the MPTP/NSE-hαSyn transgenic mice, but the total distance moved was significantly increased in the osmotin-administered group compared to the MPTP/NSE-hαSyn transgenic mice (Figure 1 ).

In addition, to evaluate the effect of osmotin protein on exercise and neuromuscular strengthening, pole test and wire-hang test were performed.

As a result, in the rod test, the total time (T-LA) taken for mice in the MPTP/α-synuclein group to come down to the floor was significantly increased compared to the control and wild type group (WT), but the osmotin of the present invention In the group administered protein, the total time taken to come down to the floor (T-LA) was statistically significantly reduced.

In the hanging test, compared to the control and wild-type groups, the hanging time was reduced in the MPTP/α-synuclein group, and in contrast, the hanging time was statistically significantly increased in the group administered the osmotin protein of the present invention. From these results, it was determined that nerve roots were reduced in MPTP/NSE-hαSyn transgenic mice, and osmotin had a neuroprotective effect, significantly reducing motor deficits caused by MPTP/NSE-hαSyn transgenic mice. (Figure 2).

Example 2. Confirmation of control of Nurr1 (Nuclear receptor related 1 protein), VMAT2 (vesicular monoamine transporter 2), DAT (dopamine transporter), and TH (tyrosine hydroxylase) expression levels according to administration of osmotin protein

To determine whether osmotin protects against dopaminergic neuron loss in mice induced by MPTP/α-synuclein, the number of TH-positive neurons in the SNpc and the density of striatum TH-positive fibers were measured by TH fluorescence. Measured through staining. The amount of TH was significantly reduced in the MPTP/NSE-hαSyn transgenic mouse group compared to the control and wild type (WT) mice, and this decrease in the amount of TH was confirmed to be significantly recovered by administration of osmotin protein (Figure 3).

Additionally, the immune activity of TH-positive neurons was confirmed in the SNpc and striatum of mice. Compared to the number of neurons in the control group, it was confirmed that the number of TH-positive neurons was significantly reduced in MPTP-induced mice, and osmotin protein significantly increased the number of TH-positive neurons in the SPnc and striatum of the Parkinson's disease-induced group by MPTP. (Figure 4).

Similarly, changes in TH expression level were analyzed using Western blot. Compared to the results of control and wild type (WT) mice, the expression level of TH in the striatum and SNpc was significantly reduced in MPTP/NSE-hαSyn transgenic mice, and osmotin protein efficiently increased the expression level of TH (Figure 5).

In vivo , the expression levels of Nurr1, VMAT2, and DAT in the SNpc and striatum of MPTP/NSE-hαSyn transgenic mice were significantly upregulated by osmotin protein administration (Figure 6).

Meanwhile, in SH-SY5Y cells induced by MPP ⁺ , it was confirmed that the expression levels of Nurr1, DAT, VMAT2, and TH were decreased. In contrast, the osmotin protein treatment group showed concentration-dependent expression of Nurr1, DAT, VMAT2, and TH. It was confirmed that the amount was up-regulated (Figure 7).

Example 3. Confirmation of p-p38, p-JNK and p-ERK expression levels of osmotin protein and regulatory effect on neuroinflammation

To confirm the effect of osmotin protein on p38, JNK, and ERK phosphorylation, the expression levels of p-p38, p-JNK, and p-ERK were measured in in vivo and in vitro models induced by MPTP/α-synuclein. analyzed.

In MPTP/NSE-hαSyn transgenic mice, the expression levels of p-p38 and p-JNK increased, and the expression levels of p-ERK decreased. In contrast, the osmotin protein treatment group significantly decreased the expression levels of p-p38 and p-JNK and significantly increased the expression level of p-ERK compared to the MPTP/NSE-hαSyn transgenic mice (Figure 8 ).

In addition, in SH-SY5Y cells induced with MPP ⁺ /α-synuclein (A53T), the effect of osmotin was evaluated, and the results showed that it was decreased in SH-SY5Y cells induced with MPP ⁺ /α-synuclein (A53T). It was confirmed that the expression level of p-ERK/ERK increased in a concentration-dependent manner by treatment with osmotin protein, and the expression level of p-JNK/JNK increased in the MPP ⁺ /α-synuclein (A53T) induced group, whereas the expression level of p-ERK/ERK increased in a concentration-dependent manner by treatment with osmotin protein. Osmotin protein treatment group decreased in a concentration-dependent manner (Figure 9).

In addition, to determine the effect on neuroinflammatory response, the effect of osmotin on activated astrocytes (GFAP) and microglial cells (Iba-1) was evaluated.

As a result, GFAP and Iba-1 expression was significantly increased in the striatum and SNpc of MPTP/NSE-hαSyn transgenic mice, and was significantly reduced by administration of osmotin (Figure 10).

In addition, immunofluorescence analysis results supported that osmotin significantly reduces the expression of GFAP and Iba-1 in the striatum and SNpc regions of MPTP/NSE-hαSyn transgenic mice (FIG. 11).

ROS quantitative analysis was performed to confirm the possible role of oxidative stress in relation to the activity of astrocytes and microglial cells. As a result, ROS in the osmotin administered group was significantly reduced compared to MPTP/NSE-hαSyn transgenic mice (FIG. 12).

In addition, as a result of analyzing the expression of iNOS and COX-2, the expression of iNOS and COX-2 increased in mice induced by MPTP/NSE-hαSyn, and the expression of iNOS and COX-2 decreased in the osmotin treated group. (Figure 13).

Example 4. Confirmation of the regulatory effect of osmotin protein on apoptosis induced by MPTP/α-synuclein in animal and cell models

To evaluate the effect of osmotin on toxicity induced by MPP ⁺ /α-synuclein (A53T), cell viability, cytotoxicity, and caspase-3/7 activity analysis were performed using the ApoTox-Glo Triplex assay. .

As a result, MPP ⁺ treatment decreased cell viability and increased cytotoxicity and caspase-3/7 activity. In contrast, when treated with the osmotin protein of the present invention, cell survival rate was increased and cytotoxicity and caspase-3/7 activity were reduced.

In addition, the effects of osmotin on cell viability, cytotoxicity, and caspase-3/7 activity decreased by the induction of α-synuclein (A53T) were confirmed, and the results showed that osmotin decreased cell viability, cytotoxicity, and caspase-3/7 activity in a dose-dependent manner. It was confirmed that there was a significant effect on -3/7 activity (Figure 14).

It is well known that mitochondria regulate the processes of apoptosis and degeneration. To investigate whether osmotin inhibits neuronal death induced by MPTP/MPP ⁺ , pro-/anti-apoptotic markers were analyzed using Western blot.

As a result, MPTP significantly increased the amount of Bax and significantly decreased the amount of Bcl-2 and Bcl-xL, but osmotin protein decreased the amount of Bax and cytochrome c secretion, and decreased the amount of Bcl-2 and Bcl-xL. The expression of -xL was increased (Figure 15).

In addition, the expression of mitochondrial apoptosis markers was examined in MPP ⁺ -induced SH-SY5Y cells. MPP ⁺ significantly decreased the amounts of Bcl-2 and Bcl-xL and increased the expression levels of Bax and cleaved caspase-3. However, osmotin protein decreased the expression level of pro-apoptotic markers such as Bax and cleaved caspase-3 in a concentration-dependent manner, and increased the expression level of anti-apoptotic markers such as Bcl-2 and Bcl-xL ( Figure 16).

In addition, the anti-apoptotic effects of osmotin on apoptotic cells in the striatum and SNpc of NSE-hαSyn transgenic mice were confirmed, and osmotin significantly increased the expression levels of Bcl-2, Bax, cytochrome c, and PARP-1. It was confirmed that the expression level of NeuN was reduced in NSE-hαSyn mice, but osmotin protein restored the expression level of NeuN (Figure 17A, B).

The in vivo effect of osmotin on apoptosis in NSE-hαSyn mice was also confirmed in in vitro cells induced by α-synuclein (A53T). It was confirmed that overexpression of α-synuclein (A53T) significantly decreased the expression levels of Bcl-2 and Bcl-xL and significantly increased the expression levels of Bax and cytochrome c. In contrast, osmotin protein significantly increased the amount of Bcl-2 and Bcl-xL in a concentration-dependent manner and significantly reduced the expression level of Bax and cytochrome c (Figure 17C).

The effects of MPTP/α-synuclein-induced neurotoxicity on the striatum and SNpc were analyzed by Nissl staining, and the number of Nissl-stained cells in MPTP/NSE-hαSyn mice was compared with the number in control or WT. Although the nerves were significantly reduced, the osmotin protein treatment group significantly recovered Nissl-stained nerves compared to the MPTP/NSE-hαSyn mouse group (FIG. 18).

Example 5. Dendritic complex and structure control, confirming the effect of osmotin protein to increase spine density in pyramidal neurons

In order to analyze these effects by osmotin treatment, Nissl staining was performed on the experimental group. The number of Nissl-stained neurons in the CA1, CA3, and DG regions of NSE-hαSyn mice was significantly reduced compared to WT mice, and was significantly upregulated by administration of osmotin protein (FIG. 19).

To analyze the effect of osmotin protein on the spine shape modified by α-synuclein, the structure, complex, and length of dendritic cells in hippocampal CA1 pyramidal neurons were confirmed by Golgi staining. It was confirmed that the total dendrites length in NSE-hαSyn mice was significantly reduced compared to the dendrites length in WT mice. In contrast, the osmotin protein administration group increased the total dendrite length compared to the dendrite length of NSE-hαSyn mice. Additionally, it was confirmed that dendritic cell complexes were reduced in NSE-hαSyn mice compared to WT mice, and in contrast, osmotin protein significantly increased complexes in the basal and apical regions of the NSE-hαSyn mouse brain. did.

In addition, it was confirmed that the density and total number of spines in NSE-hαSyn transgenic mice were significantly reduced compared to the density and number in WT mice, and were significantly upregulated upon administration of osmotin protein (Figure 20) .

In particular, the number of filopodia spines in the NSE-hαSyn group was significantly reduced compared to the WT group, while the osmotin protein administration group had lower spine density, spines, and filopodia spines compared to the NSE-hαSyn group. The total number of them increased significantly (Figure 21).

Example 6. Confirmation of the effect of osmotin protein in alleviating cognitive deficits and restoring synaptic marker expression in NSE-hαSyn mice

To analyze the protective effect of osmotin against synaptic dysfunction in a Parkinson's disease model, immunofluorescence analysis of SYP (ophysin) and PSD-95 (postsynaptic density protein-95) was performed. Immunofluorescence results showed that the expression of SYP and PSD-95 in the frontal lobe and CA1 region of NSE-hαSyn transgenic mice was significantly reduced compared to WT mice. Downregulation of PSD-95 and SYP was significantly upregulated by injection of osmotin (Figure 22).

Immunofluorescence results were confirmed by Western blot analysis. Phosphorylation of the memory-related protein CREB, as well as expression of SYP, PSD-95, and SNAP-25, were significantly reduced in the cortex and hippocampus of NSE-hαSyn mice compared to WT mice. However, the osmotin protein administration group significantly upregulated the expression of SYP, PSD-95, SNAP-25 and phosphorylation of CREB in NSE-hαSyn mice (Figure 23).

The MWM test was performed to analyze the effects of osmotin protein on α-synuclein-induced behavioral changes in NSE-hαSyn mice, and measured the time taken to reach the platform and the target quadrant after osmotin injection. Time was significantly improved. The degree of time spent in the target zone was lower in NSE-hαSyn mice than in WT mice, and the proportion of time spent in the corresponding quadrant (zone 2) where the platform was located was significantly reduced in NSE-hαSyn mice compared to WT mice. Confirmed. However, in contrast, when osmotin protein was administered to NSE-hαSyn mice, the time frequency at the target point and the time spent in the corresponding quadrant increased (Figure 24).

[Statistical processing]

Data were analyzed using GraphPad Prism 6 software. Statistical data were expressed as mean ± standard deviation (SEM) based on at least three independent experiments with 12 mice per group. For morphological analysis, three images obtained from at least three experiments with similar conclusions were considered. The significance of the difference was determined by one-way or two-way analysis of variance (ANOVA), and Student's T-test was performed. As a result of the evaluation, p<0.05 was judged to be statistically significant.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Composition for preventing, ameliorating or treating parkinson's disease comprising osmotin protein as effective component <130> PN20442 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 243 <212> PRT <213> Nicotiana tabacum <400> 1 Met Gly Asn Leu Arg Ser Ser Phe Val Phe Phe Leu Leu Ala Leu Val 1 5 10 15 Thr Tyr Thr Tyr Ala Ala Thr Ile Glu Val Arg Asn Asn Cys Pro Tyr 20 25 30 Thr Val Trp Ala Ala Ser Thr Pro Ile Gly Gly Gly Arg Arg Leu Asp 35 40 45 Arg Gly Gln Thr Trp Val Ile Asn Ala Pro Arg Gly Thr Lys Met Ala 50 55 60 Arg Val Trp Gly Arg Thr Asn Cys Asn Phe Asn Ala Ala Gly Arg Gly 65 70 75 80 Thr Cys Gln Thr Gly Asp Cys Gly Gly Val Leu Gln Cys Thr Gly Trp 85 90 95 Gly Lys Pro Pro Asn Thr Leu Ala Glu Tyr Ala Leu Asp Gln Phe Ser 100 105 110 Gly Leu Asp Phe Trp Asp Ile Ser Leu Leu Asp Gly Phe Asn Ile Pro 115 120 125 Ile Thr Phe Pro Thr Asn Pro Ser Gly Gly Lys Cys His Ala Leu Cys 130 135 140 Thr Ala Ile Asn Gly Glu Cys Pro Ala Glu Leu Arg Val Pro Gly Gly 145 150 155 160 Cys Asn Asn Pro Cys Thr Thr Phe Gly Gly Gln Gln Tyr Cys Cys Thr 165 170 175 Gln Arg Pro Cys Gly Pro Thr Phe Phe Ser Lys Phe Phe Lys Gln Arg 180 185 190 Cys Pro Asp Ala Tyr Ser Tyr Pro Gln Asp Asp Pro Thr Ser Thr Phe 195 200 205 Thr Cys Pro Gly Gly Ser Thr Asn Tyr Arg Val Ile Phe Cys Pro Asn 210 215 220 Gly Gln Ala His Pro Asn Phe Pro Leu Glu Met Pro Gly Ser Asp Glu 225 230 235 240 Val Ala Lys

Claims (6)

A health functional food composition for preventing or improving Parkinson's disease, characterized in that it contains osmotin protein consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The health functional food composition for preventing or improving Parkinson's disease according to claim 1, wherein the osmotin protein is contained in an amount of 0.1 to 100% by weight based on the total weight of the composition. The health functional food composition for preventing or improving Parkinson's disease according to claim 1, wherein the composition is manufactured in a dosage form selected from beverages, pills, tablets, capsules, and powders. According to claim 1, wherein the osmotin protein is Nurr1 (Nuclear receptor related 1 protein), VMAT2 (vesicular monoamine transporter 2), DAT (dopamine transporter), TH (tyrosine hydroxylase), p-p38, p-JNK (c- A health functional food composition for preventing or improving Parkinson's disease, characterized in that it regulates the expression level of one or more genes or proteins selected from Jun N-terminal kinase) and p-ERK (extracellular signal-regulated kinase) proteins. A pharmaceutical composition for the prevention or treatment of Parkinson's disease, characterized in that it contains osmotin protein consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. A method for preventing or treating Parkinson's disease, comprising administering a composition containing an osmotin protein consisting of the amino acid sequence of SEQ ID NO: 1 to an animal other than a human.