KR102538278B1 - Composition for preventing or treating Huntington's disease comprising gintonin - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating Huntington's disease, comprising gintonin as an active ingredient. -By inhibiting κB and MAPKs signaling pathways, 3-NPA-induced neurological disorders and striatum toxicity were ameliorated, showing beneficial effects in STHdh cells and adeno-associated virus (AAV) vector infected Huntington's disease mouse models As confirmed, it may be useful as a composition for preventing, improving or treating Huntington's disease.

Description

Composition for preventing or treating Huntington's disease comprising gintonin as an active ingredient

The present invention relates to a composition for preventing, improving or treating Huntington's disease comprising gintonin as an active ingredient.

Huntington's disease (HD) is a genetic, progressive and degenerative neurological disorder characterized by abnormal involuntary movements, cognitive impairment and psychiatric disorders.

Huntington's disease is caused by an abnormal expansion (mutation) of the CAG (glutamine) trinucleotide repeat at position 4p 16.9 in exon 1 of the huntingtin (HTT) gene. Aggregates of mutant huntingtin (mHTT) protein are neuronal cell (neuron, neuron, neuronal cell) toxicity, transcriptional dysregulation, excitotoxicity, mitochondrial dysfunction due to altered energy metabolism, striatum/striatum/ It induces various disease mechanisms including changes in axonal transport and synaptic dysfunction within the cortex.

tetrabenazine; The only US Food and Drug Administration (FDA) approved Huntington's disease treatment] and antipsychotics (such as Olanzapine and Aripiprazole) are considered as possible effective agents for the treatment of complications and psychosis. However, these drugs carry the risk of potentially serious side effects including anoxia, depression, dizziness, fatigue or parkinsonism. In addition, symptomatic treatment may have beneficial effects on motor function, quality of life, and safety, and thus may be beneficial for some individuals. Since there is no established treatment that can delay or stop the progression of Huntington's disease, the development of effective and safe drugs for the treatment of Huntington's disease is important. However, the precise mechanisms underlying neuronal death in Huntington's disease, as well as innovative drugs, are unclear.

Oxidative stress is characterized by overproduction of reactive oxygen species (ROS), which leads to mitochondrial DNA mutations, impairment of the mitochondrial respiratory chain, alteration of membrane permeability, and disruption of Ca2 ⁺ homeostasis and the mitochondrial defense system. It can lead to mitochondrial damage including dysfunction. These changes can mediate or amplify the development of Huntington's disease, neuronal dysfunction and lead to neurodegeneration. Therefore, antioxidants that modulate underlying causative factors such as mitochondrial dysfunction in oxidative stress may be excellent therapeutic candidates for the treatment of Huntington's disease. Kelch like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) protects against oxidative damage induced by injury and inflammation It is important in regulating the expression of protective antioxidant proteins. For example, mice lacking Nrf2 are congenitally susceptible to oxidative stress-induced diseases, including drug-induced toxicity and neurological diseases, whereas activators of Keap1-Nrf2 signaling are used in various in vivo and in vitro disease models, including Huntington's disease. reduces oxidative stress in According to in vitro and in vivo Huntington's disease studies, antioxidants including creatine and sulforaphane are known to exert beneficial effects. However, innovative candidates for antioxidant effects have not yet been reported.

3-nitropropionic acid (3-NPA), an irreversible inhibitor of complex II in mitochondria, is known to exhibit neurotoxicity in both animals and humans. Administration of 3-NPA impairs cellular energy metabolism by inhibiting succinate dehydrogenase (SDH) in specific brain regions, including the striatum and cortex. , ATP), produce oxidative stress and neuronal damage, mimic many of the histopathological and neurochemical features of Huntington's disease, and are known to lead to gait abnormalities and Huntington's disease-like striatal lesions, leading to Huntington's disease research. has been used in animal models for

On the other hand, since ancient times, ginseng has been generally used as a tonic for longevity/life extension, or as an adaptogenic agent to improve biological functions against various stress, fatigue, disease, cancer, and diabetes. improve Ginseng has been used as a tonic or adaptogen for more than a thousand years not only in Korea but also in China and Japan. Recently, ginseng is one of the most famous medicinal herbs consumed in the world.

Among ginseng components, ginsenosides, called ginseng saponin, were isolated in the early 1960s and known as a representative component in physiological/pharmacological studies. In addition, recent studies have revealed that ginseng contains other unknown components such as polysaccharides, polyacetylenes, and water-soluble proteins.

Gintonin (GT) is a novel glycolipoprotein that is composed of sugar, fat and protein among the components of ginseng (Korean Patent No. 10-0973202). Unlike Saeed), it was found that the activity appears through the membrane signal transducton pathway through interaction with cell membrane proteins that have not yet been identified/identified. For example, in mouse EAT (Ehrlich Ascites Tumor) cells The processing of nin is released into the cytosol through signal transduction pathways linked to phospholipase C (PLC), such as the signal transduction pathways that occur when G protein coupled receptors (GPCRs or 7TM receptors) are activated. Ca ²⁺ (Free Ca ²⁺ ) is temporarily increased, and when treated with frog eggs (Xenopus oocytes), PTX (pertussis toxin)-insensitive G protein → PLC (phospholipase C) → IP3 (inositol 1) ,4,5-triphosphate) → It was found that the calcium-dependent chloride ion channel (Ca ²⁺ activated Chloride Channel, CaCC) is activated through the release of calcium from the endoplasmic reticulum (ER), which is a calcium store. However, the mechanisms involved in the prophylactic and/or therapeutic effects of gintonin and its anti-inflammatory effects on Huntington's disease are still unclear.

Accordingly, the present inventors confirmed that neurological disorders and striatum toxicity induced by 3-nitropropionic acid (3-NPA) can be improved in a mouse model of gintonin. The beneficial effect of gintonin was confirmed in STHdh cells (a cell model of Huntington's disease) and a mouse model infected with mutant huntingtin (mHTT) by an Adeno-associated viral (AAV) vector. Accordingly, the present invention was completed by confirming that gintonin can be used as a new preventive or therapeutic agent for Huntington's disease by Keap1-Nrf2 signal activation.

Korean Patent Registration No. 10-0973202 Korean Patent Publication No. 10-2019-0124082

Choi, et. al., 2015. Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions., Front. Pharmacol. 6, 245.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating Huntington's disease.

Another object of the present invention is to provide a health functional food composition for preventing or improving Huntington's disease, comprising gintonin as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating damage and death of nerve cells caused by 3-NPA.

Another object of the present invention is to provide a health functional food composition for preventing or improving the damage and death of nerve cells caused by 3-NPA.

In order to achieve the above purpose,

The present invention provides a pharmaceutical composition for preventing or treating Huntington's disease comprising gintonin as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving Huntington's disease, comprising gintonin as an active ingredient.

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating damage and death of nerve cells caused by 3-NPA containing gintonin as an active ingredient.

Furthermore, the present invention provides a health functional food composition for preventing or improving nerve cell damage and death caused by 3-NPA containing gintonin as an active ingredient.

The composition containing gintonin of the present invention activates the Keap1-Nrf2 signaling pathway and inhibits the NF-κB and MAPKs signaling pathways, thereby improving 3-NPA-induced neurological disorders and striatum toxicity, and improving STHdh As it was confirmed that the present invention exhibits beneficial effects in a Huntington's disease mouse model infected with cells and adeno-associated virus (AAV) vectors, it may be useful as a composition for preventing, treating, or improving Huntington's disease.

Figure 1 is a schematic image of the experimental design showing the treatment time point of 3-NPA and gintonin (gintonin, GT).
Figure 2 shows the effects of gintonin (GT) treatment concentrations on neurological disorders, lethality, and striatal neuronal cell death caused by 3-NPA intoxication: neurological score for nerve damage in mice (neurological score; A), survival rate (B) and average body weight (C).
Figure 3 is a graph of the neurological damage of mice according to pre-treatment (pre-), co-treatment (co-), onset-treatment (onset-) and progress treatment (progression-) to confirm the time period of gintonin treatment in 3-NPA poisoning. Neurological score (A), survival rate (B) and mean body weight (C) are shown.
Figure 4 is an image of striatal lesions stained with cresyl violet dye for striatal neuron death according to 3-NPA and / or gintonin treatment (A), a result of quantifying the number of mice having striatal lesions (B), and striatal The result of measuring the average lesion area in (C) and the graph (D) quantifying the protein expression change of β-III-tubulin by analyzing the striatum of each group by Western blot.
5 shows the effects of gintonin pretreatment on mitochondrial dysfunction and apoptosis in the striatum after 3-NPA intoxication. Here, Figures 5A to 5D are images comparing SDH activity levels by group through histochemical analysis, Figure 5E is a graph quantifying the SDH activity (%) of the striatum compared to the sham group, and Figures 5F to 3I are histochemical 5J is a graph quantifying the number (%) of positive cells as normalized mitoSOX intensity, and FIGS. 5K to 5N and 5O to 5R are TUNEL staining (KO), respectively. ) and an image showing the distribution of apoptosis cells through FJC staining (OR), and FIG. 5S is an image showing the protein expression of activated caspase-3 (cleaved caspase-3) by analyzing the striatum of each group by Western blot. It is a graph that quantifies the change.
Figure 6 shows the effect of gintonin pretreatment on microglia activation and inflammatory response mRNA expression in the striatum after 3-NPA intoxication. Here, FIGS. 6A to 6D are images showing the results of immunostaining using an anti-Iba-1 antibody for each group, and FIGS. 6E and 6L are Western blot analysis of striatal tissue for each group to show Iba-1 ( E) and iNOS (L) are graphs quantifying the protein expression, and FIGS. 6F to 6K are OX-42 (F), IL-1β (G), and IL-6 by analyzing the striatal tissue for each group by real-time PCR. (H), it is a graph quantifying mRNA expression of TNF-α (I), COX-2 (J) and iNOS (K).
Figure 7 shows the effect of gintonin pretreatment on astrocyte activation and GFAP mRNA expression in the striatum after 3-NPA intoxication. Here, FIGS. 7A to 7D are images showing the results of immunostaining using an anti-GFAP antibody for each group, and FIGS. 7E and 7F are the results of analyzing GFAP protein expression by analyzing the striatal tissues of each group by Western blot (E ) and a graph quantifying it (F), and FIG. 7G is a graph quantifying the mRNA expression of GFAP by analyzing striatal tissues by real-time PCR.
Figure 8 shows the effect of gintonin pretreatment on acute apoptosis in the striatum after 3-NPA intoxication. Here, FIGS. 8A to 8D and FIGS. 8F to 8I are images (AD) showing the results of histochemical staining of the striatal region for each group with FJC (Fluoro-Jade® C) and immunofluorescence with anti-Iba-1 antibody, respectively. 8E and 8J are graphs quantifying the degree of staining with the FJC (E) and the anti-Iba-1 antibody (J), respectively.
Figure 9 shows the effect of gintonin (GT) pretreatment on the activation of the Keap1-Nrf2 signaling pathway in the striatum after 3-NPA intoxication. Here, FIGS. 9A to 9E show the mRNA expression levels of Keap1 (A), Nrf2 (B), NQO1 (C), GCLs (D), and HO-1 (E) by analyzing striatal tissues for each group by real-time PCR. 9F and 9G are graphs of quantification, and FIGS. 9F and 9G show the results of measuring the protein expression levels of Nrf2 and HO-1 by analyzing group-specific striatum by Western blot (F), and each protein for total histones or GAPDH 9H and 9I are graphs quantified by ratio (G), and FIGS. 9H and 9I are images (H) showing the results of immunostaining of striatal tissue by group using anti-Nrf2 antibody and anti-HO-1 antibody and the degree of staining. This is the quantified image (I).
Figure 10 shows the effects of gintonin (GT) pretreatment on the regulation of MAPKs and NF-κB signaling pathways in the striatum after 3-NPA intoxication. Here, FIGS. 10A to 10F show the results of measuring the protein expression levels of p-ERK, p-JNK, p-p38, NF-κB/p65, and p-IκBα by analyzing group-specific striatal tissues by Western blot (A) and a graph (BF) quantifying each protein as a ratio to total GAPDH.
Figure 11 shows the effect of gintonin (GT) on cell death and modified HTT (mutant huntingtin, mHTT) aggregates in the STHdh cell line. Here, Figure 11A is a graph showing the cell death rate according to the concentration and time of gintonin treatment in STHdh ^Q7/Q7 and STHdh ^Q111/Q111 cells, and Figure 11B is a graph showing EM48, active caspase in STHdh ^Q111 cells through Western blot analysis. This is the result of measuring the protein expression levels of cleaved caspase-3 and β-III-tubulin, and FIG. 11C is a graph in which each protein is quantified as a ratio to total GAPDH.
Figure 12 shows the protective effect of gintonin (GT) against neurological damage and striatal toxicity in AAV-DJ-82Q vector infected Huntington's disease mouse model. Here, Figure 12A is a graph showing the results of measuring the average latency to descend to the bottom of the pole through a pole test, and Figure 12B is a graph showing the average latency to descend from the rod to the floor through a rotarod test. A graph showing the result of measuring latency to fall, FIG. 12C shows weight change, and FIG. 12D shows the protein expression levels of EM48, Nrf2 and HO-1 by analyzing striatal tissue by group through Western blot. 12E is a graph quantifying the ratio of each protein to total histones or GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and FIG. 12F shows the striatal regions of each group using Nrf2 and HO-1 antibodies 12G is an image quantifying the degree of immunostaining, and FIG. 12H is a result of evaluating the protein expression levels of EM48, Nrf2, and HO-1 by analyzing STHdh ^Q111 cells by Western blot. 12I is a graph quantifying the ratio of each protein to total histones or GAPDH.
Figure 13 shows the protective effect of gintonin (GT) against neurological damage and striatal toxicity in AAV-DJ-82Q vector infected Huntington's disease mouse model. 13A to 13D are images showing the results of immunostaining using the EM48 antibody for each group, and 13E is a graph quantifying them.
14 is a result of evaluating the protein expression levels of Nrf2 and HO-1 by analyzing H ₂ O ₂ treated STHdh ^Q111 cells by Western blot to confirm the effect of Nrf2 inhibitor and siRNA-Nrf2 treatment (A and HO-1). C) and graphs (B and D) quantifying each protein as a ratio to total histones or GAPDH.

Hereinafter, the present invention will be described in detail.

Pharmaceutical composition for preventing or treating Huntington's disease

The present invention provides a pharmaceutical composition for preventing or treating Huntington's disease comprising gintonin as an active ingredient.

In the pharmaceutical composition according to the present invention, the gintonin may be used by receiving a glycolipoprotein (Patent Registration No. 10-0973202) extracted and isolated from ginseng.

In the pharmaceutical composition according to the present invention, the Huntington's disease may be caused by 3-NPA, and may be caused by neurological disorders and striatum toxicity caused by the 3-NPA. The Huntington's disease may include Huntington's chorea.

In the pharmaceutical composition according to the present invention, the gintonin is any one selected from the group consisting of neurodegeneration, mitochondrial dysfunction, increased apoptosis, apoptosis, microglia activation, increased expression of inflammatory mediators, and behavioral disorders in the striatum. It may be to suppress the above symptoms.

In one embodiment of the present invention, cell death, cell damage, mitochondrial dysfunction, apoptosis, infiltration and activation of microglia, inflammatory factors, behavioral disorders (motor control disorders), etc. are induced in the striatum by 3-NPA. It was confirmed that, upon treatment with gintonin, these symptoms were suppressed and improved, and it was confirmed that there was an effect of protecting the striatum from toxicity induced by 3-NPA (see Experimental Examples 2 to 7).

In the pharmaceutical composition according to the present invention, the gintonin may exhibit an effect of preventing or treating Huntington's disease by inhibiting infiltration and activation of microglia through activation of the Keap1-Nrf2 signal transduction pathway. In addition, the gintonin may inhibit NF-κB and MAPKs signal transduction pathways to prevent or treat Huntington's disease.

In one embodiment of the present invention, gintonin activates the Keap1-Nrf2 signaling pathway and inhibits the NF-κB and MAPKs signaling pathways, thereby showing a protective effect of reducing 3-NPA-induced striatal toxicity. It was confirmed (see Experimental Examples 8 to 9).

In the pharmaceutical composition according to the present invention, the gintonin may inhibit aggregation of modified huntingtin (mHTT).

In one embodiment of the present invention, gintonin inhibits the formation of modified huntingtin (mHTT) aggregates in the STHdh cell line as an in vitro model of Huntington's disease by activating the Keap1-Nrf2 signal transduction pathway and is infected with the AAVDJ-82Q vector for Huntington's disease. It was confirmed that symptoms related to Huntington's disease, such as Huntington's disease-like striatal toxicity and motor dysregulation, induced by AAV-mediated overexpression of human mutant HTT in a mouse model could be alleviated (see Experimental Examples 10 to 11).

The pharmaceutical composition may further include a pharmaceutically acceptable carrier. In the above, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause an allergic reaction such as gastrointestinal disorder, dizziness, or similar reaction when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid, and carriers for parenteral administration such as water, suitable oil, saline, aqueous glucose and glycol, etc. There are carriers and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995).

The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, etc., and these solid preparations contain one or more compounds of the present invention in at least one excipient, for example, starch, calcium carbonate, water It is prepared by mixing sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, or syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

The pharmaceutical composition may further include a carrier, excipient or diluent. Carriers, excipients, or diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "administration" means introducing a predetermined substance into a subject by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, but not limited thereto.

The term "individual" refers to all animals such as rats, mice, livestock, and the like, including humans. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the sex and age of the patient. , weight, health condition, type of disease, severity, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other medical fields It can be easily determined by a person skilled in the art according to well-known factors. It is generally about 0.001 to 100 mg/kg/day, preferably 0.01 to 35 mg/kg/day. Based on an adult patient weighing 70 kg, it is generally 0.07 to 7000 mg/day, preferably 0.7 to 2500 mg/day, and the recommended dose may be administered once a day, several times It can also be administered in divided doses.

The composition of the present invention can be used alone or in combination with methods using surgery, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of Huntington's disease.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Huntington's disease, comprising gintonin as an active ingredient and increasing succinate dehydrogenase in Huntington's disease patients in whom succinate dehydrogenase is inhibited in the striatum. .

In addition, the present invention includes gintonin as an active ingredient, and the production or expression of Iba-1, OX-42, IL-1β, IL-6, TNF-α, COX-2 and iNOS in the striatum is inhibited in patients A pharmaceutical composition for preventing or treating Huntington's disease for increasing the production or expression of Iba-1, OX-42, IL-1β, IL-6, TNF-α, COX-2 and iNOS is provided.

In addition, the present invention contains gintonin as an active ingredient, inhibits the production or expression of Keap1 in patients with increased production or expression of Keap1 in the striatum, and is selected from the group consisting of Nrf2, NQO1, GCLs and HO-1 Provided is a pharmaceutical composition for preventing or treating Huntington's disease for increasing the production or expression of one or more species.

In addition, the present invention includes gintonin as an active ingredient and treats p-ERK, p-JNK, p-p38, NF-κB/p65 and p-IκBα in patients with increased production or expression in the striatum. , To inhibit the production or expression of p-JNK, p-p38, NF-κB/p65 and p-IκBα, providing a pharmaceutical composition for preventing or treating Huntington's disease.

In addition, the present invention contains gintonin as an active ingredient and treats EM48 and activated caspase-3 in patients with increased production or expression of EM48 and cleaved caspase-3 in the striatum. Provided is a pharmaceutical composition for preventing or treating Huntington's disease for suppressing production or expression.

In the present invention, the patient may be a human or an animal.

Health functional food composition for preventing or improving Huntington's disease

The present invention provides a health functional food composition for preventing or improving Huntington's disease, comprising gintonin as an active ingredient.

The term "health functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, pills, liquids, powders and granules using raw materials or ingredients having useful functionalities for the human body. Here, 'functionality' means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be manufactured by a method commonly used in the conventional technical field, and can be prepared by adding raw materials and components commonly added in the conventional technical field at the time of manufacture. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The health functional food composition of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of using food-derived ingredients as raw materials and has no side effects that may occur when taking drugs for a long time, and has excellent portability. , The health functional food of the present invention can be consumed as an adjuvant to enhance the effect of a treatment for Huntington's disease.

In addition, the present invention provides a health food composition for preventing or improving Huntington's disease comprising gintonin as an active ingredient.

In the health functional food composition or health food composition according to the present invention, the Huntington's disease may be caused by 3-NPA, and may be caused by neurological disorders and striatum toxicity caused by the 3-NPA. . The Huntington's disease may include Huntington's chorea.

In the health functional food composition or health food composition according to the present invention, the gintonin is a group consisting of neurodegeneration, mitochondrial dysfunction, increased apoptosis, apoptosis, microglia activation, increased expression of inflammatory mediators, and behavioral disorders in the striatum. It may be to suppress any one or more symptoms selected from.

In one embodiment of the present invention, cell death, cell damage, mitochondrial dysfunction, apoptosis, infiltration and activation of microglia, inflammatory factors, behavioral disorders (motor control disorders), etc. are induced in the striatum by 3-NPA. It was confirmed that, upon treatment with gintonin, these symptoms were suppressed and improved, and it was confirmed that there was an effect of protecting against toxicity of the striatum induced by 3-NPA (see Experimental Examples 2 to 7).

In the health functional food composition or health food composition according to the present invention, the gintonin may prevent or improve Huntington's disease by suppressing the infiltration and activation of microglia through activation of the Keap1-Nrf2 signal transduction pathway. In addition, the gintonin may inhibit NF-κB and MAPKs signaling pathways to prevent or improve Huntington's disease.

In one embodiment of the present invention, by activating the gintonin Keap1-Nrf2 signaling pathway and inhibiting the NF-κB and MAPKs signaling pathways, the protective effect of reducing the toxicity of the striatum induced by 3-NPA can be shown. It was confirmed (see Experimental Examples 8 to 9).

In the health functional food composition or health food composition according to the present invention, the gintonin may inhibit aggregation of modified huntingtin (mHTT).

In one embodiment of the present invention, the formation of modified huntingtin (mHTT) aggregates is inhibited in STHdh cell line as an in vitro model of Huntington's disease by activating the gintonin Keap1-Nrf2 signal transduction pathway, and Huntington's disease infected with AAVDJ-82Q vector It was confirmed that HD-related symptoms, such as HD-like striatal toxicity and motor dysregulation, induced by AAV-mediated overexpression of human mutant HTT in a mouse model could be alleviated (see Experimental Examples 10 to 11).

The health functional food composition or health food composition according to the present invention can be added to health functional food or health food such as food or beverage for the purpose of preventing or improving related diseases through the effect of preventing or improving Huntington's disease. there is.

There is no particular limitation on the type of food. Examples of foods to which gin and tonin according to the present invention can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, and dairy products including ice cream. , various soups, beverages, alcoholic beverages and vitamin complexes, dairy products and milk-processed products, etc., and include both health foods and health functional foods in a conventional sense.

The health food and health functional food composition according to the present invention may be added to food as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the gin and tonin according to the present invention may be appropriately determined depending on the purpose of use (prevention or improvement). In general, the amount of gintonin in health food and health functional food may be added in an amount of 0.1 to 90 parts by weight based on the total weight of food. However, in the case of long-term intake for the purpose of health maintenance or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

The health food and health functional food composition of the present invention is not particularly limited in other ingredients except for containing gin and tonin according to the present invention as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates are added like conventional beverages. It can be contained as a component. Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the health functional food composition of the present invention.

In addition to the above, the health food and health functional food composition containing gintonin according to the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, and enhancers (cheese, chocolate) etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the health food and health functional food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.

These components may be used independently or in combination. The ratio of these additives is not so critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the health food and health functional food composition containing the active substance of the present invention.

In addition, the present invention provides a health functional food composition for preventing or improving Huntington's disease, comprising gintonin as an active ingredient and increasing succinate dehydrogenase in Huntington's disease patients in whom succinate dehydrogenase is inhibited in the striatum. do.

In addition, the present invention includes gintonin as an active ingredient, and the production or expression of Iba-1, OX-42, IL-1β, IL-6, TNF-α, COX-2 and iNOS in the striatum is inhibited in patients Provided is a health functional food composition for preventing or improving Huntington's disease to increase the production or expression of Iba-1, OX-42, IL-1β, IL-6, TNF-α, COX-2 and iNOS.

In addition, the present invention contains gintonin as an active ingredient, inhibits the production or expression of Keap1 in patients with increased production or expression of Keap1 in the striatum, and is selected from the group consisting of Nrf2, NQO1, GCLs and HO-1 Provided is a health functional food composition for preventing or improving Huntington's disease for increasing the production or expression of one or more species.

In addition, the present invention includes gintonin as an active ingredient and treats p-ERK, p-JNK, p-p38, NF-κB/p65 and p-IκBα in patients with increased production or expression in the striatum. , To inhibit the production or expression of p-JNK, p-p38, NF-κB/p65 and p-IκBα, it provides a health functional food composition for preventing or improving Huntington's disease.

In addition, the present invention contains gintonin as an active ingredient and treats EM48 and activated caspase-3 in patients with increased production or expression of EM48 and cleaved caspase-3 in the striatum. Provided is a health functional food composition for preventing or improving Huntington's disease for suppressing production or expression.

Composition for preventing, improving or treating damage and death of nerve cells induced by 3-NPA

Furthermore, the present invention provides a pharmaceutical composition for preventing or treating damage and death of nerve cells caused by 3-NPA containing gintonin as an active ingredient.

In one embodiment of the present invention, the damage and death of the nerve cells may be caused by striatum toxicity induced by 3-NPA.

In one embodiment of the present invention, the pharmaceutical composition may prevent or treat diseases caused by damage and death of nerve cells caused by 3-NPA. In this case, the disease may include cognitive impairment, movement disorder, personality disorder, or Huntington's disease, preferably Huntington's disease.

In the pharmaceutical composition according to the present invention, the gintonin may activate the Keap1-Nrf2 signal transduction pathway to prevent or treat damage and death of nerve cells caused by 3-NPA. In addition, the gintonin may inhibit NF-κB and MAPKs signaling pathways to prevent or treat 3-NPA-induced neuronal damage and death.

In one embodiment of the present invention, gintonin protects from 3-NPA-induced striatal toxicity, resulting in cell death, cell damage, mitochondrial dysfunction, apoptosis, infiltration and activation of microglia, inflammatory factors, and behavioral disorders (movement It was confirmed that there is an effect of suppressing and improving symptoms such as dysregulation) (see Experimental Examples 2 to 7).

In one embodiment of the present invention, it was confirmed that gintonin can protect primary apoptosis by 3-NPA and secondary apoptosis by activation of microglia in the striatum (see Experimental Example 7).

In one embodiment of the present invention, it was confirmed that gintonin activates the Keap1-Nrf2 signaling pathway and inhibits the NF-κB and MAPKs signaling pathways, thereby exhibiting a protective effect of reducing 3-NPA-induced striatal toxicity. (See Experimental Examples 8 to 9).

In one embodiment of the present invention, gintonin inhibits the formation of modified huntingtin (mHTT) aggregates in the STHdh cell line as an in vitro model of Huntington's disease through activation of the Keap1-Nrf2 signaling pathway, and in AAVDJ-82Q vector-infected cells. It was confirmed that Huntington's disease-related symptoms, such as Huntington's disease-like striatal toxicity and motor dysregulation, induced by AAV-mediated overexpression of modified huntingtin (mHTT) in the Huntington's disease mouse model can be alleviated (see Experimental Examples 10 to 11) .

Furthermore, the present invention provides a health functional food composition for preventing or improving nerve cell damage and death caused by 3-NPA containing gintonin as an active ingredient.

In one embodiment of the present invention, the damage and death of the nerve cells may be caused by striatum toxicity induced by 3-NPA.

In one embodiment of the present invention, the health functional food composition may prevent or improve diseases caused by damage and death of nerve cells caused by 3-NPA. In this case, the disease may include cognitive impairment, movement disorder, personality disorder, or Huntington's disease, preferably Huntington's disease.

In the health functional food composition according to the present invention, the gin and tonin may activate the Keap1-Nrf2 signal transduction pathway to prevent or improve neuronal damage and death caused by 3-NPA. In addition, the gintonin may inhibit NF-κB and MAPKs signaling pathways to prevent or improve 3-NPA-induced damage and death of nerve cells.

In one embodiment of the present invention, gintonin protects from 3-NPA-induced striatal toxicity, resulting in cell death, cell damage, mitochondrial dysfunction, apoptosis, infiltration and activation of microglia, inflammatory factors, and behavioral disorders (movement It was confirmed that there is an effect of suppressing and improving symptoms such as dysregulation) (see Experimental Examples 2 to 7).

In one embodiment of the present invention, it was confirmed that gintonin can protect primary apoptosis by 3-NPA and secondary apoptosis by activation of microglia in the striatum (see Experimental Example 7).

In one embodiment of the present invention, it was confirmed that gintonin activates the Keap1-Nrf2 signaling pathway and inhibits the NF-κB and MAPKs signaling pathways, thereby exhibiting a protective effect of reducing 3-NPA-induced striatal toxicity. (See Experimental Examples 8 to 9).

In one embodiment of the present invention, gintonin inhibits the formation of modified huntingtin (mHTT) aggregates in the STHdh cell line as an in vitro model of Huntington's disease through activation of the Keap1-Nrf2 signaling pathway, and in AAVDJ-82Q vector-infected cells. It was confirmed that Huntington's disease-related symptoms, such as Huntington's disease-like striatal toxicity and motor dysregulation, induced by AAV-mediated overexpression of human modified huntingtin (mHTT) in the Huntington's disease mouse model could be alleviated (see Experimental Examples 10 to 11). ).

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Preparation of gin and tonin

Gintonin (GT) was prepared according to the method of Choi et al. (Front. Pharmacol. 6;245, 2015). Briefly, 1 kg of 4-year-old ginseng (Korea Ginseng Corporation, Daejeon, Republic of Korea) was ground into small pieces (> 3 mm) and refluxed 8 times with 70% fermented ethanol at 80° C. for 8 hours to obtain an extract. The extract was concentrated, and the concentrate (340 g) was dissolved in distilled cold water at a ratio of 1:10, and stored at 4° C. for 24-96 hours, followed by water precipitation. The supernatant and precipitate fraction obtained by water fractionation (water precipitation method) of ethanol extract of ginseng were separated by centrifugation (3000 rpm, 20 minutes), and the precipitate was freeze-dried to obtain gintonin.

Gintonin is composed of carbohydrates, lipids, and ginseng proteins, and the ratios of total carbohydrates, lipids, and proteins in gintonin were about 30%, 20.2%, and 30.3%, respectively, and other trace components were included.

The lipid composition of gintonin based on LC-MS/MS analysis was as follows: fatty acids (7.53% linoleic acid, 2.82% palmitic acid and 1.46% oleic acid); lysophospholipids and phospholipids (0.60%); and phosphoric acid (1.75%). The total lipid content of gintonin is about 14.2%. Qualitative analysis showed that gintonin also contained diacylglycerols and triacylglycerols.

<Experimental Example 1> Preparation and Experimental Method of Animal Model

1-1. Preparation of experimental animals

Male adult C57BL/6NTac mice (Narabiotec Co., Ltd., Seoul, Republic of Korea; body weight 23-25 g; seeded mice were from Taconic Biosciences Inc. (Cambridge, IN, USA)) were seeded 1 week before the experiment. They were acclimatized in a breeding facility maintained at a temperature of 23 ± 2 °C with a 12-hour light/dark cycle (light from 08:00 to 20:00) with food and water provided ad libitum.

1-2. ethical approval

All experimental procedures were reviewed and approved by the Animal Research Ethics Committee of Kyung Hee University (KHUASP(SE)-14-040). Appropriate randomization of laboratory animals and data handling in this process were performed in accordance with the recent recommendations of the NIH Workshop on Preclinical Models of Neurological Diseases (Landis et al., 2012, Nature 490, 187-191).

1-3. Treatment of 3-NPA and gintonin

The prepared experimental animals were treated with 3-nitropropionic acid (3-NPA) and gintonin (GT) in the following manner.

First, 3-NPA was prepared and processed according to a known method. Briefly, 3-NPA (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in saline (50 mg/ml concentration, pH 7.4) and passed through a 0.2-μm filter at 12-hour intervals for 2 days. (8:30 am and 8:30 pm) 60 mg/kg on the first day and 80 mg/kg on the second day, twice daily (i.e., 60-60-80-80 dose regimen) intraperitoneally (i.p.) administration (FIG. 1).

In the case of gintonin, it was dissolved in saline and orally administered (per oral, p.o.) at a dose of 25, 50 or 100 mg/kg. Pre-treatment and co-treatment of gintonin were defined as daily administration before, 5 days before, and 1 hour before the first 3-NPA intoxication, respectively. Onset-treatment and progression-treatment were the time when nerve damage was first observed (1 hour before the third 3-NPA intoxication; average score, 0–1) and the time when nerve damage increased after the onset phase, respectively. It was defined as the time point (1 h before the fourth (last) 3-NPA intoxication; average score, 2-4) (Figure 1).

Each experiment was repeated at least three times using the same protocol, and the severity of nerve damage was assessed immediately before every administration and at the end of the experiment.

1-4. experimental group

Experiments were performed using mice divided into the following five groups.

(1) To select the most effective concentration of gintonin (GT) in 3-NPA intoxication, mice were co-treated with sham, 3-NPA and 3-NPA+GT (25, 50 and 100 mg/kg/day, p.o.) and randomly divided into groups.

(2) To investigate the therapeutic time-window of gintonin in 3-NPA intoxication, mice were sham, 3-NPA and 3-NPA + GT (100 mg / kg / day pre-treatment, simultaneous treatment, Onset treatment and progression treatment) were randomly divided into groups.

(3) To investigate the histopathological and molecular mechanisms of gintonin, mice were randomly divided into sham, 3-NPA, 3-NPA+GT (pre-treated with 100 mg/kg/day, p.o.) and GT groups.

(4) To investigate the acute protective effect of gintonin in 3-NPA poisoning, mice were randomly assigned to sham, 3-NPA, 3-NPA+GT (pre-treatment with 100 mg/kg/day, p.o.) and GT groups. Divided.

(5) Normal, hydrogen peroxide (H ₂ O ₂ ) and H ₂ using STHdh ^Q7/Q7 (WT cells) and STHdh ^Q111/Q111 cells to investigate the effect of gintonin in STHdh cells as an in vitro model of Huntington's disease. It was divided into O ₂ +GT (0.1, 1 or 10 ul/ml) groups.

(6) To investigate the effect of gintonin in an AAV vector-infected mouse model of Huntington's disease, an AAV vector containing N171-18Q wild-type HTT (18Q; WT vector) and N171-82Q mutant HTT (82Q) serotype DJ ( AAV-DJ) mice were randomly divided into sham, AAV-DJ-18Q and AAV-DJ-82Q groups.

1-5. Semi-quantitative behavioral assessment after 3-NPA intoxication

Specific neurological behavioral changes caused by 3-NPA in mice were observed once a day to evaluate the degree of neurological damage. The criteria for behavior change were (1) reduced activity, (2) grasping of the hind limbs, (3) dystonia of the hind limbs, (4) kyphosis of the thoracic spine, and (5) recovery to a normal posture, each of which was 0 according to the degree. , 1 and 2 steps were measured, and each index step was added (a total of 10 steps) and compared. The higher the score, the more severe the neurological behavioral symptoms. In addition, the survival rate and body weight were confirmed 24 hours after the last (fifth) administration of 3-NPA.

1-6. AAV vector generation

To generate AAV-HTT-N171-82Q, an N171-82Q mouse model of Huntington's disease was constructed using the HD-N171-82Q cDNA construct in a pBluescript cloning vector. Human HTT and the first three exons (171 amino acids) of human HTT with 82 CAG gene repeats (HTT-N171-82Q) were obtained from Dr. David R. Borchelt (Santa Fe Health Care Alzheimer's Disease Research Center, University of Florida). A structure was provided and used. The HTT-N171-82Q construct was double digested with EcoRI/XhoI and inserted into the multiple cloning site of an AAV-MCS expression vector (Cell Biolabs, Inc. cat# VPK-410). Expression of the vector was performed by the KIST Virus Facility (http://virus.kist.re.kr) under the cytomegalovirus (CMV) immediate early enhancer and promoter as previously described (Jang et al. ., 2018, Front Cell Neurosci. 12, 157). As a control vector, HTT cDNA containing 18Q CAG repeats was used. The production titer was 1.3×10 ¹³ genome copies/ml (GC/ml) for AAV-DJ-HTT-N171-82Q and 1.6×10 ¹³ GC/ml for AAV-DJ-HTT-N171-18Q. was

1-7. AAV vector infection by stereotactic brain surgery

AAV-DJ vector infection was performed through stereotaxic brain surgery according to a previously known method (Jang et al., 2018, Front Cell Neurosci. 12, 157). Briefly, mice (4 weeks old; 14-15 g body weight) were deeply anesthetized (2-3% isoflurane, 60% O ₂ , 40% N ₂ O), and placed in a stereotaxic device (myNeuroLab, St. Louis, MO; USA) was fixed. 2 μl of AAV-DJ-18Q (7.9 × 10 ¹² GC/ml) and AAV-DJ-82Q (1.23 × 10 ¹² GC/ml) were injected into a 30-gauge, sharp-tipped Hamilton syringe (Sigma-Aldrich, Saint Louis). , MO, USA) was used to induce infection with the AAV-DJ vector by injecting into the mid-coronal of the bilateral striatum.

1-8. Behavioral assessment after AAV vector infection

To investigate whether injection of the AAV-DJ-82Q vector causes neurological damage (decreased motor control and imbalance), body weights were collected weekly for 10 weeks from 1 day before injection of the AAV-DJ-82Q and AAV-DJ-18Q vectors. were measured and pole and rota-rod behavioral tests were performed.

1-9. STHdh cell culture

Wild-type (STHdh ^Q7/Q7 ) and mutant (STHdh ^Q111/Q111 ) cells of the STHdh cell line (a cell line of conditionally immortalized progenitor progenitor neurons) were cultured in 10 CT-75cm ² flasks in a 5% CO ₂ incubator at 33°C. Cultured in DMEM supplemented with % FBS and 1% P/S and 1% G418 (geneticin). After 7 days, these cells were detached from the cell culture flask using trypsin and seeded in a 24-well plate at a density of 2.5×10 ⁵ cells/well for analysis.

Meanwhile, Nrf2 siRNA was transfected with siRNA transfection reagent (Santa Cruz) according to the manufacturer's protocol. Briefly, STHdh ^Q111/Q111 cells with 60% confluency were incubated with 50 nM Nrf2 siRNA or 50 nM control siRNA (scRNA) duplex/transfection reagent mix and/or jinto for 6 hours. Cultured with serum-free medium containing nin (1 μg/ml). 24 hours after transfection, the medium was changed to complete medium and the cells were further cultured for 24 hours. These cells were then further treated with H ₂ O ₂ (200 μM) for 5 hours or the treatment was omitted. Also, instead of Nrf2 siRNA, 50 μM Nrf2 inhibitor (ML385) was treated for 12 hours.

1-10. Preparation of frozen sections for histopathological analysis

Histopathological changes in the striatum after 3-NPA intoxication are described in protocols (Jang and Cho, 2016, Mol. Neurobiol. 53, 2619-2635; Jang et al., 2014, Brain Behav. Immun. 38, 151-165; Jang et al. al., 2013, Evidence Based Complement Altern. Med. 2013, 237-207). The specific method is as follows.

Mice (sham, n=5; 3-NPA, n=8; 3-NPA+GT25, n=8; 3-NPA +GT50, n=8; 3-NPA+GT100, n=8) were anesthetized with diethyl ether, and then 0.1 M phosphate buffer (PB, pH 7.4) and cold 4% paraformaldehyde solution were perfused into the heart. . Refer to the mouse brain atlas (Franklin and Paxinos, 2008) and use a cryosection machine (CM3050S; Leica, Wetzlar, Germany) to take coronal sections (30 μm thick) of the brain including the striatum. ), and tissue sections were obtained in 0.1 M phosphate buffer. Striatal tissue sections (210 μm apart) per average 300 μm were stained with cresyl violet dye.

1-11. SDH activity assay

After the brain was rapidly frozen without fixation, cryosections (10 μm thick) of the region including the striatum were prepared (sham, n = 3; 3-NPA, n = 5; 3-NPA + GT100, n = 5; and GT100 , n=3), according to previously known methods (Jang and Cho, 2016, Mol. Neurobiol. 53, 2619-2635; Jang et al., 2014, Brain Behav. Immun. 38, 151-165; Jang et al. ., 2013, Evidence Based Complement Altern. Med. 2013, 237-207) SDH activity was evaluated. Quantification of SDH expression levels was measured in the cerebral cortex and lateral striatum using the NIH Image J program (Brouillet et al., 1998, J. Neurochem. 70, 794-805). Measurement regions (regions of interest, ROI) were observed as digitized images, and SDH activity (% compared to sham) was analyzed by calculating the color of each section in the ROI as a chroma value.

1-12. MitoSOX activity assay

MitoSOX activity was measured according to a conventional method (Wojtala et al., 2014, Methods Enzymol. 542, 243-262). Briefly, tissue sections from each group (sham, n=3; 3-NPA, n=5; 3-NPA+GT100, n=5; and GT100, n=3) were free floating. Incubated for 10 minutes, the probe was allowed to enter the cells and start the reaction in 0.5 ml of assay buffer containing 5 mM MitoSOX Red (Molecular Probes, Eugene, OR, USA) inside the mitochondria at 37°C. Tissue sections were then washed twice with PBS and covered with coverglass.

1-13. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)

To evaluate the level of apoptosis in the striatum after 3-NPA intoxication, terminal deoxynucleotidyl transferase dUTP nick end labeling (Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL) was performed.

Specifically, brain tissue slices (30 μm thick) were taken from each group (sham, n=3; 3-NPA, n=5; 3-NPA+GT100, n=5; and GT100, n=3), and ApopTag® The degree of apoptosis was evaluated using the Peroxidase In Situ Apoptosis Detection Kit (S7100) (Millipore, Billerica, MA, USA).

1-14. Fluoro Jade C (FJC) staining

Fluoro-Jade C (FJC) staining was performed according to a conventional method (Schmued et al., 1997, Brain Res. 751, 37-46). The specific method is as follows. The obtained free-floating sections from each group were immersed in 70% ethanol for 2 minutes and washed with distilled water (DW) for 2 minutes. Sections were then incubated in 0.06% potassium permanganate solution for 20 minutes, washed with distilled water, and immersed in 0.001% FJC dye (Millipore) solution for 10 minutes on a shaker. Then, the sections were washed with distilled water for 3 minutes, dried in air for 30 minutes, immersed in 100% xylene for 2 minutes, and then coverslipped with DPX mounting medium (Sigma-Aldrich). .

1-15. Immunohistochemical analysis

Immunohistochemical evaluation was performed using conventional methods (Jang and Cho, 2016, Mol. Neurobiol. 53, 2619-2635; Jang et al., 2014, Brain Behav. Immun. 38, 151-165; Jang et al., 2013, Evidence Based Complement Altern. Med. 2013, 237-207). The specific method is as follows.

Brain tissue sections (30 μm thick) from each group (sham, n=5; 3-NPA, n=8; 3-NPA+GT100, n=8; and GT100, n=5) were treated with rabbit anti-ionized calcium -Binding adapter molecule 1 (rabbit anti-Iba-1; 1:2,000; WAKO, Chuo-Ku, Japan) and rabbit anti-glial fibrillary acidic protein (rabbit anti-GFAP; 1:5,000; DACO, Carpinteria, CA, USA). Each section was incubated with biotinylated rabbit/mouse IgG antibody (1:200; Vector Laboratories, Burlingame, CA USA), and avidin-biotinylated horseradish peroxidase-complex (abidin- After incubation with biotinylated horseradish peroxidase complex; 1:200; Vector Laboratories), it was visualized by staining with 3,3'-diamino-benzidine. Immunostained sections were analyzed using the DP70 image analysis system (Olympus).

In addition, immunofluorescence staining was performed as in the conventional method (Lee et al., 2016a). Specifically, frozen striatal tissue sections (n=5 per brain in each group) were incubated with primary antibodies, then stained with FJC dye (Millipore) with a mixture of secondary antibodies, and confocal imaging system (LSM 5 PASCAL; Carl Zeiss , Germany). Primary antibodies were rabbit anti-Nrf2 (rabbit-anti-Nrf2; 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-ham oxygenase-1 (mouse anti-HO-1; 1:1000 Enzo Life Sciences, Farmingdale, NY, USA) and mouse anti-HTT (1:400; catalog MAB5374, clone EM48; Millipore) antibodies.

1-16. Image analysis of tissue sections

Stained sections of tissue sections were captured using a DP70 image analysis system (Olympus, Tokyo, Japan) and analyzed using the NIH Image J program (http://rsbweb.nih.gov/ij/). The average area of the lesion is based on previous literature (Jang and Cho, 2016, Mol. Neurobiol. 53, 2619-2635; Jang et al., 2014, Brain Behav. Immun. 38, 151-165; Jang et al., 2013, Evidence Based Complement Altern. Med. 2013, 237-207), it was measured as a relative ratio to the total area of the striatum. The lesion area of each tissue section was analyzed using a confocal imaging system (LSM 5 PASCAL; Carl Zeiss, Germany).

1-17. Western blot analysis

To investigate protein expression after 3-NPA intoxication, previous literature (Jang and Cho, 2016, Mol. Neurobiol. 53, 2619-2635; Jang et al., 2014, Brain Behav. Immun. 38, 151-165; Jang et al. Western blot analysis was performed as described in Al., 2013, Evidence Based Complement Altern. Med. 2013, 237-207; Lee et al., 2016, Mol. Neurobiol. 53, 1419-1445). The specific method is as follows.

Twenty-four hours after the last 3-NPA poisoning, mice (n=3 per group) were anesthetized, and striatum harvested and immersed in lysis buffer. Proteins (30 μg) from each striatum were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked. The PVDF membrane contains mouse anti-β-III-tubulin (1:2,000; Sigma-Aldrich), rabbit anti-cleaved caspase-3; 1: 1,000; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-Iba-1 (1:1,000; WAKO), mouse anti-GFAP (1:500; Millipore) , mouse anti-iNOS; 1:200; Santa Cruz Biotechnology), rabbit anti-Nrf2 (rabbit-anti-Nrf2; 1:500; Santa Cruz Biotechnology), mouse anti-HO- 1 (mouse anti-HO-1; 1:1,000; Enzo Life Sciences), rabbit-anti-ap-JNK/p-ERK/p-p38 (rabbit-anti-p-JNK/p-ERK/p-p38; 1:1,000; Cell Signaling Technology), rabbit anti-NF-κB/p65 (rabbit-anti-NF-κB/p65; 1:1,000; Cell Signaling Technology) and rabbit anti-p-IκBα (rabbit-anti-p- IκBα; 1:1,000; Cell Signaling Technology) and mouse anti-HTT (1:400; catalog MAB5374, clone EM48; Millipore) antibodies were probed. Subsequently, it was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, and bands were identified using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). For normalization of antibody band signals, membranes were stripped and reconfirmed with mouse anti-glyceraldehyde 3-phosphate dehydrogenase (mouse anti-GAPDH; 1:10,000; Cell Signaling Technology). After Western blotting was performed three times, the density of each band was converted to a numerical value by subtracting the background value from the area of the film immediately adjacent to the stained band using the Photoshop CS2 program (Adobe, San Jose, CA, USA). Protein expression levels were quantified by the ratio (%) of each sample protein to GAPDH.

1-18. Real-time polymerase chain reaction (PCR) analysis

To investigate the mRNA level of inflammatory mediators, the existing literature (Jang and Cho, 2016, Mol. Neurobiol. 53, 2619-2635, Mol. Neurobiol. 53, 2619-2635; Jang et al., 2014, Brain Behav. Immun. 38, 151-165, Brain Behav. Immun. 38, 151-165 Jang et al., 2013, Evidence Based Complement Altern. Med. 2013, 237-207, Evidence Based Complement Altern. Med. Real-time polymerase chain reaction (PCR) analysis was performed as described in Lee et al., 2016, Mol. Neurobiol. 53, 1419-1445). The specific method is as follows.

Twelve hours after the last 3-NPA intoxication, mice (n=3 per group) were anesthetized, and the striatum was isolated and frozen. Real-time PCR analysis was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The reaction was performed in duplicate with a solution in a total volume of 10 μl each containing 10 μM primer, 4 μl cDNA and 5 μl SYBR Green PCR Master Mix. The mRNA level of each target gene was normalized to that of GAPDH mRNA. Fold-induction was calculated using the 2-ΔΔCT method as in the conventional method (Livak and Schmittgen, 2001, Methods 25, 402-408). All real-time PCR experiments were performed in triplicate. Table 1 below shows sequence information of primers used for PCR analysis.

Forward primer (5´→3´) Reverse primer (5´→3´) OX-42 GAC CGG TGC AGG ATC ATA CAG CCT TTC GTC CGC TTC AGA GT IL-1β TTG TGG CTG TGG AGA AGC TGT AAC GTC ACA CAC CAG CAG GTT IL-6 TCC ATC CAG TTG CCT TCT TGG CCA CGA TTT CCC AGA GAA CAT G TNF-α AGC AAA CCA CCA AGT GGA GGA GCT GGC ACC ACT AGT TGG TTG T COX-2 CAG TAT CAG AAC CGC ATT GCC GAG CAA GTC CGT GTT CAA GGA iNOS GGC AAA CCC AAG GTC TAC GTT TCG CTC AAG TTC AGC TTG GT Keap1 GAT CGG CTG CAC TGA ACT G GGA CTC GCA GCG TAC GTT Nrf2 TCT CCT CGC TGG AAA AAG AA AAT GTG CTG GCT GTG CTT TA NQO1 CAT TCT GAA AGG CTG GTT TGA CTA GCT TTG ATC TGG TTG TCA G GCLs ACA AGC ACC CCC GCT TCG GT CTC CAG GCC TCT CTC CTC CC HO-1 CCT GGA GCA GGA CAT GGC AAT CAT CCC TTG CAC GCC GAPDH AGG TCA TCC CAG AGC TGA ACG CAC CCT GTT GCT GTA GCC GTA T β-III-tubulin CTC AGG GGC CTT TGG ACA TC CAG GCA GTC GCA GTT TTC AC Apaf-1 TCT GAT GCT GCG CAA ACA C CAT CCT GCT CTT TTG CGA CTT C Cleaved caspase 3 TGT CAT CTC GCT CTG GTA CG AAA TGA CCC CTT CAT CAC CA Actin CTG TCT GGC GGC ACC ACC AT GCA ACT AAG TCA TAG TCC GC

1-19. statistical analysis

Statistical analysis was performed using the SPSS 21.0 package for Windows (SPSS Inc, Chicago, IL, USA). Two-sample comparisons were performed using Student's t-test and multiple comparisons were performed using two-way ANOVA with post hoc tests. All data are presented as mean ± S.E.M., and unless otherwise specified, statistical differences are recognized at the 5% level (#p<0.05 and ##p<0.01 compared to the sham group; *p<0.05 and **p<0.01 is shown as the 3-NPA group contrast).

<Experimental Example 2> Protective effect of gin and tonin on 3-NPA-induced neurological behavior and striatal cell death according to concurrent treatment with gin and tonin

To investigate whether gintonin (GT) has beneficial effects on neurological disorders intoxicated by 3-NPA and to determine the effective dose of gintonin, mice were treated with GT (25, 25, 50, and 100 mg/kg/day) were orally administered (simultaneous treatment) (Fig. 2A-C).

As a result, at the end of the experiment (24 hours after the last (4th) 3-NPA intoxication (60-60-80-80 mg/kg)), all mice in the 3-NPA group exhibited clasping of the hind limbs, freely It showed severe neurological impairment, including reduced general activity in a moving environment, hindlimb tension, kyphotic posture, and imbalanced adjustment for other postural problems, resulting in a neurological score of 8.2±0.6. On the other hand, mice in the 3-NPA+GT group showed 7.0±0.7, 6.0±0.4, and 5.1±0.3 when treated with GT 25, 50, and 100 mg/kg, respectively, in a dose-dependent manner compared to the 3-NPA group. scores were significantly reduced (Fig. 2A).

At the end of the experiment, the mice in the 3-NPA group showed a survival rate of 50.0%, and the mice in the 3-NPA+GT group were dosed with 60.0%, 70.0%, and 70.0% at the GT 25, 50, and 100 mg/kg treatment concentrations, respectively. dependently showed better survival rates (Fig. 2B).

In addition, the average body weight of the 3-NPA group was reduced to 19.1 ± 1.4 g compared to the sham group (24.8 ± 1.2 g), and this weight loss was significantly improved by GT treatment (100 mg/kg) to 22.4 ± 1.2 g. (Fig. 2C).

From the above results, it was confirmed that pretreatment with GT (100 mg/kg/day) was most effective against 3-NPA-induced neurological damage.

<Experimental Example 3> Effects of gintonin prior, simultaneous, onset, and progressing treatment time zones

To investigate the treatment time window of gintonin (GT) for 3-NPA-addicted neurological damage, we orally pretreated, co-treated, onset, and progressed mice with gintonin orally. The treatment concentration was 100 mg/kg/day, which was shown to be more effective than 25 and 50 mg/kg/day in the above experimental examples (FIGS. 3A to 3C).

The neurological scores observed in the gintonin pre-treatment, co-treatment, and onset treatment groups were 4.0±0.8, 5.0±1.2, and 7.0±0.4, respectively, which were significantly lower in a dose-dependent manner compared to the 3-NPA group (8.6±0.9) ( Figure 3A).

The survival rate of the 3-NPA group was 50% at the end of the experiment and increased over time by gintonin pre-treatment (80%), simultaneous treatment (60%) and onset treatment (60%) (Fig. 3B).

In addition, pre- and co-treatment with gintonin significantly improved the average body weight lost by 3-NPA intoxication (Fig. 3C).

From these results, it was confirmed that the pretreatment with gintonin had a wide treatment window.

<Experimental Example 4> Inhibitory effect of gintonin on apoptosis of striatal cells

Since pretreatment with gintonin (GT) 100 mg/kg/day was most effective in ameliorating the severity of 3-NPA-induced neurological damage, using the same protocol, the neuroprotective effect was associated with a reduction in striatal cell death. was further investigated. The level of striatal cell death was measured 24 h after the last 3-NPA intoxication using Cresyl violet staining.

Figure 4A shows representative striatal images for each group of sham, 3-NPA, 3-NPA+GT (25, 50, 100 mg/kg/day) and GT alone (100 mg/kg/day). The striatal lesions were colored relatively pale.

66.7% (n=4/6) of surviving mice in the 3-NPA group had symmetric striatal lesions, whereas 50.0% (n=4) of the groups pretreated with 50 and 100 mg/kg/day gintonin, respectively /8) and 33.3% (n = 3/9) (Fig. 4B).

The average lesion area of the 3-NPA group constituted 79.8±4.9% of the total striatum, whereas the 3-NPA+GT group had 53.8±3.5% and 39.8±4.0% of gintonin 50 and 100 mg/kg/day treatment, respectively. % was significantly decreased (Fig. 4C).

In addition, the expression of β-III-tubulin protein, a specific marker for striatal neurons, was significantly reduced in the 3-NPA group compared to the sham group, whereas pretreatment with gintonin (100 mg/kg/day) The decrease in expression was significantly inhibited (Fig. 4D). Gintonin itself did not affect neuronal cell death and β-III-tubulin protein expression.

These results indicate that pretreatment with gintonin can alleviate 3-NPA-induced striatal damage.

<Experimental Example 5> Protective effect of gintonin against mitochondrial dysfunction and apoptosis in 3-NPA-induced striatum

Since 3-NPA irreversibly inhibits SDH, a key enzyme located in the inner mitochondrial membrane of the striatum, it plays a role in converting succinate to fumarate. Accordingly, the present inventors investigated whether gintonin (GT) could block the decrease in SDH activity during the last 24 hours after 3-NPA-intoxication through histochemical analysis of unfixed frozen striatal regions.

The intensity of SDH activity was significantly reduced in the striatum of the 3-NPA group (54.6±3.8%) compared to the sham group (95.6±3.6%), whereas the reduced SDH activity was significantly recovered by pretreatment with GT (72.1±4.8%). (FIGS. 5A to 5E).

In addition, since 3-NPA-induced mitochondrial perturbation increases reactive oxygen species (ROS) production through depletion of SDH activity, ROS production was measured using MitoSOX staining.

As shown in Figures 5F to 3J, the intensity of MitoSOX increased in the striatum of the 3-NPA group (286.4 ± 9%) compared to the sham group (100 ± 4.8%), whereas this increase in MitoSOX intensity was observed with gintonin (100 mg /kg/day) was suppressed to 186.8±10.3%.

On the other hand, since 3-NPA induces apoptosis through SDH deficiency and ROS generation in the striatum, we investigated whether or not gintonin has an anti-apoptotic effect in the striatum 24 hours after the last 3-NPA intoxication (Figs. 5K to 5S). ).

The number of apoptotic cells analyzed by TUNEL staining (Fig. 5K to Fig. 5N) and FJC staining (Fig. 5O to Fig. 5R) was significantly increased in the striatum in the 3-NPA group, whereas this increase was not due to gintonin pretreatment (100 mg/kg). /day) was found to be significantly inhibited. These results were consistent with changes in the expression of active caspase-3 analyzed by Western blot analysis (Fig. 5S).

On the other hand, gintonin itself did not induce SDH, MitoSOX and apoptosis in the striatum compared to that in the sham group (Figs. 5A to 3S).

As a result, it was confirmed that the protective activity of gintonin against mitochondrial dysfunction and apoptosis in the striatum can contribute to the inhibition of neurological damage and striatal toxicity caused by 3-NPA intoxication.

<Experimental Example 6> Inhibitory effect of gin and tonin on microglial infiltration, inflammation and acute cell death in the striatum after 3-NPA poisoning

Residental microglia infiltrate into the lesions of neurological diseases including Huntington's disease and their in vivo models (Block and Hong, 2007; Lobsiger and Cleveland, 2007). Therefore, we investigated whether pretreatment with gintonin (GT) modulated immunopathological symptoms 24 h after the last 3-NPA intoxication (FIG. 6).

In the striatum of the sham group, Iba-1 (marker for microglia)-immunoreactive microglia showed a typical morphology of resting cells with small cell bodies and thin projections, whereas in the 3-NPA group, large cell bodies were observed. and showed an active form with short, wide and thick protrusions (FIGS. 6A and 6B). However, compared to the 3-NPA group, pretreatment with gintonin inhibited Iba-1-immunoreactive microglia (Fig. 6C), and immunoblot analysis of Iba-1 protein expression (Fig. 6E) and OX-42 mRNA Real-time PCR analysis of expression (FIG. 6F) showed the same results.

Astrocytes can be activated by products of dead cells or infiltrating/activated microglia lineages in or around lesions of neurodegenerative disorders. However, in the 3-NPA-induced acute striatal toxicity, GFAP-immunoreactive astrocytes were not significantly activated in or around the striatal lesion compared to the sham and gintonin groups (FIGS. 7A to 7D), and the mRNA of GFAP and protein expression did not change (FIGS. 7E-7G).

Infiltrating and activated microglia can release mediators that are either detrimental or beneficial for neuronal survival. Real-time PCR analysis was used to investigate whether the inhibitory effect of gintonin on microglia activation was involved in the expression of inflammatory mediators in the striatum after 3-NPA intoxication. As a result, little or no expression of IL-1β, IL-6, TNF-α, COX-2 and iNOS mRNA was detected in the striatum of the sham and gintonin-only groups, but at 12 hours after the last 3-NPA intoxication Their expression was significantly enhanced in the striatum (Figs. 6G-6K). The relative inductions of these factors for the Sham group were as follows: IL-1β (7.3-fold), IL-6 (6.6-fold), TNF-α (4.5-fold), COX-2 (8.0-fold) and iNOS (5.2-fold). ). On the other hand, compared to the 3-NPA group, pretreatment with GT (100 mg/kg/day) significantly inhibited the induction of expression of each factor: IL-1β (54%), IL-6 (67%), and TNF. -α (64%), COX-2 (66%) and iNOS (65%) (Figures 6G-6K). Similar to these results, the increased protein expression of iNOS (a representative inflammatory mediator) was also significantly reduced by gintonin (Fig. 6L).

On the other hand, gintonin alone did not increase or decrease microglia infiltration and inflammation (Fig. 6A-L).

As a result, it was confirmed that the anti-inflammatory effect of gintonin can contribute to alleviation of striatal toxicity and neurological dysfunction induced by 3-NPA.

<Experimental Example 7> Inhibitory effect of gintonin on acute cell death in the striatum after 3-NPA poisoning

Striatal neurons can be damaged primarily by 3-NPA or secondarily by a series of cellular processes involving microglia activation. Therefore, to differentiate the effects of gintonin (GT) on the two cell death mechanisms, we further investigated the correlation between striatal neuron death and microglia activation 12 h after tertiary 3-NPA-intoxication. According to previous studies by the present inventors, primary cell death by 3-NPA-intoxication was clearly observed, but activation of microglia was not clear in the early stage.

The number of early apoptotic cells in the striatum by FJC staining was significantly reduced in the 3-NPA+GT group (24.7±1.5/striatal section) compared to the 3-NPA group (19.7±0.9/striatal section) (FIG. 8A to Figure 8E). The intensity of the Iba-1-immune response increased by 3-NPA-intoxication did not show a significant difference between the 3-NPA and 3-NPA+GT groups (FIGS. 8F to 8J).

These results suggested that gintonin protects against primary cell death in the early stages after 3-NPA-intoxication.

<Experimental Example 8> Activation effect of gintonin on the Keap1-Nrf2 pathway in the striatum after 3-NPA poisoning

We further investigated whether gintonin (GT) could modulate the Keap1-Nrf2 pathway.

Keap1, an inhibitory protein of Nrf2, was significantly increased (3.3-fold) in the striatum after 3-NPA intoxication compared to the sham group, whereas the increase in expression was significantly reduced (32.1%) by gintonin pretreatment (FIG. 9A) .

The mRNA expression level of Nrf2 was significantly increased by 2.5 times compared to the sham group. This increase in expression in the 3-NPA group was further increased (196%) by pretreatment with gintonin (FIG. 9B).

As a result of real-time PCR analysis, the product of Nrf2 mechanism, nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase-1 (nicotinamide-adenine dinucleotide phosphate:quinone oxidoreductase-1, NQO-1), γ-glutamate cysteine ligase regulatory sub The mRNA expressions of γ-glutamate cysteine ligase regulatory subunits (GCLs) and heme oxygenase-1 (HO-1) were 2.7-fold and 5.8-fold higher than those of the sham group, respectively, in the striatum of the 3-NPA group. and 4.4-fold, and their expression in the gintonin group was further increased by 165%, 164%, and 184%, respectively (FIGS. 9C to 9E).

To further clarify the antioxidant effect of gintonin, we investigated the protein expression levels of Nrf2 and HO-1 as representative products of the Nrf2 pathway through western blot analysis. Cytoplasmic expression of -1 increased in a concentration-dependent manner compared to the 3-NPA group (FIGS. 9F and 9G).

This increase in expression was also shown in the same pattern in the immunofluorescence staining results (FIGS. 9H and 9I).

As a result, it was confirmed that gintonin stabilizes the Keap1-Nrf2 pathway.

<Experimental Example 9> Inhibitory effect of gin and tonin on MAPKs and NF-κB signaling pathways in the striatum after 3-NPA intoxication

The Nrf2 signaling pathway is involved in the inhibition of MAPKs and NF-κB and their inflammatory effects. Therefore, we analyzed whether gintonin (GT) could regulate MAPKs and NF-κB pathways in the striatum after 3-NPA intoxication.

As a result, in the case of the 3-NPA group, compared to the sham group, the expression of p-ERK, p-JNK, p-p38, NF-κB/p65 and p-IκBα in the striatum 24 hours after the last 3-NPA-intoxication was increased. 0.62, 0.77, 0.67, 0.65 and 0.73-fold, respectively, but these increases in phosphorylation were 83%, 68%, 78%, 78%, It was significantly inhibited by 35% and 39% (FIGS. 10A-10F). Gintonin itself did not increase or decrease activation of MAPKs or NF-κB pathways (FIGS. 10A to 10F).

These results indicate that gintonin pretreatment can reduce striatal toxicity induced by 3-NPA intoxication through inhibition of MAPKs and NF-κB pathways related to Nrf2 pathway activation.

<Experimental Example 10> STHdh ^Q111 Protective effects of gintonin in cells

The neuroprotective effect of gintonin (GT) against 3-NPA poisoning in mice confirmed through the above experimental examples (Figs. 2 to 10) indicates that gintonin can have a positive effect on HD patients. Accordingly, the effect of gintonin in STHdh ^Q111 cells was investigated. A safe dose of gintonin was determined by MTT assay, and H ₂ O ₂ treated STHdh ^Q111 cells were treated with 0.1, 1 or 10 g/ml gintonin for 24 and 48 hours.

As a result of lactate dehydrogenase (LDH) assay, the level of apoptosis in both STHdh ^Q7 (WT cells) and STHdh ^Q111 cells after H ₂ O ₂ treatment was significantly protected by gintonin in a time-dependent and concentration-dependent manner. It was found (Fig. 11A).

Then, the present inventors investigated whether this protective effect of gintonin was due to the reduction of neuronal cell death, apoptosis, and modified HTT aggregation in STHdh ^Q111 cells through Western blot analysis.

As a result, as expected, the decrease in the protein expression level of β-III-tubulin in H ₂ O ₂ -treated STHdh ^Q111 cells was suppressed by gintonin treatment. Enhanced protein expression levels of activated caspase-3 and EM48 in H ₂ O ₂ treated STHdh ^Q111 cells were also suppressed by gintonin treatment ( FIGS. 11B and 11C ).

As a result, the results confirmed in the above experimental examples of the present invention indicate that gintonin can exert a protective effect on the cell line of Huntington's disease.

<Experimental Example 11> Protective effect of gin and tonin in AAV/Q82-induced Huntington's disease mouse model

As confirmed through the above experimental examples, gintonin (GT) can exert a protective effect against striatal toxicity induced by 3-NPA in mice and H ₂ O ₂ -treated STHdh ^Q111 cells (FIGS. 2 to 11). .

In this regard, we confirmed the protective effect of gintonin against Huntington's disease-like striatal toxicity induced by AAV-mediated overexpression of human mutant HTT in the striatum.

As a result of the pole test, the average latency to descend to the bottom of the pole was compared to the sham group (8.0±0.5 seconds) or the AAV-DJ-18Q injection group (4.1±0.2 seconds at 4 weeks after injection). increased to 6.6±0.2 seconds after AAV-DJ-82Q injection, whereas this increase in average descent time to the bottom of the pole was partially suppressed by gintonin treatment from 8.0±0.5 seconds to 5.3±0.2 seconds (Fig. 12A). .

As a result of the rotarod test, the average reaction time (latency to fall) at 7 weeks after AAV-DJ-82Q injection was 294.3 ± 1.8 seconds to 282.0 ± 1.7 seconds, and the sham group (293.9 ± 2.1 seconds to 294.5 ± 2.1 seconds) or AAV-DJ-18Q injection group (294.1 ± 1.5 seconds to 294.5 ± 1.3 seconds) (Fig. 12B). However, this decrease in average reaction time was partially suppressed by gintonin treatment from 294.5±1.4 seconds to 287.3±1.3 seconds (FIG. 12B).

Body weight decreased significantly from the 8th week after AAV-DJ-82Q injection, but this weight change was somewhat alleviated by gintonin treatment (FIG. 12C). At the end of the experiment, all mice were alive.

Next, the present inventors found that the beneficial effect of gintonin on the motor control ability of the AAV-DJ-Q82-induced Huntington's disease mouse model was related to the reduced expression of modified HTT and the activation of the Nrf2 pathway by gintonin treatment. It was investigated whether this

In Western blot analysis, the protein expression of EM48 (a marker for mutant HTT) was significantly increased in the AAV-DJ-82Q injection group, and this increased expression was significantly decreased in the gintonin-treated group (Fig. 12D and Fig. 12E ). These results correspond to those of EM48 immunoreactivity by immunofluorescence staining (FIGS. 13A to 13E).

In addition, the nuclear translocation of Nrf2 and the cytoplasmic expression of HO-1 were significantly increased in the striatum after gintonin treatment compared to the AAV-DJ-82Q injection group (Fig. 12D and Fig. 12E). This expression pattern corresponded to the results by immunofluorescence staining (Fig. 12F and Fig. 12G).

To further clarify the antioxidant effect of gintonin, the effect of gintonin on H ₂ O ₂ treated STHdh ^Q111 cells was further investigated. As a result, the nuclear translocation of Nrf2 and the cytoplasmic expression level of HO-1 in H ₂ O ₂ -treated STHdh ^Q111 cells were enhanced by gintonin treatment, whereas these enhanced expression levels were significantly neutralized by siRNA-Nrf2 ( 12H-12I), or was significantly neutralized by an Nrf2 inhibitor (ML385) (FIGS. 14A and 14B). Meanwhile, siRNA-Nrf2 itself did not significantly affect nuclear translocation of Nrf2 and cytoplasmic expression of HO-1 in H ₂ O ₂ -treated STHdh ^Q111 cells (FIGS. 14C and 14D).

As a result, it was confirmed that gintonin could alleviate the motor control disorder induced by AAV-DJ-82Q infection through its antioxidant action.

Overall, it was confirmed that gintonin can protect primary apoptosis by 3-NPA and secondary apoptosis by activating microglia in the striatum after 3-NPA intoxication, and gintonin transmits Keap1-Nrf2 signaling. It may contribute to protection from 3-NPA-induced striatal toxicity by inhibiting the NF-κB and MAPKs signaling pathway by activating the pathway. In addition, the beneficial effect of gintonin on Huntington's disease was confirmed both in vitro (STHdh cells) and in vivo (AAV vector-infected mouse model of HD), through activation of the Keap1-Nrf2 signaling pathway, in an in vitro model of Huntington's disease. As a result, it was confirmed that the formation of modified HTT aggregates in the STHdh cell line could be inhibited and the symptoms of the AAVDJ-82Q vector-infected HD mouse model could be alleviated. Thus, it was confirmed that gintonin may be useful as a therapeutic agent for treating Huntington's disease.

So far, the present invention has been looked at with respect to its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is particularly indicated in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

Claims (12)

Inhibition of succinate dehydrogenase in the striatum, including gintonin as an active ingredient; Formation of aggregates of mutant huntingtin; And microglia are activated; as a pharmaceutical composition for the prevention or treatment of Huntington's disease,
The gintonin is administered to a patient group in which Nrf2 is underexpressed and mutant Huntington protein is expressed,
The Huntington's disease is caused by succinate dehydrogenase inhibition, altered huntingtin aggregate formation, or 3-NPA,
The gintonin suppresses 3-NPA-induced reduction of β-III-tubulin protein expression in the striatum and increased ROS generation due to mitochondrial perturbation, thereby inhibiting neuronal cell death in the striatum,
Preparing a 70% ethanol extract of the ginseng and tonin from ginseng; Obtaining a precipitate through water precipitation by mixing the concentrate of the extract with water; And freeze-drying the precipitate to obtain gintonin;
The gintonin (1) inhibits microglia activation in the striatum; (2) suppress behavioral disorders caused by neurological damage; (3) suppress the aggregation of huntingtin; (4) A pharmaceutical composition characterized by increasing succinate dehydrogenase in the striatum. According to claim 1,
The pharmaceutical composition, characterized in that the gintonin is a glycolipoprotein extracted and isolated from ginseng. delete delete According to claim 1,
The pharmaceutical composition, characterized in that the gintonin exhibits an effect of preventing or treating Huntington's disease by activating the Keap1-Nrf2 signal transduction pathway. delete According to claim 1,
The pharmaceutical composition, characterized in that the gintonin increases the succinate dehydrogenase in Huntington's disease patients in which succinate dehydrogenase is inhibited in the striatum. According to claim 1,
The gintonin suppresses the production or expression of Keap1 in patients with increased production or expression of Keap1 in the striatum, and increases the production or expression of one or more selected from the group consisting of Nrf2, NQO1, GCLs, and HO-1. Characterized by a pharmaceutical composition. According to claim 1,
The gintonin inhibits the production or expression of EM48 and activated caspase-3 in patients with increased production or expression of EM48 and cleaved caspase-3 in the striatum. To, the pharmaceutical composition. As a method of inhibiting nerve cell damage and death by treating nerve cells in which cell damage and death were induced by 3-NPA with gintonin in vitro to reduce striatal toxicity,
The gintonin inhibits 3-NPA-induced β-III-tubulin protein expression reduction and increased ROS generation due to mitochondrial perturbation, thereby suppressing neuronal cell death,
Preparing a 70% ethanol extract of the ginseng and tonin from ginseng; Obtaining a precipitate through water precipitation by mixing the concentrate of the extract with water; And freeze-drying the precipitate to obtain gintonin;
Characterized in that the gintonin inhibits both primary apoptosis by 3-NPA and secondary apoptosis by activating microglia. According to claim 10,
Wherein the gintonin activates the Keap1-Nrf2 signaling pathway and inhibits the NF-κB and MAPKs signaling pathways. As a method of inhibiting apoptosis and aggregation of modified huntingtin by treating the STHdh cell line with gintonin in vitro,
The gintonin inhibits 3-NPA-induced decrease in the expression of β-III-tubulin protein, thereby suppressing neuronal cell death,
Preparing a 70% ethanol extract of the ginseng and tonin from ginseng; Obtaining a precipitate through water precipitation by mixing the concentrate of the extract with water; And freeze-drying the precipitate to obtain gintonin;

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