KR20200034149A - Composition for preventing or treating brain damage comprising gintonin - Google Patents

Abstract

The present invention relates to a composition for preventing or treating brain damage diseases containing gintonin as an active component. The gintonin promotes glycogenolysis of brain astrocytes to increase production of ATP and absorption of glutamate in brain astrocytes, and also protects brain astrocytes from hypoxic and reoxygenation stress, thereby being able to be usefully used for preventing or treating brain damage diseases.

Description

Composition for preventing or treating brain damage comprising gintonin

The present invention relates to a composition for the prevention or treatment of brain injury diseases, which contains gintonin as an active ingredient.

Brain damage refers to a condition in which the brain is unable to function properly and can be caused by oxygen starvation or a direct blow to the brain. Brain damage can be caused by numerous diseases, such as open head injury, obstructive head injury, slowing damage, exposure to toxic substances, lack of oxygen, tumors, infections, and strokes. Among the brain injury diseases, hypoxic brain injury, ischemic brain disease, stroke or cerebral ischemia may be included, and the ischemic brain disease is also referred to as cerebral infarction, and may be subdivided into thrombosis, embolism, transient ischemic attack, dysplasia or small infarction. . The cerebral ischemia is known to be an excessive influx of calcium into the cells, free radical-related neuronal damage, and glutamate-receptor-mediated neuronal damage. That is, when cerebral ischemia occurs, depolarization of neurons occurs, and synaptic glutamate is released, thereby increasing the concentration of extracellular glutamate. In addition, reversal of active transport caused by a decrease in ATP (Adenosine Triphosphate) accelerates the accumulation of extracellular glutamate.

Astrocytes are the most numerous cells in the brain and are known to perform various functions in normal or abnormal conditions (Trends Mol Med 13 (2): 54-63. 2007). Compared to other brain cells, the unique characteristic of brain astrocytes is that they can store glycogen, which can be used for energy metabolism in the brain, while performing advanced brain functions such as cognitive processes. (Front Integr Neurosci 9:70, 2016) and during pathophysiological conditions such as hypoxia (Neurosci Lett 637: 18-25, 2015) are reported to be used to assist neurons. According to the hypothesis of the astrocyte-neuron lactate shuttle, brain astrocytes metabolize glycogen to lactic acid, and lactic acid is absorbed into neurons for oxidative metabolism (Dev Neurosci 20 (4-5): 291-299, 1998). Therefore, the main function of glycogen of cerebral astrocytic cells can be seen as acting as an energy reservoir in normal conditions and abnormal conditions such as hypoxia, ischemia and hypoglycemia, where glucose supply to the brain through the blood vessels is impaired (Nature Neuroscience 10 (11): 1377-1386, 2007).

Gintonin is an exogenous ligand for the G protein-coupled LPA receptor from Panax ginseng from which saponin has been removed, and ginseng major latex-like protein 151 and lipids. Consists of a complex, the main active ingredient is LPA C _{18: 2} (lysophosphatidic acid 18: 2) (Acta Crystallogr D Biol Crystallogr D 71: 1039-1050, 2015). Gintonin is known to use the G protein-linked LPA receptor signaling pathway to induce in vitro and in vivo effects in the nervous system (Neurochem Res. 25 (5): 573-82, 2000).

Korean Patent Publication No. 10-2011-0005144

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain injury diseases, including gintonin (Gintonin) as an active ingredient.

Another object of the present invention is to provide a food composition for the prevention or improvement of brain damage diseases, including gintonin (Gintonin) as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating brain injury disease, comprising administering a pharmaceutically effective amount of Gintonin to an individual other than a human.

Another object of the present invention is to provide a composition for the protection of astrocytes (astrocytes) comprising gintonin as an active ingredient.

Another object of the present invention is to provide a method for promoting astrocytic glycogenolysis of astrocytic cells, including the step of treating Gintonin in astrocytes in vitro.

Another object of the present invention is to provide a method for increasing ATP production in brain astrocytes, comprising the step of treating in vitro astrocytes with gintonin (Gintonin).

Another object of the present invention is to provide a method for increasing the uptake of glutamate (glutamate) in the brain astrocytes, including the step of treating in vitro a gintonin (Gintonin) to astrocytes.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of brain injury diseases, including gintonin (Gintonin) as an active ingredient.

In one embodiment of the invention, the gintonin may be to promote astrocytic glycogenolysis of astrocytic cells.

In one embodiment of the present invention, the gintonin may be to increase production of ATP and absorption of glutamate in cerebral stellate cells.

In one embodiment of the present invention, the gintonin may be to protect cerebral stellate cells from hypoxia and reoxygenation stress.

In one embodiment of the present invention, the brain injury disease may be hypoxic brain injury, ischemic brain disease, stroke, cerebral infarction, cerebral ischemia, thrombosis, embolism, transient ischemic attack, dysplasia or small infarction, but is not limited thereto. no.

In one embodiment of the present invention, the gintonin may be administered at a concentration of 0.1 to 10 μg / mL, preferably at a concentration of 0.1 to 1 μg / mL, more preferably 0.3 to It may be a concentration of 1 μg / mL.

In addition, the present invention provides a food composition for the prevention or improvement of brain damage disease, which contains Gintonin (Gintonin) as an active ingredient.

In addition, the present invention provides a method of preventing or treating brain injury diseases, comprising administering a pharmaceutically effective amount of Gintonin to an individual other than a human.

In addition, the present invention provides a composition for protecting astrocytes (astrocytes) containing Gintonin as an active ingredient.

In addition, the present invention provides a method for promoting astrocytic glycogenolysis of astrocytic cells, comprising the step of treating Gintonin in astrocytes in vitro.

In addition, the present invention provides a method of increasing ATP production in brain astrocytes comprising the step of treating Gintonin in astrocytes in vitro.

In addition, the present invention provides a method for increasing the uptake of glutamate (glutamate) in the brain astrocytes, comprising the step of treating in vitro in the brain astrocytes.

Gintonin according to the present invention promotes astrocytic glycogenolysis of brain astrocytes, thereby increasing the production of ATP and absorption of glutamate in brain astrocytes, and protecting brain astrocytes from hypoxia and reoxygenation stress. It can be usefully used for the prevention or treatment of damaged diseases.

1A and 1B show the concentrations of gintonin (GT) (0.1, 03. 1 and 3 μg / mL, A) or time (0.5, 1, After treatment according to 2 and 8 hours, B), the amount of glycogen is measured ( ^* p <0.01, ^** p <0.001, compared with the control (Con); ^# p <0.05, 1 and 3 μg / mL compared to gintonin; and data are expressed as mean ± SEM (n = 4).
1C and 1D show Ki16425 (10 μM), an antagonist of the LPA1 / 3 receptor, phospholipase C (PLC) inhibitor U73122 (5) to identify the signal modulators related to gintonin-mediated glycogenolysis. μM), IP ₃ (inositol 1,4,5-triphosphate) receptor antagonist 2-APB (2-aminoethoxydiphenyl borate, 100 μM) and intracellular Ca ²⁺ chelate BAPTA-AM (50 μM) after treatment , It is a result of measuring the amount of glycogen ( ^* p <0.01, compared with Ki16425 not treated; ^** p <0.001, compared with the control (Con); ^# p <0.05, ^## p <0.01, ^{# ##} p <0.001, compared with treatment with 1 μg / mL of gintonin alone; and data are expressed as mean ± SEM (n = 4)).
1E is a result of measuring the amount of glycogen after treatment with 0.1 μg / mL of gintonin alone or simultaneously with ATP (10 μM) or glutamate (10 μM), and FIG. 1F is 1 μg / mL of gintonin alone or It is the result of measuring the amount of glycogen after treating ATP (100 μM) or glutamate (100 μM) at the same time ( ^* p <0.01, ^** p <0.001, compared with control (Con); ^# p <0.05, soil Compared with Nin, ATP or glutamate alone; and data are expressed as mean ± SEM (n = 4).
Figure 2A is a result of measuring the amount of glycogen after treatment with K252a, an inhibitor of phosphorylase kinase, before processing of Gintonin or LPA, and Figure 2B is a glycogen phosphorylase ) Is the result of measuring the amount of glycogen after treatment with the inhibitor of isofagomine ( ^* p <0.01, ^** p <0.001, not treated with gintonin or compared to the control (Con); ^# p <0.05, compared with gintonin or LPA alone treatment; and data are expressed as mean ± SEM (n = 4-5).
2C shows that after treatment with gintonin according to concentrations (1, 3 and 10 μg / mL, a) and time (0.5, 1, 2, 8 and 12 hours, b), glycogen phosphorylase Immunoprecipitation for isoenzyme BB, brain form, GPBB) is performed and immunoblotting is performed to measure the amount of GPBB (input is 10% cell lysate; ^# p <0.05, with and without gintonin treatment) Comparison; and data are expressed as mean ± SEM (n = 4-5).
2D shows the results of immunoblotting for glycogen synthase (GS) and phosphorylated glycogen synthase (pGS) after treatment with gintonin and the measurement of the amount of pGS / GS ( ^* p <0.01 , Compared with the case where no gintonin was treated; and data are expressed as mean ± SEM (n = 4-5).
FIG. 3 shows treatment (B, D, F) with or without treatment of gintonin under normal oxygen (A, B), hypoxic (C, D) and reoxygenation (E, F) conditions (A, C, E). , Comparison of cell morphology and cell distribution (Scale bar: 100 μm).
Figure 4A is a result of measuring the amount of glycogen after treating gintonin according to concentrations (0, 0.1, 0.3, 1 and 3 μg / mL) under hypoxic conditions for 12 hours, and 4B is 0.5, 1, 2 And the result of measuring the amount of glycogen after treatment with gintonin under hypoxic conditions for 12 hours ( ^* p <0.05, ^** p <0.01, compared with control (Con) (A) or gintonin treated Comparison with the case (B); ^# p <0.05, ^## p <0.01, compared with the case where no gintonin was treated; and data are expressed as mean ± SEM (n = 4-5)).
Figure 4C is a result of measuring the amount of glycogen after treatment with gintonin in concentrations (0, 0.1, 0.3 and 1 μg / mL) under reoxygenation conditions for 30 minutes after hypoxic treatment for 12 hours, 4D It is the result of measuring the amount of glycogen after treatment with gintonin under reoxygenation conditions for 30 minutes or 8 hours after hypoxic treatment for an hour ( ^*** p <0.001, compared with normal oxygen condition (Con); ^## p <0.01, compared with the case where no gintonin was treated (C); ^** p <0.01, compared with the normal oxygen condition (Con) (D); ^*** p <0.001, compared with the normal oxygen condition (Con) ( D); and data are expressed as mean ± SEM (n = 4-5)).
5A and 5B show the amount of ATP after treatment with gintonin at normal oxygen conditions according to concentration (0.1, 0.3, 1 and 3 μg / mL, A) or time (0.5, 2 and 8 hours, B). Results (Isophagomine (300 μM), an inhibitor of glycogen phosphorylase, was treated for 30 minutes; and Gintonin at B was treated at 0.3 μg / mL) ( ^* p <0.01, compared to control; ^# p <0.05, compared with treatment with 0.3 μg / mL of gintonin; and data are expressed as mean ± SEM (n = 4-5).
5C is a result of measuring the amount of ATP after treating gintonin at a low oxygen condition for 12 hours according to concentrations (0, 0.1, 0.3, 1 and 3 μg / mL, C), and 5D is time (0.5, 1 , 2 and 12 hours, D) is the result of measuring the amount of ATP after treatment with gintonin (0.3 μg / mL) in hypoxic conditions ( ^* p <0.01, compared with the control group; ^** p <0.001, 0.3 Comparison with μg / mL of gintonin treatment; ^## p <0.01, ^### p <0.005, comparison with no gintonine treatment (C); ^# p <0.05, ^## p <0.01, Comparison with no gintonin treatment (D); and data are expressed as mean ± SEM (n = 4-5).
Figure 5E is the result of measuring the amount of ATP after treatment with gintonin according to the concentration (0, 0.1, 0.3, 1 and 3 μg / mL) in hypoxic conditions for 2 hours after hypoxic treatment for 12 hours, 5F Is the result of measuring the amount of ATP after treatment with gintonin (0.3 μg / mL) under reoxygenation conditions according to the time (0, 0.5, 2 and 8 hours) after hypoxic treatment for 12 hours ( ^* p <0.01, ^** p <0.001, compared with control; ^## p <0.01, ^### p <0.005, compared with no gintonin treatment; ^# p <0.005, compared with isopagomin treatment; and data Is expressed as mean ± SEM (n = 4-5).
6A and 6B are intracellular after treatment with gintonin at normal oxygen conditions according to concentration (0.1, 0.3, 1 and 3 μg / mL, A) or time (5, 15, 30, 60 and 120 min, B). It is the result of measuring the amount of glutamate (TBOA, an inhibitor of glutamate transporter, was treated with 30 or 100 μM; and in the B, gintonin was treated with 0.3 μg / mL) ( ^* p <0.01, ^** p <0.005, Comparison with the control (Con); ^# p <0.01, compared with the case where 0.3 μg / mL of gintonin was treated; and data are expressed as mean ± SEM (n = 4-5)).
Figure 6C is the result of measuring the amount of glutamate in the cell after treatment with gintonin according to concentrations (0, 0.1, 0.3, 1 and 3 μg / mL, C) under hypoxic conditions for 12 hours, and 6D is the time (0 , 0.5, 1, 2 and 12 hours, D) is the result of measuring the amount of glutamate in the cell after treatment with gintonin (0.3 μg / mL) under hypoxic conditions ( ^* p <0.01, ^** p <0.005 , Comparison with the control group; ^# p <0.05, ^## p <0.01, ^### p <0.001, compared with the case where no gintonin was treated; and data are expressed as mean ± SEM (n = 4-5)) .
Figure 6E is the result of measuring the amount of glutamate in the cell after treatment with gintonin in concentrations (0, 0.1, 0.3, 1 and 3 μg / mL) under conditions of reoxygenation for 30 minutes after hypoxic treatment for 12 hours. , 6F is the amount of glutamate in the cell after treatment with gintonin (0.3 μg / mL) under reoxygenation conditions according to the time (0, 5, 15, 30, 60 and 120 minutes) after hypoxic treatment for 12 hours. Results are ( ^* p <0.01, ^** p <0.005, compared with control; ^# p <0.05, ^## p <0.01, ^### p <0.001, compared with no gintonin treatment; and data are average ± SEM (n = 4-5)).
7A is a result of measuring cell viability through XTT assay after treating gintonin under normal oxygen conditions for 30 minutes according to concentrations (0.1, 0.3, 1 and 3 μg / mL), and 7B is hypoxic for 12 hours. After treatment, gintonin was treated for 30 minutes according to concentrations (0.1, 0.3, 1 and 3 μg / mL), and then the cell viability was measured through XTT assay. 7C was treated with isopagomine under hypoxic conditions. after the results of measurement of cell viability through the after XTT assay for 30 minutes according to the dust the non-concentration (0.1, 0.3, 1 and ^{3 μg / mL) (* p} <0.05, the control and comparison; ^# p < 0.05, ^## p <0.01, compared with the case where no gintonin was treated; and data are expressed as mean ± SEM (n = 4-5).
7D to 7F show concentrations of gintonin in hypoxic conditions for 30 minutes (D), 2 hours (E) or 8 hours (F) after hypoxic treatment for 12 hours (0.1, 0.3, 1 and 3 μg / mL). Cell viability was measured through XTT assay after treatment according to ( ^* p <0.05, ^** p <0.01, compared with control group; ^# p <0.05, ^## p <0.01, without gintonin treatment) Comparison with case; and data are expressed as mean ± SEM (n = 4-5).
Figure 8 is a schematic diagram showing the intracellular and intercellular effects of gintonin-mediated glycogenolysis by the LPA receptor cell membrane signaling pathway in primary cultured brain astrocytes of the cerebral cortex, and the intracellular effects are hypoxic and reoxygenated. Increased ATP production, increased glutamate uptake and improved cell viability in stress induced by, and the effect between cells is linked to astrocyte-neuron lactate shuttle, which is hypoxic or It is intended to use lactic acid as energy under reoxidation stress, and weakens glutamate neurotoxicity by removing extracellular glutamate after synaptic transmission or under hypoxic conditions.
9A and 9B are results of confirming whether or not glucose is absorbed using fluorescence glucose 2-NBDG after treatment with (A) or (A) treatment with gintonin (10 μg / mL) in cerebral stellate cells under normal oxygen conditions. (Scale bar: 50 μm), 9C is 2-NBDG (or 2-) after treatment with gintonin (0.1, 0.3, 1 and 3 μg / mL), glutamate (Glu, 100 μM) and LPA (1 μM) It is a result of measuring the absorption amount of DG).
10 is a result of measuring cell proliferation through measurement of chemiluminescence by adding BrdU for 2 hours after treatment with gintonin in normal oxygen (A) and hypoxic (B) conditions according to concentration ( ^* p <0.01, ^{* *} p <0.005, compared with the control group; ^# p <0.05, ^## p <0.01, compared with the case of not treated with gintonin in hypoxic conditions; and data are expressed as mean ± SEM (n = 4-5)) .

The present invention provides a pharmaceutical composition capable of preventing and treating brain injury diseases, and the pharmaceutical composition may further include a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not cause an allergic reaction or similar reaction, such as gastrointestinal disorder, dizziness, etc., when administered to a human. Pharmaceutically acceptable carriers include, for example, oral administration carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, etc. Carriers and the like, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-parabens and chlorobutanol. As other pharmaceutically acceptable carriers, reference may be made to those described in the following documents (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 may be prepared in various parenteral or oral dosage forms according to a known method, and isotonic aqueous solutions or suspensions are preferred as injectable dosage forms as representatives of parenteral dosage forms. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be formulated for injection by dissolving it in saline or buffer. In addition, oral dosage forms include, but are not limited to, powders, granules, tablets, pills and capsules.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means the introduction of a predetermined substance to an individual in a suitable manner, and the route of administration of the composition can 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, and rectal administration, but are not limited thereto.

The term, “individual,” refers to all animals, including humans, mice, mice, and livestock. Preferably, it can be a mammal, including a human.

The term, “pharmaceutically effective amount” means an amount 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 the patient's gender and age. Factors and other medical fields, including weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of discharge, duration of treatment, combination or drug used concurrently It can be easily determined by those skilled in the art according to the well-known factors. For the administration, the recommended dosage may be administered once a day, or may be divided into several times.

The food composition of the present invention may include all foods in a conventional sense, and can be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention means food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6727 of the Health Functional Food Act, and "functional food" means It means to intake for the purpose of obtaining useful effects for health purposes such as adjusting nutrients for structure and function or physiological action.

The term "health functional food" of the present invention refers to food produced and processed by a method of extracting, concentrating, refining, mixing, or the like, as a raw material for a specific component for the purpose of health supplementation or by extracting, concentrating, refining, mixing, etc. A food designed and processed to fully exert bioregulatory functions such as biodefense, biorhythm control, and disease prevention and recovery by ingredients, and the composition for health foods prevents disease and recovers disease It can perform the functions related to.

There are no restrictions on the types of foods in which the composition of the present invention can be used. In addition, the composition of the present invention may be prepared by mixing a known additive with other suitable auxiliary ingredients that may be included in food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gums, ice cream, various soups, beverages, teas, drinks, alcoholic beverages, and There are vitamin complexes, etc., and can be prepared by adding the extract according to the present invention and its fractions as a main component, and adding it to juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (eg, formulas, infants, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (eg ramen, noodles, etc.), health supplements , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.) ), Beverages (eg fruits, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates, etc., as additional components, like a conventional beverage. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin , Alcohol, carbonic acid used in carbonated beverages, and the like. In addition, it may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Experimental materials and methods

1.1. Experimental material

Gintonin from which ginseng saponin has been removed was prepared according to the method previously described in Panax ginseng (Neurosci Lett. 603: 19-24, 2015). Gintonin was dissolved in demineralized water and diluted in media before use in the experiment. Dulbecco's Minimum Essential Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Invitrogen (Camarillo, CA, USA). LPA (1-oleoyl-2-hydroxy- sn -glycero-3-phosphate) is Avanti Polar Lipids, Inc. (AL, USA), Ki16425 and isofagomine were purchased from Cayman Chemicals (Ann Arbor, MI, USA). Other reagents, including ATP, BAPTA-AM (1,2-Bis (2-aminophenoxy) ethane-N, N, N ', N'-tetraacetic acid tetrakis-acetoxymethyl ester), glutamate and K252a, are Sigma-Aldrich (St Louis, MO, USA). TBOA (DL- threo- β-benzyloxyaspartate) was purchased from Tocris Bioscience (Bristol, UK).

1.2. Primary culture of mouse astrocytes

The primary culture of cerebral astrocytes is Kim et al . It was prepared from the cerebral cortex of 1-day-old ICR (CD-1®) mice according to the method (Neurosci Lett. 603: 19-24, 2015). Cells were inoculated in a culture dish coated with poly-L-lysine hydrobromide (100 μg / mL; Sigma-Aldrich), 10% FBS, 100 units / mL penicillin and 100 μg / mL strepto The cells were incubated in a 37 ° C, 5% CO ₂ incubator using DMEM medium containing mycin. When primary cultured cells grew to 100% confluence (days 5-7), cells were collected with a 0.05% trypsin / EDTA (ethylenediaminetetraacetic acid) solution (Life technologies, Carlsbad, CA, USA) and pretreated poly- Inoculated into culture dish coated with L-lysine hydrobromide (100 μg / mL; Sigma-Aldrich) and cultured using DMEM medium containing 10% FBS, 100 units / mL penicillin and 100 μg / mL streptomycin. It was then used in other experiments.

1.3. Measurement of glycogen decomposition of brain astrocytes

After primary cultured cells were overgrown, the glycogen assay was performed as previously described (J Neurosci 12 (12): 4923-4931, 1992). Before the glycogen assay was performed, it was replaced with serum-free DMEM medium containing 5 mM glucose. After incubation for 4 hours with the exchanged medium, the cells were treated with medium with or without gintonin at 37 ° C. The level of intracellular glycogen was measured according to the method suggested by the manufacturer using the Glycogen Assay Kit (Cayman Chemicals). The amount of glycogen in all samples was normalized to the amount of protein in each sample.

1.4. Induction of hypoxia or re-oxygenation

Cerebral astrocytes were exposed to hypoxic conditions using the BD GasPak ^TM EZ anaerobic system (Stroke 24 (6): 855-63, 1993). Cells were washed after exposure to hypoxic conditions for 0, 0.5, 1, 2 or 12 hours. In the case of reoxygenation conditions, the medium was replaced with fresh medium, and then cells were treated with 0 to 0.3 μg / mL of gintonin for 0, 0.5, 2 or 8 hours in normal culture conditions according to the experimental procedure.

1.5. Intracellular ATP production and intracellular glutamate measurement

The amount of ATP in the cells was determined by performing according to the manufacturer's protocol using the ATP Assay Kit (Abcam, Cambridge, UK). Cerebral astrocytes were inoculated at 1 × 10 ⁶ cells / well in a 6-well plate and allowed to adhere overnight. Cells were incubated in medium exchanged for 4 hours and then treated with medium and with or without gintonin under normal oxygen, hypoxia and reoxygenation conditions according to the given time and concentration. Thereafter, the cells were washed with cold phosphate-buffered saline (PBS), and cells were collected in the ATP Assay Buffer using a cell scraper. ATP values were corrected for the amount of protein in the sample and normalized to control ATP values.

The amount of glutamate in the cell was measured using the Glutamate Assay Kit (Abcam) according to the manufacturer's instructions. To perform the intracellular glutamate assay, cells were prepared as above and treated with medium with or without gintonin, then 100 μM glutamate was added to the medium and terminated after 7 minutes. The glutamate values were corrected for the amount of protein in the sample and normalized to the glutamate value of the control.

1.6. XTT (2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-carboxanilide) assay and cell morphology

Cell viability was determined by XTT Assay Kit (Daeillab Service, Seoul, Korea). Cells were seeded in 96-well plates, attached overnight, and then treated with gintonin under normal oxygen, hypoxia and reoxygenation conditions. After 2 hours of XTT treatment, it was measured at 450 nm with a Microplate Reader (SpectraMax®Plus 384 Microplate Reader).

To observe cell morphology, cells were seeded at 5 x 10 ⁵ cells / well in a cover glass on a 6-well plate. After treatment with gintonin under normal oxygen, hypoxia or reoxygenation conditions, the cells were fixed with 4% paraformaldehyde for 1 hour and washed with PBS. Images were observed with an AE2000 Inverted Phase Contrast Microscope (× 20 objective).

1.7. Immune blot of phosphorylated glycogen phosphorylase

Cell lysates were separated by 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to NC membranes (nitrocellulose membranes, Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% bovine serum albumin (BSA) in TTBS (20 mM Tris [pH 7.6], 137 mM NaCl, and 0.1% Tween 20) for 1 hour, and then, anti-GPBB (glycogen phosphorylase isoenzyme BB) Antibody (Abcam), anti-pGS (phospho-glycogen synthase), anti-GS (glycogen-synthase) antibody (Cell signaling), or mouse monoclonal β-actin antibody (Sigma-Aldrich, St Louis, MO, USA) After reacting at 4 ℃ overnight. Next, the membrane was washed several times with TTBS at intervals of 5 minutes, and then an anti-rabbit horseradish peroxidase (HRP) -conjugated secondary antibody (Millipore) was added and reacted at room temperature for 2 hours. Protein bands were visualized by chemiluminescence using the ECL Kit (Abfrontier, Seoul, Korea).

1.8. Immunoprecipitation method of phosphorylated glycogen phosphorylase

Cell lysates were modified Radioimmunoprecipitation Assay Buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM ethylene glycol tetraacetic acid, protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail ( phosStop; Roche, USA)). Subsequently, 3 μg of anti-phosphoserine antibody (Abcam) was added to 350 μg of the cell lysate and reacted overnight at 4 ° C., and anti-phosphoserine antibody with protein A-agarose beads (GE Healthcare) was added and precipitated at 4 ° C. overnight. . Immune blot was performed with anti-GPBB antibody (Abcam). Each phosphorylation intensity was normalized to an untreated control.

1.9. Data analysis

Differences between groups were determined by one-way or two-way ANOVA (analyses of variance) with unpaired Student's t-tests, Bonferroni post-hoc test, and were considered significant at p <0.05. Data are expressed as mean ± SEM (standard error of the mean).

Example 2. Effect of Gintonin or LPA (Lysophosphatidic acid) on Glycogenolysis in Cortical Primary Cerebral Astrocytes and its Signaling Pathway

Astrocytes store glycogen in the brain for energy metabolism, and the ligand of the G protein-coupled receptor linked to [Ca ²⁺ ] _i transients promotes glycogen degradation in brain astrocytes (Dev Neurosci 20 (4-5): 291-299, 1998), the present inventors conducted experiments to confirm the effect of gintonin on glycogenolysis in primary astrocytic cells of cultured mouse cortex. As a result, it was confirmed that glycogenolysis is promoted in a concentration-dependent manner when gintonin (GT) or LPA C _{18: 1} is treated with cerebral stellate cells for 30 minutes (FIGS. 1A and 1D). In addition, gintonin (1 μg / mL) promoted glycogen degradation of astrocytic cells in a time-dependent manner (FIG. 1B). Interestingly, the glycogen decomposition of gintonin-mediated cerebral astrocytic cells was found to occur to a maximum extent after 30 minutes of treatment with gintonin, and the degradation was reduced by 2 hours, and then the glycogen degradation was increased by 8 hours. It was confirmed (Fig. 1B).

Next, the present inventors examined the effect of gintonin on glycogen decomposition of astrocytic cells in the case of treatment with or without treatment of Ki16425, an antagonist of the LPA1 / 3 receptor. As a result, when Ki16425 was treated, it was confirmed that glycogenolysis promoted by gintonin or LPA was significantly reduced (FIGS. 1C and 1D). In addition, in brain astrocytes, a phospholipase C (PLC) inhibitor U73122, an IP ₃ (inositol 1,4,5-triphosphate) receptor antagonist, 2-APB (2-aminoethoxydiphenyl borate), and intracellular Ca ²⁺ chelate BAPTA- Even in the case of AM treatment, glycogen degradation promoted by gintonin was inhibited (FIG. 1C). By the above results, it was confirmed that gintonin promotes glycogen decomposition of astrocytic cells by the LPA1 / 3 receptor signaling pathway.

In addition, the present inventors performed an experiment to confirm the effect of ATP or glutamate on glycogen decomposition of brain stellate cells (Dev Neurosci 20 (4-5): 291-299, 1998; and News Physiol Sci 14 (5): 177-182, 1999). As a result, it was confirmed that when ATP or glutamate was treated for 30 minutes, concentration-dependently promoted glycogen decomposition of astrocytic cells (FIGS. 1E and 1F). Particularly, when co-treatment with 0.1 μg / mL of gintonin with 10 μM of ATP or glutamate each showed a synergistic effect on glycogen decomposition of astrocytic cells compared to treatment with each alone (FIG. 1E). . However, when co-treatment of 1 μg / mL of gintonin with 100 μM of ATP or glutamate did not show a synergistic effect on glycogen decomposition (FIG. 1F), this is seen as a saturation effect according to a high concentration of ligand. . Interestingly, gintonin had no significant effect on glucose uptake in experiments with fluorescent glucose 2-NBDG (FIG. 9).

Example 3. Effect of Gintonin on Glycogenase-Related Enzymes and Glycogen Synthase-Related Enzymes in Primary Cerebral Astrocytes under Normal Oxygen Conditions

The present inventors have shown that K252a, an inhibitor of phosphorylase kinase, and isopagomine, an inhibitor of glycogen phosphorylase, in glycogenolysis of brain stellate cells promoted by gintonin or LPA ( isofagomine) was performed (Neurochem Int 36 (4-5): 435-440, 2000). As a result, it was confirmed that the two inhibitors significantly reduced the glycogen decomposition of gonocytes promoted by gintonin or LPA (FIGS. 2A and 2B). In addition, the present inventors conducted an experiment to evaluate the phosphorylation degree of glycogen phosphorylase on the basis that phosphorylation of glycogen phosphorylase by phosphorylase kinase is an important starting step of glycogen decomposition. As a result, it was confirmed that phosphorylation of glycogen phosphorylase isoenzyme BB (brain form, GPBB) was enhanced in concentration and time-dependent manner when gintonin was treated on brain astrocytes (FIGS. 2C and 2D). However, treatment with Ki16425, an antagonist of the LPA1 / 3 receptor, reduced phosphorylation of glycogen phosphorylase (FIG. 2C b).

Next, the present inventors conducted an experiment to confirm the effect of gintonin on phosphorylation of glycogen synthase, on the basis that increased phosphorylation of glycogen synthase is associated with a decrease in glycogen decomposition. As a result, it was confirmed that phosphorylation of glycogen synthase is reduced when gintonin is treated on astrocytic cells compared to untreated control cells (FIG. 2D). It is linked to the glycogen decomposition of astrocytic cells by the activity of Gintonin LPA receptor-PLC (phospholipase C)-intracellular IP ₃ receptor-[Ca ²⁺ ] _i transient signaling pathway, the pathway is glycogen synthesis It appears to phosphorylate glycogen phosphorylase.

Example 4. Effect of gintonin on cell morphology and distribution of astrocytic cells under normal oxygen, hypoxia or reoxygenation conditions

The present inventors carried out an experiment to check whether normal brain astrocytes were cultured under normal oxygen, hypoxia and reoxygenation conditions, and if there was a change in cell morphology and cell distribution when gintonin was treated. As a result, gintonin had no effect on the cell morphology under normal oxygen conditions, and the cell number was slightly increased (Figs. 3A and 3B). In the case of no treatment with gintonin, hypoxic conditions for 12 hours (Figure 3C) and reoxygenation conditions for 2 hours after hypoxic conditions for 12 hours (Figure 3E) induced changes in cell morphology and cell distribution during culture. Became. Cells under these conditions were not evenly distributed and tended to be dense, and empty spaces between cells were observed compared to cells under normal oxygen conditions, and it was shown that the connections between cells were small (FIGS. 3C and 3E). However, in the case of treatment with gintonin for 2 hours under hypoxic conditions and reoxygenation conditions, the cell distribution and cell morphology were recovered again to a degree similar to that observed in the oxygenated oxygen condition (FIGS. 3D and 3F). From the above results, it was confirmed that gintonin has an effect of protecting against damage to brain astrocytes induced by hypoxia and reoxygenation.

Example 5. Effect of Gintonin on Glycogenesis of Cerebral Astrocytes in Hypoxia and Reoxygenation Conditions

Existing reports that gintonin had the effect of preventing changes in cell morphology and cell distribution induced by hypoxia and reoxygenation stress in brain astrocytes, and glycogen decomposition of cerebral astrocytes tends to increase in hypoxic conditions (Stroke 24 On the basis of (6): 855-63, 1993), the present inventors conducted experiments to confirm how ginstonin affects the glycogen decomposition of astrocytic cells under hypoxic and reoxygenation conditions. As a result, glycogen decomposition of gintonin-mediated cerebral astrocytic cells showed a dual effect in hypoxic and reoxygenation conditions. Particularly, glycogen decomposition of gintonin-mediated cerebral astrocytic cells after treatment with gintonin in hypoxic conditions It increased at 30 minutes and 1 hour, slightly increased to 2 hours, but at 12 hours, glycogen decomposition was reduced to increase the amount of glycogen (FIGS. 4A and 4B). This dual effect of gintonin is shown to increase glycogen decomposition of astrocytic cells in the early stages after hypoxia and increase the amount of glycogen in the later stages when gintonin acts on astrocytic cells. Similarly, when gintonin was treated to astrocytic cells, treated with hypoxia for 12 hours, and exposed to reoxygenation conditions for 30 minutes, glycogen decomposition was accelerated in a concentration-dependent manner (FIG. 4C), 12 hours When exposed to hypoxia conditions for 8 hours after being treated with hypoxia, the amount of glycogen was increased compared to astrocytic cells that were not treated with gintonin (FIG. 4D). From the above results, it was confirmed that the glycogen decomposition of progressive nin-mediated brain astrocytes under hypoxia and reoxygenation conditions showed a dual effect depending on the hypoxia or reoxygenation period.

Example 6. Effect of gintonin on ATP production under normal oxygen, hypoxia and reoxygenation conditions

The present inventors conducted an experiment to confirm that glycogen decomposition of gintonin-mediated cerebral astrocytic cells is linked to ATP production under normal oxygen, hypoxia and reoxygenation conditions. As a result, ATP production was increased in a concentration-dependent manner when gintonin was treated in cerebral stellate cells under normal oxygen conditions (FIG. 5A), and over time, ATP production was most pronounced at 30 minutes after gintonin treatment. Increased, then slightly elevated (Fig. 5B). However, enhancement of ATP production mediated by gintonin in cerebral astrocytic cells was inhibited by isofagomine, an inhibitor of glycogen phosphorylase (FIG. 5A).

Hypoxia significantly reduces ATP production, which means that metabolic stress can damage astrocytic cells under hypoxic conditions. It was confirmed that ATP levels were significantly increased when gintonin was treated under these hypoxic conditions, whereas it was confirmed that isopagomin inhibited ATP production by gintonin (FIG. 5C). Moreover, ATP production was further reduced when the period of hypoxia was prolonged, whereas it was confirmed that ATP depletion was prevented and ATP production was maintained at a control level when gintonin was treated in a hypoxic state (FIG. 5D).

In addition, the present inventors performed an experiment to treat hypoxic conditions for 12 hours and then to treat for 2 hours for reoxygenation conditions and to confirm the effect of gintonin on ATP levels. As a result, after hypoxic treatment, the conditions of reoxygenation decreased ATP levels compared to the control group, but when treated with gintonin, ATP levels increased and showed a maximum increase effect at a concentration of 1 μg / mL (FIG. 5E). The effect of this gintonin was not increased in a concentration-dependent manner as in hypoxia. In the results over time, gintonin promoted ATP production in a time-dependent manner and appeared to be saturated at 2 hours (FIG. 5F). As in normal oxygen conditions, the treatment of isopagomine under hypoxic conditions and reoxygenation conditions inhibited ATP production by gintonin (FIGS. 5C and 5E). From the above results, an increase in the production of gintonin-mediated ATP is highly related to the glycogenation of astrocytic cells, and ATP depletion observed after exposure to hypoxia and reoxygenation stress when gintonin is treated on astrocytic cells. It was confirmed to weaken.

Example 7 Effect of Gintonin on the Level of Glutamade in Cells under Normal Oxygen, Hypoxia and Reoxygenation Conditions

Glutamate is a major neurotransmitter, a substance secreted by neurons during normal synaptic activity in the brain, and brain ischemia has been reported to induce the release of large amounts of glutamate in neurons (J Cereb Blood Flow Metab) 5 (3): 413-419, 1985; and Brain Res Bull 25 (2): 319-324, 1990). Since accumulation of glutamate outside neurons induces excitatory neurotoxicity in the nervous system, one of the main roles of astrocytic cells is to reduce the concentration of extracellular glutamate through absorption of glutamate (Acta Pharmacol Sin 30 (4): 379- 387, 2009). Cerebral astrocyte-mediated glutamate uptake requires a constant supply of ATP, thereby maintaining the equilibrium of normal glutamate. On the basis of this, the present inventors conducted experiments to confirm whether the increase in ATP production by ginstonin under normal oxygen, hypoxia and reoxygenation conditions is associated with an increase in intracellular glutamate levels.

As a result, it was confirmed that the level of glutamate in the cells of the brain astrocytes was increased in a time-dependent manner when gintonin was treated under normal oxygen conditions (FIG. 6B). Interestingly, the level of intracellular glutamate by gintonin increased maximally at a concentration of 0.3 μg / mL, and the glutamate level was slightly decreased at high concentrations (1 and 3 μg / mL) of gintonin (FIG. 6A). When 0.3 μg / mL of gintonin was treated with astrocytic cells over time, it was found to be almost saturated after 30 minutes (FIG. 6B). However, when co-treatment with TBOA, an inhibitor of glutamate transporter, along with gintonin (0.3 μg / mL), an increase in intracellular glutamate induced by gintonin was inhibited (FIG. 6A). From the above results, it was confirmed that an increase in the concentration of glutamate in the cell of gintonin-mediated is related to the glutamate transporter.

Cerebral stellate cells exposed to hypoxic conditions had lower levels of intracellular glutamate compared to cells under normal oxygen conditions, and intracellular glutamate levels compared to non-gintonin-treated cells when treated with gintonin (0.3 μg / mL). This was increased to the maximum (Figure 6C). In addition, gintonin increased intracellular glutamate levels for 1 hr 12 hours compared to the control (Figure 6D). The intracellular glutamate levels in hypoxic post-oxygenated conditions were also lower in intracellular glutamate levels than in the control group, which is a normal oxygen condition, but the levels of intracellular glutamate were significantly increased in the concentration- and time-dependent manner compared to the control group when gintonin was treated ( 6E and 6F). From these results, it is shown that the elevation of intracellular ATP observed in glycogenolysis of gintonin-mediated cerebral astrocytic cells is linked to glutamate uptake through glutamate transporters.

Example 8. Effect of gintonin on cell viability of astrocytic cells under normal oxygen, hypoxia and reoxygenation conditions

The present inventors investigated the cell viability and cell proliferation rate with the number of cells based on metabolic activity in order to confirm the glycogenolysis effect of gintonin-mediated cerebral astrocytic cells on the cell viability of cerebral astrocytic cells under normal oxygen, hypoxia and reoxygenation conditions. Evaluation XTT assay and BrdU (bromodeoxyuridine) assay were performed. As a result, the cell survival rate and the cell proliferation rate increased concentration-dependently when gintonin was treated with astrocytic cells under normal oxygen conditions (FIGS. 7A and 10A). In addition, when treated with gintonin even in hypoxic conditions for 12 hours, cell viability and cell proliferation rate increased in a concentration-dependent manner (FIGS. 7B and 10B). However, in the case of treatment with the isopagomine inhibitor of glycogen phosphorylase, the increase in cell viability by gintonin was attenuated (Fig. 7C), which suggests that glycogenolysis of gintonin-mediated cerebral astrocytic cells results in cell survival. It seems to play an essential role.

However, in hypoxic conditions after hypoxic treatment, the cell viability by gintonin showed a slightly different pattern than that observed in normal oxygen and hypoxic conditions. In hypoxic conditions for 30 minutes and 2 hours after hypoxic treatment, maximum cell viability was seen when 0.3 μg / mL of gintonin was treated (FIGS. 7D and 7E). It has been shown that in hypoxic conditions for 8 hours after hypoxic treatment, gintonin improves cell viability in a concentration-dependent manner. Even under reoxygenation conditions, isopagomine attenuated the increased cell viability by ginstonin (data not shown). Interestingly, the cell viability under hypoxic conditions was low compared to reoxygenated conditions. From the above results, it was confirmed that gintonin may have a better protective effect when applied during the hypoxia post-oxygenation process.

Thus, gintonin has an effect of restoring a decrease in cell viability induced by hypoxia, and glycogenolysis of gintonin-mediated brain astrocytes after exposure to hypoxia and reoxygenation stress may contribute to the survival of astrocytic cells. Was confirmed.

Claims (14)

A pharmaceutical composition for the prevention or treatment of brain injury diseases, which includes Gintonin as an active ingredient. According to claim 1,
The gintonin is a composition characterized in that it promotes the glycogenolysis of astrocytic cells (astrocytic glycogenolysis). According to claim 1,
The composition is characterized in that to increase the production of ATP and the absorption of glutamate in the brain astrocytes. According to claim 1,
The gintonin is a composition characterized in that it protects the brain stellate cells from hypoxia and reoxygenation stress. According to claim 1,
The brain injury disease is hypoxic brain injury, ischemic brain disease, stroke, cerebral infarction, cerebral ischemia, thrombosis, embolism, transient ischemic attack, dysplasia or small infarction. According to claim 1,
The gintonin is a composition characterized in that it is administered at a concentration of 0.1 to 10 μg / mL. Food composition for the prevention or improvement of brain damage diseases, including gintonin (Gintonin) as an active ingredient. The method of claim 7,
The gintonin is a composition characterized in that it is administered at a concentration of 0.1 to 10 μg / mL. The method of claim 7,
The brain injury disease is hypoxic brain injury, ischemic brain disease, stroke, cerebral infarction, cerebral ischemia, thrombosis, embolism, transient ischemic attack, dysplasia or small infarction. A method of preventing or treating a brain injury disease, comprising administering a pharmaceutically effective amount of Gintonin to an individual other than a human. Gintonin (Gintonin) composition for protecting astrocytes (astrocyte) containing as an active ingredient. A method for promoting astrocytic glycogenolysis of brain astrocytes, comprising the step of treating Gintonin in astrocytes in vitro. A method for increasing ATP production in brain astrocytes comprising the step of treating Gintonin in astrocytes in vitro. A method for increasing the uptake of glutamate in brain astrocytes, comprising the step of treating Gintonin in brain cells in vitro.

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