KR102627493B1 - Composition for preventing, improving or treating neurodegenerative disorders trimethyltin mediated comprising Ishige okamurae extract and uses thereof - Google Patents

Abstract

The present invention relates to a composition containing an extract of Ishige okamurae for preventing, improving or treating neurodegeneration, particularly neurodegeneration caused by trimethyltin.
In addition, the present invention relates to a method for protecting brain cells treated with trimethyltin, which includes the step of treating Ishige okamurae extract, and a composition for protecting brain cells treated with trimethyltin, which includes the shell extract.
The shell extract of the present invention protects brain cells damaged by trimethyltin treatment by i) reducing the expression of cleaved caspase-3, ii) reducing the expression of cleaved PARP, and iii) expressing BDNF (brain-derived neurotrophic factor). increased, iv) decreased ERK phosphorylation, v) decreased p38 phosphorylation, vi) decreased JNK phosphorylation, vii) increased heme oxygenase-1 (HO-1) expression, and viii) increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression. can be provided. Therefore, the shell extract of the present invention can be effectively used as a composition for preventing, improving or treating trimethyltin-mediated neurodegeneration, and as a method or composition for protecting trimethyltin-treated brain cells.

Description

Composition for preventing, improving or treating neurodegenerative disorders trimethyltin mediated comprising Ishige okamurae extract and uses thereof}

The present invention relates to a composition containing an extract of Ishige okamurae for preventing, improving or treating neurodegeneration, particularly neurodegeneration caused by trimethyltin.

In addition, the present invention relates to a method for protecting brain cells treated with trimethyltin, which includes the step of treating Ishige okamurae extract, and a composition for protecting brain cells treated with trimethyltin, which includes the shell extract.

Neurodegeneration is the progressive functional or structural loss of nerve cells that leads to many incurable neurodegenerative diseases, such as Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, fatal familial insomnia, and Alzheimer's disease. These neurodegenerative diseases have been proven to have many similar phenotypes at the subcellular level, providing an opportunity to develop treatments that simultaneously improve neurodegenerative diseases. For this reason, several model systems, such as TMT-induced neurodegeneration and glutamate excitotoxicity, are frequently used to investigate the effects of therapeutic candidates for neurodegenerative diseases.

Trimethyltin (TMT) is an organotin compound widely used in agriculture and industry. Systemic administration of TMT to humans induces neurodegeneration in the central nervous system (CNS) leading to clinical symptoms such as hyperactivity and cognitive impairment. Therefore, TMT has been used as a potential model neurotoxin to investigate brain dysfunction and neurodegeneration. Several underlying mechanisms of TMT-mediated neurotoxic effects have been proposed, including intracellular calcium overload, glutamate excitotoxicity, and oxidative stress. However, the detailed cellular and molecular mechanisms of TMT-induced neurodegeneration remain to be elucidated.

Ishige okamurae ( Ishige okamurae ) is a type of edible brown algae that is widespread throughout East Asia. Brown algae, including shellfish, are attracting attention as functional materials used in medicinal foods as their physiological activities have been suggested to include hypoglycemic, anti-inflammatory, and antiviral effects.

Republic of Korea Patent Application No. 10-2020-0109571 Republic of Korea Patent Application No. 10-2018-0096791

Accordingly, the present inventors confirmed that Ishige okamurae extract (IOE) can be applied to neurodegeneration other than Alzheimer's disease (AD) using a TMT injection animal model and in vitro glutamate excitotoxicity, and developed the present invention. Completed.

Accordingly, the purpose of the present invention is to provide a composition for preventing, improving, or treating trimethyltin-mediated neurodegeneration, including an extract of Ishige okamurae .

Another object of the present invention is a method of protecting brain cells treated with trimethyltin, which includes the step of processing an extract of Ishige okamurae , or a method of protecting brain cells treated with trimethyltin, which includes an extract of Ishige okamurae . To provide a composition for cell protection.

The present invention provides a pharmaceutical composition for preventing or treating trimethyltin-mediated neurodegeneration containing an extract of Ishige okamurae .

According to a preferred embodiment of the present invention, the extract may be extracted using water, an organic solvent, or a mixture thereof as an extraction solvent.

According to a preferred embodiment of the present invention, the trimethyltin-mediated neurodegeneration causes hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease ( It may be one or more selected from the group consisting of Alzheimer's disease.

In addition, the present invention provides a health functional food composition containing an extract of Ishige okamurae for preventing or improving trimethyltin-mediated neurodegeneration.

Additionally, the present invention provides a method for protecting brain cells treated with trimethyltin, which includes the step of treating Ishige okamurae extract.

According to a preferred embodiment of the present invention, the shell extract may be processed to induce one or more characteristics selected from the group consisting of the following eight characteristics:

i) Reduced expression of cleaved caspase-3;

ii) reduced expression of cleaved PARP;

iii) increased expression of brain-derived neurotrophic factor (BDNF);

iv) decreased ERK phosphorylation;

v) decreased p38 phosphorylation;

vi) decreased JNK phosphorylation;

vii) increased expression of heme oxygenase-1 (HO-1); and

viii) Increased expression of Nrf2 (nuclear factor erythroid 2-related factor 2).

According to a preferred embodiment of the present invention, the brain cells may be HT22 cells.

The present invention provides a composition for protecting brain cells treated with trimethyltin containing an extract of Ishige okamurae .

The shell extract of the present invention protects brain cells damaged by trimethyltin treatment by i) reducing the expression of cleaved caspase-3, ii) reducing the expression of cleaved PARP, and iii) expressing BDNF (brain-derived neurotrophic factor). increased, iv) decreased ERK phosphorylation, v) decreased p38 phosphorylation, vi) decreased JNK phosphorylation, vii) increased heme oxygenase-1 (HO-1) expression, and viii) increased nuclear factor erythroid 2-related factor 2 (Nrf2) expression. can be provided. Therefore, the shell extract of the present invention can be effectively used as a composition for preventing, improving or treating trimethyltin-mediated neurodegeneration, and as a method or composition for protecting trimethyltin-treated brain cells.

Figure 1 shows the method and results of memory impairment test according to oral administration of mouse shell extract (IOE) (A). The number of entries in the Y-maze test did not differ between groups (B), but TMT-induced spontaneous alterations (%) were recovered in IOE mice compared to the TMT mouse group (C). (D) shows the path trace of each group. Data shown are means ± SD. Con: control (untreated), a: significant difference (p < 0.05) compared to b.
Figure 2 shows the method and results of testing long-term memory impairment following oral administration of Ishige okamurae extract (IOE) to mice (A). Escape latency of the Morris water maze test (A) and time in W zone (%) (B) on day 5 were shown (n = 5). (C) shows the movement patterns of each group during the probe test. Data shown are means ± SD. Con: control (untreated), a: significant difference (p < 0.05) compared to b.
Figure 3 shows the expression of cleaved Caspase-3 and cleaved PARP (A) and BDNF (B) in brain tissue of the control group (Con), trimethyltin administration (TMT), and trimethyltin and plaque extract administration (TMT+IOE) groups. (n = 5). The expression level of each gene was estimated and normalized to that of GAPDH. Data shown are means ± SD. a: significant difference (p <0.05) compared to b.
Figure 4 shows representative CA1 subfields of the hippocampal region stained with H & E of mouse brains from the control (Con), trimethyltin administered (TMT), and trimethyltin and plaque extract administered (TMT+IOE) groups. The enlarged area of the square in the upper panel is shown in the lower panel (A) and indicated the number of viable cells and enriched nuclei (n = 5) (B). Arrows indicate enriched nuclei. Data are expressed as mean ± SD. a: significant difference (p <0.05) compared to b. Magnifications 200x (top panel) and 400x (bottom panel). Scale bar = 50 μm.
Figure 5 shows ERK (A), p-38 MAPK (B) and JNK ( C) shows the phosphorylation level of ERK (p-ERK/t-ERK), p38 (p-p38/p38), and JNK (p-JNK/JNK) in each group. Data shown are means ± SD. a: significant difference (p <0.05) compared to b.
Figure 6 shows the expression levels of Nrf2 and HO-1 in the brains of mice in the control group (Con), trimethyltin administration (TMT), and trimethyltin and plaque extract administration (TMT+IOE) groups (n = 5) (A), respectively. Relative expression rates of groups Nrf2 (Nrf2/GAPDH) and HO-1 (HO-1/GAPDH) are shown (B). Data shown are means ± SD. a: significant difference (p <0.05) compared to b.
Figure 7 shows the cytotoxicity of plaque extract (IOE) on HT22 cells. Glutamate-mediated cytotoxicity was inhibited by treatment with non-toxic levels of IOE. Cell viability was measured with the WST-1 assay kit (B) and cell counting (C). Data values shown are means ± SD. Untreated cells (IOE- and Glu-) were used as controls. The experiment was repeated three times with similar results. a: significant difference compared to b or c (p < 0.05) b: significant difference compared to a or c (p < 0.05), *: significant difference compared to control group (p < 0.01). Glu: glutamate.
Figure 8 shows the effect of IOE on glutamate-mediated ROS production in HT22 cells (A). Expression of apoptosis-related genes (Bax, Bcl-1 and cleaved caspase-3) (B, C) (n = 3), ratio of Bax/Bcl-2 and cleaved caspase-3 (cleaved caspase-3) /β actin) relative expression. Data shown are means ± SD. Glu: glutamate. Untreated cells (IOE- and Glu-) were used as controls. The experiment was repeated three times with similar results. a: significant difference (p <0.05) when compared to b or c. b: significant difference (p <0.05) when compared to a, c and d.
Figure 9 shows the results of measuring phosphorylation of ERK (A), p38 MAPK (B), and JNK (C) in HT22 cells pretreated with IOE with or without glutamate (5mM) for 2 hours (n = 3). . Each data value is expressed as mean ± SD. Glu: glutamate. Untreated cells (IOE- and Glu-) were used as controls. The experiment was repeated three times with similar results. a: significant difference (p <0.05) when compared to b or c. b: significant difference (p <0.05) when compared to a or c.
Figure 10 shows that Nrf2 and HO-1 inhibition by glutamate was restored by IOE (2 hours pretreatment) and MAPK inhibitors (PD98059, SB203580, and SP600125, 1 hour pretreatment). Glutamate-induced expression of Nrf2 (DF) and HO-1 (AC) in HT22 cells by qRT-PCR (n = 3), PD98059 (A, D), SB203580 (B, E) and SP600125 (C and F) The effect of Nrf2 and HO-1 on the expression of Nrf2 and HO-1 was estimated by qRT-PCR (n = 3). Untreated cells (IOE, Glu and inhibitor-) were used as controls. The experiment was repeated three times with similar results. Data shown are means ± SD. a: significant difference (p <0.05) when compared to b or c. b: significant difference (p <0.05) when compared to a or c.
Figure 11 shows that glutamate-induced ROS overproduction and cytotoxicity were recovered by IOE and MAPK inhibitors in HT22 cells. ROS overproduction (AC) and cell viability (DF) were estimated after pretreatment of HT22 cells with IOE (2 h) and MAPK inhibitors (1 h) (PD98059; MEK inhibitor, SB203580; p38 MAPK inhibitor, SP600125; JNK inhibitor). did. Untreated cells (IOE, Glu and inhibitors) were used as controls. Data shown are means ± SD. Glu: glutamate. The experiment was repeated three times with similar results. a: significant difference (p < 0.05) compared to b or c or d b: significant difference (p < 0.05) compared to a, c or d; c: significant difference (p < 0.05) compared to a, b or d.
Figure 12 is a schematic diagram showing the anti-neurodegenerative effect mediated by plaque extract (IOE).

Hereinafter, the present invention will be described in more detail.

In the present invention, we investigated the inhibitory role of Ishige okamurae , an edible brown algae, on the neurodegeneration process by confirming the effect of Ishige okamurae on neurodegeneration induced by trimethyltin (TMT) in vivo. did. The TMT injection mouse model prepared in the present invention refers to a neurodegenerative mouse model, and the neurodegenerative mouse model includes hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. It may be a mouse model of Huntington's disease or Alzheimer's disease. Oral administration of plaque extract (IOE) to TMT-injected mice disrupted TMT-mediated short-term and long-term memory impairments examined in the Morris water maze test and Y-maze test. IOE attenuated TMT-mediated cell death and the expression of brain-derived neurotrophic factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in mouse brain. Additionally, TMT-induced MAPK (mitogen-activated protein kinases) phosphorylation in mouse brain tissue and HT22 cells was attenuated by IOE treatment. Overall, the IOE of the present invention can provide an attenuating effect on neurodegenerative processes such as TMT-mediated neuronal dysregulation by regulating the MAPK/Nrf-2/HO-1 antioxidant pathway.

Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating trimethyltin-mediated neurodegeneration containing an extract of Ishige okamurae .

According to a preferred embodiment of the present invention, the extract may be extracted using water, an organic solvent, or a mixture thereof as an extraction solvent. Preferably, it is water, C1 to C4 lower alcohol, or a mixture thereof, more preferably, it is water, methanol, ethanol, propanol, or a mixture thereof, and most preferably, 1 to 100% ethanol is used as the solvent. It is most desirable to do so.

According to a preferred embodiment of the present invention, the trimethyltin-mediated neurodegeneration causes hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease ( It may be one or more selected from the group consisting of Alzheimer's disease.

'Prevention' of the present invention refers to any action that suppresses or delays the onset of neurodegenerative diseases due to the shell extract of the present invention.

'Improvement' or 'treatment' of the present invention refers to any action that improves or benefits parameters related to neurodegenerative diseases, such as the degree of symptoms, due to the shell extract of the present invention.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the composition, one or more buffering agents (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch (corn starch, wheat starch, rice starch, potato). starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl. -It is prepared by mixing cellulose or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.

Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, or preservatives. You can. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate can be added as a disintegrant, and anti-coagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, and packaging agents. , pigments and preservatives may be additionally included.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, or suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.

The composition of the present invention can be administered orally or parenterally. When administered parenterally, it can be administered intraperitoneally, rectally, intravenously, intramuscularly, subcutaneously, intrauterineally, or intracerebrovascularly; Alternatively, it can be formulated in the form of a nasal inhalant according to methods known in the art.

The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.

For inhalation administration, the compounds used according to the invention may be packaged in pressurized packs or using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. For pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type of patient's disease, severity, activity of the drug, sensitivity to the drug, and administration time. , route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the field of medicine. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. That is, the total effective amount of the composition of the present invention can be administered to the patient in a single dose, or by a fractionated treatment protocol in which multiple doses are administered over a long period of time. . Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.

In addition, the present invention provides a health functional food composition containing an extract of Ishige okamurae for preventing or improving trimethyltin-mediated neurodegeneration.

Since the shell extract is the same as that included in the pharmaceutical composition, the description is replaced with the above description.

The health functional food composition according to the invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, mamalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , vegetable proteins, retort foods, frozen foods, and various seasonings (e.g., soybean paste, soy sauce, sauce, etc.) can be produced by adding the shell extract of the present invention. In addition, nutritional supplements are not limited to this, but can be prepared by adding the shell extract of the present invention to capsules, tablets, pills, etc. In addition, the health functional food is not limited to this, but for example, the shell extract of the present invention itself is manufactured in the form of tea, juice, and drinks and liquefied, granulated, encapsulated, and powdered so that it can be consumed (health drink). It can be consumed. Additionally, in order to use the shell extract of the present invention in the form of a food additive, it can be prepared and used in powder or concentrate form. Additionally, it can be prepared in the form of a composition by mixing the shell extract of the present invention with a known active ingredient known to have an effect in preventing or improving neurodegenerative diseases.

When the shell extract of the present invention is used as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like ordinary drinks. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.

In addition, the shell extract of the present invention can be contained as an active ingredient in a health functional food composition for preventing or improving neurodegenerative diseases, but the amount is not particularly limited to an amount effective to achieve the effect of preventing or improving neurodegenerative diseases. It is preferably 0.01 to 100% by weight based on the total weight of the entire composition. The health functional food composition of the present invention can be prepared by mixing shell extract with other active ingredients known to be effective in treating neurodegenerative diseases.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , may contain glycerin, alcohol, or carbonating agent. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Additionally, the present invention provides a method for protecting brain cells treated with trimethyltin, which includes the step of treating Ishige okamurae extract.

Since the shell extract is the same as that included in the pharmaceutical composition, the description is replaced with the above description.

According to a preferred embodiment of the present invention, the shell extract may be processed to induce one or more characteristics selected from the group consisting of the following eight characteristics:

i) Reduced expression of cleaved caspase-3;

ii) reduced expression of cleaved PARP;

iii) increased expression of brain-derived neurotrophic factor (BDNF);

iv) decreased ERK phosphorylation;

v) decreased p38 phosphorylation;

vi) decreased JNK phosphorylation;

vii) increased expression of heme oxygenase-1 (HO-1); and

viii) Increased expression of Nrf2 (nuclear factor erythroid 2-related factor 2).

According to a preferred embodiment of the present invention, the brain cells may be HT22 cells.

Additionally, the present invention provides a composition for protecting brain cells treated with trimethyltin containing an extract of Ishige okamurae .

Since the shell extract is the same as that included in the pharmaceutical composition, the description is replaced with the above description.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

Experiment preparation

<1-1> Preparation of shell extract

Ishige okamurae was extracted using 70% ethyl alcohol, and the supernatant was concentrated with a vacuum evaporator (Heidolph Instruments GmbH & Co., Schwabach, Germany) and freeze-dried with a freeze dryer (ilShinBioBase, Seoul, Korea). The yield was 13.7% (w/w).

<1-2> Preparation of mouse model

Mice were fed IOE at a dose of 20 mg/kg bw/day for 3 weeks as described in Example <1-3> below. The human equivalent dose was calculated using body surface area, and the IOE (20 mg/kg bw/day) concentration of TMT-injected mice was 36.45 mg/60 kg bw/day, which can be estimated as human intake. This dose in humans (36.45 mg/60 kg bw/day) can be met with available supplements.

<1-3> Preparation of mouse experimental group

Four-week-old male C57BL/6 mice (NARA Biotec; Seoul, South Korea) were housed in cages with controlled light cycle (12 h light/12 h dark), temperature (23 ± 3 °C), and humidity (55 ± 10%). Mice were allowed free access to standard food and water (Orient Bio, Seoul, South Korea). After acclimatization for 1 week, mice were divided into control group (solvent injection, n = 5), TMT (TMT injection, n = 5). ), TMT+IOE (TMT injection+oral IOE gavage 20mg/kg bw/day, n = 5). The TMT+IOE group was treated with IOE for 21 days. TMT (Sigmaldrich, Seoul, South Korea) was injected intraperitoneally into mice (2.5 mg/kg/bw) only once after completing IOE administration (Figure 1 A). Control mice were inoculated with the same volume of PBS. All experimental procedures were carried out by Incheon National University Laboratory Animal Center. It was performed in accordance with the Care and Use Guidelines and was approved by the Incheon National University Institutional Animal Care and Use Committee (INU-ANIM-2018-11).

Effect of IOE on TMT-mediated cognitive impairment

<2-1> Y-maze test

We aimed to determine the effect of plaque extract (IOE) on trimethyltin (TMT)-mediated cognitive impairment. The Y-maze test is a behavioral test that measures the willingness to explore a new environment, so it can be used to assess short-term memory in mice.

Specifically, the Y-maze test was initiated 3 days after TMT injection (Figure 1A). Each mouse was placed at the end of one arm and allowed to move freely through the maze for 8 min. Eight item sequences were recorded manually using the SMART 3.0 video tracking system (Harvard Apparatus, Holliston, MA, USA). Alteration was calculated as the number of successful entries into the arm in three sets (Equation 1). When an animal first enters the order A, B, C, it is counted as one shift (actual shift), but an animal that enters the order B, A, B does not count as a shift.

[Equation 1]

Possible shifts = total number of arm entries: 2

Alternating motion (%) was calculated using the following [Equation 2].

[Equation 2]

Shift operation (%) = (actual shift) / (possible shift) × 100

The results showed that the number of entries did not differ between groups, as shown in [Figure 1B], suggesting that TMT did not affect the basic locomotive ability of mice. However, spontaneous changes (%) in the TMT-injected mouse group (68.46 ± 15.37%) were significantly reduced compared to the control group (100.20 ± 13.50%) (p < 0.05). However, TMT had no effect on spontaneous alterations in the IOE group (99.80 ± 12.58%) (Fig. 1C), suggesting that oral administration of IOE may attenuate TMT-mediated spatial working memory impairment. Additionally, although unbalanced movement paths were identified in TMT-injected mice compared with control mice, IOE mice showed movement patterns similar to control mice (Figure 1 D), suggesting that TMT-mediated abnormal behavior could be suppressed by oral administration of IOE. represents.

<2-2> Morris water maze test

The Morris Water Maze Test (MWM) test was used to evaluate the effect of IOE on TMT-mediated impairment on spatial and long-term learning memory.

Specifically, the MWM test equipment consisted of a circular pool (90 cm diameter, 60 cm height) filled with white ink (Wilton Industries, Inc., Woodridge, IL, USA) in water (22 ± 2 °C) up to 30 cm high, with four were divided into equal quadrants. A white escape platform was set up in the center of section W. Mice were allowed to swim and their latency was monitored up to 60 s. They were allowed to stay on the platform for 5 s. Training session (5 days after TMT injection) ), mice were required to swim to escape during three trials per day. A probe test (10 days after TMT injection) was performed to estimate spatial memory and long-term memory for 60 s without a platform, and time spent in the W zone was calculated as SMART Recorded using a 3.0 video tracking system (Harvard Apparatus, Holliston, MA, USA).

As a result, as can be seen in [Figure 2A], the time to reach the platform (escape latency) of control mice tended to decrease during the MWM test. However, the escape latency of TMT mice (55.71 ± 4.58%) did not decrease during the MWM test and was significantly higher on day 3 of the training test compared to control mice (34.93 ± 9.79%) (p < 0.05). This suggests impairment of long-term learning and spatial memory in TMT mice. In contrast, the escape latency of TMT+IOE mice (34.21 ± 8.05%) was significantly lower (p < 0.05) than that of TMT mice (55.71 ± 4.58%) at day 3 in the training test, which suggests that TMT-IOE mice (34.21 ± 8.05%) are significantly lower (p < 0.05) than TMT mice (55.71 ± 4.58%) at day 3 in the training test. Mediated impairment appears to be attenuated by oral administration of IOE. Additionally, TMT mice (4.32 ± 1.40%) stayed for a shorter time in the W area compared to control mice (17.57 ± 4.21%), whereas TMT+IOE mice (21.09 ± 2.87%) stayed longer in the W area when compared to TMT mice. A lot of time was spent (Figure 2B, C). Taken together, these results strongly suggest that TMT-mediated cognitive deficits, such as memory and learning impairments, can be suppressed by oral administration of IOE.

Effect of IOE on TMT-mediated mouse brain neuron injury

We aimed to evaluate the neuroprotective effects of IOE on TMT-injected mouse brain.

Specifically, mouse brain tissue was preserved in formaldehyde solution (10% w/v) for tissue fixation. Hematoxylin-Eosin (H&E) was applied to tissue sections (5 μm) to observe the hippocampal area. The number of neurons and nuclear agglutination-positive cells were counted under a microscope (Nikon, Tokyo, Japan).

For Western blot analysis, after glutamate (5mM) administration for 16 h, cells were washed with PBS and HT22 cells were lysed with protein extraction buffer (20mM Tris-HCl (pH 7.4), 70μM EDTA). and 150mM NaCl, Nonidet P-40 (1%, w/v) phosphatase inhibitor cocktail (Cell Signaling, Danvers, MA, USA)) on ice for 1 h. After centrifugation (13,000 × g) for 10 min at 4 °C, the supernatant was used to measure each protein. At the end of the animal experiment, mouse brain tissue was separated and homogenized with the protein extraction buffer described above using a homogenizer (Huanyu Instrument, Zhejiang, China). 20 μg of each protein sample was used for separation on SDS-PAGE. After completing SDS-PAGE, the gel was separated and transferred to a nitrocellulose membrane. After incubation with 5% non-fat milk to block non-specific signals, the membranes were incubated with each antibody (1:1500) for 12 h at 4 °C. Each protein was detected using rabbit anti-cleaved caspase 3 (9664S) or rabbit anti-brain-derived neurotrophic factor (BDNF) (NBP1-46750) - Novus (Centennial, CO, USA). Tween 20 buffer (20mM Tris, 136mM NaCl, (1%, w/v), Tween 20; pH 7.4), horseradish peroxidase (HRP)-conjugated rabbit or mouse secondary antibody (1:3000) (Abcam , Cambridge, UK) and then incubated for an additional 2 hours at room temperature. Each band was visualized using enhanced chemiluminescence (ECL) detection reagent (Bio-Rad, Hercules, CA, USA).

As a result, as can be seen in [Figure 3], the expression of cleaved caspase-3 and cleaved PARP, which are markers of cell death, were significantly increased in the brains of TMT-injected mice compared to control mice (p <0.05). Interestingly, oral administration of IOE significantly reversed the elevated expression of cleaved caspase-3 and cleaved PARP detected in TMT mice (Figure 3 A). In addition, we found that the expression of BDNF (brain-derived neurotrophic factor), which plays a role in supporting the survival of existing neurons, was significantly decreased (p < 0.05) in the brain tissue of TMT mice compared to the control group. However, the expression level of BDNF in brain tissue of TMT+IOE mice was not significantly different from that of control mice (p > 0.05) (Figure 3B). The neuroprotective effect of IOE on brain tissue was further confirmed by H&E staining.

Additionally, as can be seen in [Figure 4], the number of surviving neurons in the CA1 region of the hippocampus was decreased in TMT mice compared to control mice (p < 0.05), and the number of pyknotic nuclei was decreased compared to control mice. It was significantly increased in the CA1 region of TMT mice (p < 0.05). However, this neuronal damage was significantly abolished in the CA1 region of TMT+IOE mice (p <0.05). Taken together, this suggests that TMT-induced neuronal death can be attenuated by oral administration of IOE.

Effect of IOE on TMT-induced abnormal phosphorylation of MAPK in mouse brain

MAPKs are well-known intracellular signaling molecules that control several important cellular regulations such as apoptosis and gene expression. Additionally, abnormal activation of MAPK has been reported to be closely related to neurodegenerative processes. Therefore, to elucidate the cellular mechanisms responsible for the IOE-mediated neuroprotection mechanism, we compared the effect of IOE on the activation/phosphorylation of MAPKs in brain tissues of mouse groups by Western blot analysis.

Western blot was performed using rabbit anti-ERK (9101S), rabbit anti-phospho-p38 (9212S), rabbit anti-p38 (9211S), rabbit anti-phospho-JNK (9251S), or rabbit anti-JNK (9252S). This was performed in the same manner as <Example 3> above.

As a result, as shown in [Figure 5], it was confirmed that the phosphorylation of ERK (534.65 ± 74.80), p38 MAPK (316.96 ± 56.51), and JNK (236.48 ± 54.78) was increased in the brain tissue of TMT mice compared to control mice. (ERK: 100 ± 33.46, p38 MAPK: 100 ± 33.46, JNK: 100 ± 42.76). However, oral administration of IOE significantly reversed the abnormal phosphorylation of ERK, p38 MAPK and JNK found in the TMT mouse group (p < 0.05). These results suggested that TMT-mediated abnormal phosphorylation of MAPK in mouse brain tissue could be attenuated by oral administration of IOE.

Effect of IOE on TMT-induced abnormal expression of Nrf2/HO-1

Since oxidative stress is one of the important causes of TMT-induced neurodegenerative process, we sought to determine whether oral administration of IOE could control the antioxidant molecular system in the brain of TMT-injected mice.

Western blot was performed as described above using rabbit anti-HO-1 (43966S)-Cell Signaling (Danvers, MA, USA) and mouse anti-NRF2 (M200-3) (Medical & Biological Laboratories Co., Nagoya, Japan). Example 3> was performed in the same manner.

As a result, as shown in [Figure 6], among several antioxidant mechanisms, HO-1 (heme oxygenase-1), which plays a protective role against oxidative stress, and Nrf2 (nuclear factor erythroid 2-related factor 2), the main transcription factor of HO-1 ) was confirmed to have recovered in the TMT+IOE mouse brain. The expression of Nrf2 and HO-1 was significantly reduced in brain tissue of the TMT mouse group (Nrf2: 44.52 ± 20.28, HO-1: 41.80 ± 16.79) (p < 0.05). Control mouse group (Nrf2: 100 ± 19.49, HO-1: 100 ± 43.92); However, it was abolished by oral administration of IOE (Nrf2: 111.92 ± 33.50, HO-1: 92.64 ± 17.96) (p < 0.05). These data strongly suggested that TMT-mediated neurodegeneration may occur by blocking intracellular signaling responsible for the expression of HO-1, which is important in cellular antioxidant processes in the mouse brain. Interestingly, TMT injection increased phosphorylation of MAPK (Figure 5) and decreased expression of Nrf2 and HO-1 (Figure 6). However, in the TMT+IOE mouse group, these TMT-induced abnormal cellular processes in the mouse brain were abolished. This suggested that TMT-mediated oxidative stress could be inhibited by oral administration of IOE by controlling the MAPK/Nrf2/HO-1 pathway in mouse brain.

Effect of IOE on glutamate-mediated excitotoxicity

Since glutamate excitotoxicity is considered to be the cause of several neurodegenerative diseases that induce oxidative stress in neurons, resulting in neuronal damage, we sought to further confirm IOE-derived anti-neurodegenerative effects on a cellular model of glutamate excitotoxicity in animal experiments. did.

Specifically, HT22 cells were purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (HyClone, Logan, UT, USA) containing 10% fetal bovine serum (FBS) (Corning, NY, USA) and 1% streptomycin/penicillin. ) were cultured in a CO ₂ incubator at 37°C. HT22 cells (10 ⁴ cells/well) were seeded in 96 well plates and cultured for 24 hours. IOE (0-0.1 mg/mL) was treated for 2 h and then glutamate (5mM) was added to the culture plate. It was further incubated at 37°C in a CO ₂ incubator for 14 hours. To measure cell viability, the WST-1 Assay Kit (Dozen, Seoul, Korea) was used, HT22 cells were photographed using a microscope (Nikon, Tokyo, Japan), and the number of viable HT22 cells was estimated.

As a result, non-toxic levels of IOE were determined in mouse hippocampal neurons, HT22, using a cell viability assay kit. Treatment of HT22 cells with IOE up to a concentration of 0.2 mg/mL did not alter HT22 cell viability (p <0.05) (Figure 7A). Based on these results, glutamate-mediated cytotoxicity was attenuated by IOE treatment in a dose-dependent manner (control: 100 ± 7.62, glutamate: 9.64 ± 1.24, glutamate+IOE 0.05: 44.53 ± 9.36, glutamate+IOE 0.1: 103.19 ± 4.27). (p < 0.05) (Figure 7B). The effect of IOE on glutamate-mediated cytotoxicity was further confirmed by counting the number of HT22 cells. As shown in Figure 7C, a large number of viable HT22 cells were restored using IOE administration (control: 43.93 ± 2.55, glutamate: 21.68 ± 2.27, glutamate+IOE 0.05:32 ± 3.93, glutamate+IOE 0.1:39.62 ± 1.78) (p < 0.05). This suggests that glutamate-induced excitotoxicity in HT22 neurons may be attenuated by IOE.

Effect of IOE on glutamate-mediated reactive oxygen species overproduction

Excessive glutamate is known to cause cellular stress by increasing intracellular reactive oxygen species (ROS), which leads to cellular oxidative stress.

Specifically, the effect of IOE on glutamate-induced intracellular ROS levels was estimated in HT22 cells. HT22 cells ( ¹⁰ cells/well) were cultured in black plates (96 wells, SPL Life Sci ences, Gyeonggi, Korea) for 24 h. HT22 cells were treated with glutamate (5mM) and IOE for 16 hours at 37°C. After washing with phosphate-buffered saline (PBS), 30 μM DCFH-DA (2′-7′dichlorofluorescin) solution (Sigma-Aldrich Co., St. Louis, MO, USA) was added and CO at 37 °C for 1 h. It was further cultured in an incubator. Each well was washed twice with PBS and then PBS (100 μL) was added to each well. The intensity of fluorescence was calculated at 488 nm using a microplate fluorometer (Tecan, Mannedorf, Switzerland).

As a result, as shown in [Figure 8 A], the intracellular ROS level of glutamate-treated HT22 cells increased (150.24 ± 5.70) compared to the control group (100 ± 5.64). However, it was attenuated by IOE treatment in a dose-dependent manner (IOE 0.05:139.14 ± 2.11, IOE 0.1: 114.76 ± 5.20) (p < 0.05). These results indicate that glutamate-mediated cellular stress, such as ROS overproduction, can be effectively counteracted by IOE administration.

Effect of IOE on glutamate-mediated activation of apoptosis

Because glutamate-mediated cytotoxicity and intracellular ROS levels were attenuated by IOE (Figures 7 and 8 A), we investigated whether IOE could inhibit glutamate-induced apoptosis of HT22 cells.

As a result, the expression of Bax, a pro-apoptosis molecule, was significantly increased after treating HT22 cells with glutamate (1 ± 0.0256) and attenuated by IOE treatment (0.901 ± 0.045) (Figure 8B). However, the expression of the anti-apoptotic molecule Bcl-2 was attenuated by glutamate treatment (1 ± 0.0354) but recovered after IOE treatment (0.834 ± 0.048) (Figure 8 B) (p < 0.05). Additionally, treatment with IOE significantly attenuated glutamate-induced expression of cleaved caspase-3, an intracellular apoptotic signal, in HT22 cells (p <0.05) (Figure 8 C). These results suggest that IOE has inhibitory activity against glutamate-induced HT22 cell death mainly by regulating the expression of apoptosis-related genes.

Effect of IOE on glutamate-mediated MAPK activation

To further elucidate the regulatory role of IOE in glutamate excitotoxicity in HT22 cells, we investigated the effect of IOE on glutamate-induced intracellular activation of MAPK.

As a result, as shown in [Figure 9], when HT22 cells were treated with glutamate, the activation/phosphorylation of ERK, p38 MAPK, and JNK proteins increased compared to the control group, as shown in the brain tissue of mice treated with TMT (Figure 5). Interestingly, this aberrant activation/phosphorylation of MAPK in glutamate-treated HT22 cells was attenuated by IOE treatment in a dose-dependent manner (p <0.05) (Figure 9). These results suggest that glutamate-induced MAPK dysregulation in HT22 cells can be restored by IOE treatment.

Effect of IOE or MAPK inhibitors on glutamate-mediated Nrf2 and HO-1 inhibition

After confirming the regulatory effect of IOE on glutamate-induced abnormal activation of MAPKs, we investigated the downstream pathways of MAPKs involved in oxidative stress. Oxidative stress is known to be one of the important causes of glutamate excitotoxicity, and since TMT-mediated Nrf2 and HO-1 expression was restored by IOE in mouse brain tissue (Figure 6), glutamate-induced Nrf2 and HO-1 expression in HT22 cells. We investigated the impact of IOE on We further confirmed the role of MAPKs on glutamate-induced Nrf2 and HO-1 expression by MAPK-specific inhibitors (SB203580, a p38 MAPK inhibitor; PD98059, a MEK inhibitor; SP600125, a JNK inhibitor).

For real-time PCR, HT22 cells (10 ⁵ /well) were seeded in a 6-well plate and cultured for 24 hours. After incubation with glutamate (5mM) for 16 h at 37°C with IOE or inhibitors (PD98059, SP600125 and SB203580), total RNA was isolated using TRIzol Reagent (Invitrogen, Waltham, MA, USA). 1 μg of total RNA and 10 pM of oligo dT primer were used for the synthesis of complementary DNA (cDNA). SYBR Green Realtime PCR Master Mix (Toyobo Co., Tokyo, Japan) was used in the RT-PCR detection system (CFX Connect, Bio-Rad Co., Hercules, CA, USA). The relative expression of each gene was estimated using the comparative Ct method and normalized to the expression of GAPDH. The primer sequences used are shown in [Table 1] below.

gene direction Sequence (5'→3') sequence number HO-1 Forward ACATCGACAGCCCCACCA One Reverse TGAGCAGGAAGGCGGTCT 2 Nrf-2 Forward ACTCCCAGGTTGCCCACA 3 Reverse TCCAGGGCAAGCGACTCA 4 GAPDH Forward ACGGGAAGCTCACTGGCA 5 Reverse TCCAGGGCGGCATGTCAGA 6

As a result, as shown in [Figure 9], the inhibition of Nrf2 and HO-1 expression by glutamate was recovered by IOE administration (p < 0.05). Additionally, as shown in [Figure 10], it was confirmed that glutamate-mediated expression of Nrf2 and HO-1 was restored by administration of SB203580, PD98059, and SP600125 (p <0.05). This indicates that glutamate-mediated suppression of HO-1 expression can be attenuated by the IOE and MAPK (ERK/p38 MAPK/JNK) pathways, which are critically involved in glutamate-mediated neurodegeneration by regulating the expression of Nrf2 and HO-1. .

Effects of IOE or ERK, p38 MAPK, and JNK inhibitors on glutamate-mediated ROS overproduction and reduced cell viability.

Because glutamate-induced HO-1 inhibition was restored by IOE and MAPK inhibitors (Figure 10), we further verified the anti-neurodegenerative effects of IOE and MAPK inhibitors on glutamate-induced ROS production and HT22 cell survival.

Specifically, <Example 6> and <Example 6> and <Example 6> above were performed using a MAPK (mitogen-activated protein kinase) inhibitor (SP600125, Santa Cruz, Dallas, TX, USA; SP600125 and PD98059, Cell Signaling, Boston, MA, USA). 7> ROS production and HT22 cell viability were confirmed using the same method.

As shown in [Figure 11], glutamate-induced ROS overproduction was significantly inhibited by treatment with PD98059, SB203580, and SP600125 (p < 0.05) (Figure 11 A-C). We also found that glutamate-mediated HT22 cell survival was significantly restored by treatment with these inhibitors (p < 0.05) (Figure 11 D–F). Interestingly, IOE at a dose of 0.1 mg/mL had potent inhibitory activity (up to 10 μM of each MAPK signaling inhibitor). Taken together, these data strongly suggest that IOE can protect against glutamate excitotoxicity in HT22 cells by attenuating glutamate-mediated dysregulation of the MAPK/Nrf2/HO-1 pathway (Figure 12).

SEQUENCE LISTING <110> INCHEON NATIONAL UNIVERSITY RESEARCH ＆ BUSINESS FOUNDATION <120> Composition for preventing, improving or treating neurodegenerative disorders trimethyltin mediated comprising Ishige okamurae extract and uses it <130> 1069167 <160> 6 <170> PatentIn version 3.2 <210> 1 <211> 18 <212> DNA <213> Artificial <220> <223> HO-1 primer_F <400> 1 acatcgacag ccccacca 18 <210> 2 <211> 18 <212> DNA <213> Artificial <220> <223> HO-1 primer_R <400> 2 tgagcaggaa ggcggtct 18 <210> 3 <211> 18 <212> DNA <213> Artificial <220> <223> Nrf-2 primer_F <400> 3 actcccaggt tgcccaca 18 <210> 4 <211> 18 <212> DNA <213> Artificial <220> <223> Nrf-2 primer_R <400> 4 tccagggcaa gcgactca 18 <210> 5 <211> 18 <212> DNA <213> Artificial <220> <223> GAPDH primer_F <400> 5 acgggaagct cactggca 18 <210> 6 <211> 18 <212> DNA <213> Artificial <220> <223> GAPDH primer_R <400> 6 tccaggcggc atgtcaga 18

Claims (8)

A pharmaceutical composition for preventing or treating trimethyltin-mediated neurodegeneration comprising a 70% ethanol extract of Ishige okamurae ,
The trimethyltin-mediated neurodegeneration is selected from the group consisting of hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. A pharmaceutical composition characterized by one or more of the following.
delete delete A health functional food composition for preventing or improving trimethyltin-mediated neurodegeneration containing 70% ethanol extract of Ishige okamurae ,
The trimethyltin-mediated neurodegeneration is selected from the group consisting of hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. A health functional food composition characterized by one or more of the following.
A method of protecting brain cells treated with trimethyltin, comprising the step of treating Ishige okamurae 70% ethanol extract,
The trimethyltin-treated brain cells are selected from the group consisting of hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. Indicates one or more trimethyltin-mediated neurodegeneration,
A method of protecting brain cells, characterized in that the brain cells are brain cells isolated from an individual.
The method of claim 5, wherein the ethanol extract is treated to induce one or more characteristics selected from the group consisting of the following eight characteristics:
i) Reduced expression of cleaved caspase-3;
ii) reduced expression of cleaved PARP;
iii) increased expression of brain-derived neurotrophic factor (BDNF);
iv) decreased ERK phosphorylation;
v) decreased p38 phosphorylation;
vi) decreased JNK phosphorylation;
vii) increased expression of heme oxygenase-1 (HO-1); and
viii) Increased expression of Nrf2 (nuclear factor erythroid 2-related factor 2).
The method of claim 5, wherein the brain cells are HT22 cells.
A composition for protecting brain cells treated with trimethyltin containing 70% ethanol extract of Ishige okamurae ,
The trimethyltin-treated brain cells are selected from the group consisting of hyperactivity, aggression, cognitive impairment, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease. A composition for protecting brain cells, characterized in that it exhibits one or more trimethyltin-mediated neurodegeneration.