WO2012115306A1 - Composition including vitamin c for protecting fetal nerve cells, and functional health food including same - Google Patents

Abstract

The present invention relates to a composition including vitamin C for preventing damage to or treating fetal nerve cells in a pregnant mother exposed to alcohol or PTZ, and to a method for protecting fetal nerve cells by introducing the composition into the pregnant mother. When the composition and the functional health food including the composition as an active ingredient according to the present invention are used, nerve-protecting effects are exhibited for a fetus expected to have brain-related disorders due to the intake of PTZ or alcohol by the pregnant mother, such that fetal brain disorders and brain-related birth defects can be prevented or treated.

Description

Fetal nerve cell protective composition containing vitamin C and health functional food comprising the same

The present invention relates to a composition capable of protecting the fetus from the nerve damage of the fetus by ingestion of alcohol or PTZ of the mother, and more particularly to prevent or treat neuronal cell damage of the fetus in the mother exposed to alcohol or PTZ. A composition comprising vitamin C, a dietary supplement comprising the composition, and a method for protecting nerve cells of a fetus by administering the composition to a mother or a fetus in the mother.

Alcohol is most commonly used as a psychoactive substance, and alcoholism is a medical and socioeconomic problem in many countries. Such alcohols (ethanol) are known to be able to modulate the synthesis, release, receptor binding and signaling of neurotransmitters and neuromodulators. Ethanol exposure during brain growth has been shown to cause adverse effects such as defects in neuromigration, cell loss in several brain regions, synaptic connections, and neuronal cell defects.

In fetuses (early embryos or fetuses after gestation), ethanol affects the structural and functional deformation of large areas in the vulnerable parts of the nerves during the growth of the nerves, resulting in Fetal Alcohol Syndrom (FAS). Causes In particular, neurons are more vulnerable to ethanol-induced neurodegeneration during synapse formation. In humans, this synapse formation period is known to persist from 3 months of gestation to postnatal period, at which time the brain grows rapidly. Therefore, when a pregnant woman ingests alcohol, it causes serious problems in brain growth and development of the fetus in the mother.

Pentylenetetrazol (PTZ), also known as pentetrazol or pentamethylenetetrazol, is a drug used as a circulatory and respiratory stimulant, causing convulsant cognitive deficits in rodents. It is known as a substance. This PTZ has been used as a shock therapy because it causes cramps on excessive intake.

Mechanism of action of PTZ yet, but that only some known, generally PTZ is a peak toxin-Cl to inhibit GABA _A receptor as an antagonist (antagonist) of the GABA _A receptor that binds to the Bar cucurbit rate (picrotoxin-barbiturate) binding ^site-channel It is known to prevent and cause seizures. In addition, it has been reported to induce seizures in animal models of epilepsy, and in 1997, Pohle et al. Reported that PTZ reduced neurons in the hippocampus. In addition, PTZ is also known to cause seizures-induced neuronal death by oxidative stress, and the seizure induced by PTZ in the brain of a mature rat by PTZ is caused by apoptosis of neurons. It is known to activate kephase-3, which induces.

In addition, the expression of protein kinase A-α (PKA) decreases when the hippocampus and cerebral cortex are exposed to PTZ during early embryo development. Therefore, when a pregnant woman is exposed to PTZ, it causes serious problems in brain growth and development of the fetus in the mother.

Under this background, the present inventors studied a substance which prevents neuronal cell damage of the fetus in the mother due to alcohol or PTZ, and found that when ethanol, PTZ and vitamin C are treated in the hippocampus of pregnant rats, The present invention was completed by observing the role of apoptosis of neurons by ethanol or PTZ in the expression of proteins related to apoptosis and confirming that it could be a therapeutic approach to brain injury.

It is an object of the present invention to provide a composition for protecting nerve cells of a fetus in a mother exposed to alcohol or PTZ containing vitamin C.

Another object of the present invention to provide a health functional food comprising the composition.

It is still another object of the present invention to provide a method for protecting neurons of a fetus in a mother by administering vitamin C to a mother or a fetus exposed to alcohol or PTZ.

In the case of using the composition of the present invention and the health functional food containing the composition as an active ingredient, the fetal brain disease and brain-related by showing a neuroprotective effect in the fetus expected brain-related diseases by parental PTZ or alcohol intake Malformations can be prevented or treated.

1 is a graph showing the effect of vitamin C on the survival of hippocampal neurons treated with PTZ and ethanol for 24 hours.

FIG. 2A is a photograph and graph showing Western blot results of Bcl-2 in primary culture of hippocampal neurons obtained from fetal rats at day 17.5 gestation.

Figure 2b is a photograph and graph showing the Western blot results of Bax in the primary culture of hippocampal neurons obtained from fetal rats at day 17.5 gestation.

Figure 3 is a photograph and graph showing the Western blot results of cytochrome C in the primary culture of hippocampal neurons obtained from fetal rats at day 17.5 gestation.

FIG. 4 is a photograph and graph showing Western blot results of kephase-9 in primary culture of hippocampal neurons obtained from fetal rats at day 17.5 gestation.

FIG. 5 is a photograph and graph showing Western blot results of kephas-3 in primary culture of hippocampal neurons obtained from fetal rats at day 17.5 of gestation.

2-5 show cells in normal medium (Control C); Ethanol 100 mM containing medium (E); PTZ 20 mM containing medium (PTZ); Vitamin C 1 mM containing medium (Vit-C); Ethanol 100 mM and PTZ 20 mM containing medium (E + PTZ); Ethanol 100 mM and vitamin C 1 mM containing medium (Vit-C + E); PTZ 20 mM and Vitamin C 1 mM containing medium (Vit-C + PTZ); Pictures and graphs showing Western blot results after treatment with 100 mM ethanol, PTZ 20 mM and vitamin C 1 mM containing medium (Vit-C + E + PTZ) for 24 hours, in all cases beta-actin (β- actin) was mixed.

FIG. 6A is a confocal micrograph showing the effect of PTZ, ethanol and vitamin C on cytochrome C release from the mitochondria and cytoplasm 3 activity in hippocampal neurons. A to H are all 40 times magnification, and the length of the scale bar is 20 μm.

Figure 6b is a photograph showing the mitochondrial membrane potential analysis results by flow cytometry JC-1 staining cells treated for 24 hours using PTZ, ethanol and vitamin C, respectively or in combination. Loss of mitochondrial cell membrane potential is associated with increased FL1 fluorescence. Quantification of cells with low mitochondrial membrane potential (percentage of total cells) is induced by other binding with ethanol, PTZ, vitamin C as detected by flow cytometry. The results are presented as ± standard error of three independent experiments.

As one aspect for solving the above problems, the present invention provides a composition for protecting nerve cells of the fetus in the mother exposed to alcohol or PTZ containing vitamin C.

As used herein, the term "fetal" refers to a fluid that grows in the mother's mother, and examples of the mammal include rats, rabbits, horses, cows, sheep, dogs, cats, monkeys, humans, and the like. The type of fetus of the invention is not limited. Preferably, the fetus of the invention may be a fetus of a rat or a human as a fetus of a mammal.

In addition, the fetus may be classified into embryo, which is a fetus in early pregnancy, or fetus, which is a fetus after the middle of pregnancy, depending on the mother's gestation period and fetal development. Preferably, the fetus of the present invention is a fetus at the time of neural cell development and growth, in particular an embryo of early pregnancy, known as the early stage of neurogenesis, more preferably the fetus of the present invention Human fetuses after 2 months of pregnancy, more preferably human fetuses of 2 to 8 months of pregnancy, even more preferably 3 to 5 months of pregnancy, most preferably of 3rd month of pregnancy It may be an embryo.

In one embodiment of the present invention, the effects of vitamin C in the Sprag-Dolly rats on day 17.5 of gestation corresponding to the initial gestation period were confirmed.

As used herein, the term "neuronal cell" refers to a cell that modulates the action of other cells, including muscles, and, unlike general cells, transmits a signal by an electrical method and no longer divides after the developmental stage. In this way, the nerve cells are almost impossible to recover from damage due to the limitation of the number of divisions, and therefore, it is important to allow normal division to perform normal functions when they occur. In particular, the protection of neurons exposed to ethanol or PTZ is also important in the future development of the fetus, it was first identified by the inventors that vitamin C has the effect of protecting neurons exposed by ethanol or PTZ.

The neuronal protective effect using the composition of the present invention can protect all neurons irrespective of the location and size of the neurons, preferably hippocampal neuronal cells present in the hippocampal region of the fetal brain. Can be.

In one embodiment of the present invention, the neuronal protective effect on the brain of the rat fetus, which was sacrificed and separated from the amniotic membrane, at the expense of 17.5 gestation of Sprag-Dawley rats, was confirmed in hippocampal neurons. .

In the present invention, the term "ethanol" is a type of alcohol in which one hydrogen atom is substituted with a hydroxy group (-OH) in two carbon ethane, also called ethyl alcohol. Such ethanol is a colorless flammable compound, and is widely used as a solvent, a disinfectant, a fuel, and the like. When ethanol is ingested with respect to the nervous system, the ethanol becomes excited due to the inhibition of cerebral function, and then the effect of central nervous suppression is shown. Drinking alcoholic beverages that contain ethanol too often can lead to habits and addiction.

Ethanol is one of the leading causes of preventable cause of birth defects, mental retardation, and neurodevelopmental disorders.In pregnant women, ethanol intake is associated with long-term cognitive, It damages the development of the central nervous system, which indicates neurorobehavioral disruption, growth deficits, and epilepsy. In the present invention, it was confirmed that the nerve cell damage of the fetus in the mother fed such ethanol is effectively suppressed by vitamin C.

In the present invention, the term "PTZ" is also known as pentetrazol or pentamethylenetetrazol, and is a drug that is also used as a circulatory system and a respiratory stimulator. These PTZs are known to reduce neurons in the hippocampus and cause seizures-induced neuronal death by oxidative stress, and seizures induced by PTZ in the brains of mature rats by PTZ are neurons. It is also known to activate kephase-3, which induces apoptosis of. The composition of the present invention was first confirmed that effectively inhibit the nerve damage of the fetus in the mother that can be caused by the mother ingested such PTZ.

The composition of the present invention provides increased expression of Bcl-2 protein reduced by alcohol or PTZ; Decreased expression of Bax protein increased by alcohol or PTZ; Reduced release of cytochrome C released into the cytoplasm by alcohol or PTZ; Decreased expression of increased keasephase-9 by alcohol or PTZ; Decreased expression of kephas-3 increased by alcohol or PTZ; And recovery of mitochondrial membrane potential increased by alcohol or PTZ. Thus, such a feature is to inhibit the apoptosis caused by alcohol or PTZ to support vitamin C to protect the neuronal damage caused by the apoptosis.

Specifically, in the embodiment of the present invention by culturing the hippocampal neurons of the fetus alone or in combination with ethanol, PTZ and vitamin C, the survival rate was measured. As a result, it was confirmed that the survival rate of the cells treated with vitamin C significantly increased compared to the control (Fig. 1). In addition, when PTZ and ethanol were treated with vitamin C, expression of Bax, kephas-3 and kephas-9, proteins involved in cellular apoptosis, decreased the amount of cytochrome C released into the cytoplasm, and mitochondrial membrane potential It was confirmed that the expression of Bcl-2, known to inhibit recovery and apoptosis, was increased (FIGS. 2-6).

In the case of using the composition of the present invention, it is possible to prevent neurons of the fetus in the mother to prevent related diseases caused directly or indirectly by the alcohol or PTZ of the fetus. Related diseases caused directly or indirectly by the alcohol or PTZ include both neurological diseases caused by the mother's ingestion of the substance directly or indirectly and ultimately by the fetus being exposed to the alcohol or PTZ. Examples of such disorders include Fetal Alcohol Syndrom (FAS) -related brain injury (fetal disorders in which the baby's alcohol consumption causes symptoms such as mental retardation, cerebellar disease, underweight, and short eyelid fever) or alcohol-related neurodevelopment (ARNDS), fetal developmental disorders, fetal malformations, and neonatal sudden death syndrome, and the like, but the above examples do not limit the diseases that can be treated or prevented by the compositions of the present invention. Preferably, the composition of the present invention may prevent or treat ARNDS or FAS related brain injury by reducing the expression of Bax, cytochrome C, kephas-3 and kephas-9 in the fetus and increasing Bcl-2 protein expression. have.

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, a binder, a suspending agent, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring matter, a flavoring agent, etc. may be used for oral administration. In the case of topical administration, bases, excipients, lubricants, preservatives and the like can be used. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, in the case of injections, in the form of unit dose ampoules or multiple doses. have. Others may be formulated into solutions, suspensions, tablets, herbs, capsules, sustained release preparations and the like. Examples of suitable carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate or mineral oil and the like can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.

In addition, oral, intravenous, subcutaneous, intradermal, intranasal, intraperitoneal, intramuscular, and transdermal of the composition of the present invention can be administered using a variety of methods, the dosage is depending on the age, sex, weight of the patient And may be readily determined by one skilled in the art. Preferably, the compositions of the present invention can be administered orally or parenterally.

In addition, since the dosage of the composition may be determined by one skilled in the art according to the type of drug as an active ingredient, together with various related factors such as the route of administration, the degree of disease, sex, weight, age, etc. Is not limited. Preferably, the composition of the present invention may be administered at 0.1 to 2 mM per 1 × 10 ⁵ cells, more preferably 0.3 to 1.5 mM, even more preferably 0.4 to 1.2 mM, most preferably 1 × 10 ⁵ cells. It may be administered at 1 mM per 10 ⁵ cells.

In another aspect, the present invention provides a dietary supplement comprising the composition of the present invention.

The neuronal protective composition according to the present invention can be formulated in the same manner as the pharmaceutical composition can be used as a health functional food. Examples of dietary supplements include, but are not limited to, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, ice creams, alcoholic beverages, vitamin complexes, and health supplements.

As another aspect, the present invention provides a method for protecting the nerve cells of the fetus in the mother comprising administering a neuroprotective composition of the present invention to the mother or fetus in the mother exposed to alcohol or PTZ.

Preferably, the maternal fetus may be an early embryo.

Preferably, when the composition of the present invention is administered to the mother or fetus in the early pregnancy, nerve cells of the fetus may be protected by reducing or blocking the inhibition or damage of nerve cell development by alcohol or PTZ.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Preparation of Animals

Female (n = 25) Sprag-Dolly rats (250 g, Gyeongsang National University, Neurobiology Laboratory, Jinju, South Korea) were bred in a light environment with feed from 6 to 20 o'clock in a temperature-controlled environment. The fertilized day was 0.5 days pregnant. Pregnant Sprague-Dawley rats on day 17.5 of gestation were injected intravenously with pentobarbital sodium (3 mg / 100 g b.w) and then sacrificed.

Example 2. Primary Cell Culture and Drug Treatment

Cultures were prepared from the hippocampus of prenatal rats on day 17.5 of pregnancy. Hippocampal tissues separated at 17.5 days of gestation were treated with 0.25% trypsin-EDTA for 20 minutes and dissociated by mechanical softening in frozen calcium and magnesium-free Han's balanced salt solution (pH 7.4). After the suspension was centrifuged to collect the pellets, the cells were placed in cell culture plates precoated with poly-lysine (0.02 g / l) and chamber slides (1 × 10 ⁶ cells / ml). Medium was DMEM (Dulbecco's modified Eagle medium), 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 4.2 mM sodium hydrocarbon, 20 mM HEPES, 0.3 g / L bovine serum albumin, 50 U / ml penicillin and 50 mg / l streptomycin. The culture was carried out at a temperature of 5% CO ₂ , 95% humidity, 37 ℃. Neuroroglia cells were inhibited by a medium containing 100 μΜ cytosine β-D-Arabino Furanoside for 12 hours.

After 4 days, hippocampal neurons were normal medium (Control C); Ethanol 100 mM containing medium (E); PTZ 20 mM containing medium (PTZ); Vitamin C 1 mM containing medium (Vit-C); Ethanol 100 mM and PTZ 20 mM containing medium (E + PTZ); Ethanol 100 mM and vitamin C 1 mM containing medium (Vit-C + E); PTZ 20 mM and Vitamin C 1 mM containing medium (Vit-C + PTZ); Ethanol 100 mM, PTZ 20 mM and vitamin C 1 mM containing medium (Vit-C + E + PTZ), respectively, were treated, all drug treatment groups were incubated for 24 hours at 37 ℃ in vitro culture.

Example 3. Western Blotting

The hippocampal neurons isolated from the hippocampus tissue of Example 2 were homogenized in a cell lysate (Cell signaling # 9803) containing 100 mM PMSF as a protein inhibitor, and allowed to stand on ice for 20 minutes and then ultrasonicated for 4 minutes. sonicator, crush 15 seconds, stop 10 seconds). Subsequently, the supernatant containing protein was separated through ultracentrifugation (12,000 rpm, 10 minutes, twice). Protein content was confirmed in 30 μl per sample by measuring the absorbance of 295 nm using a Bio-Rad Protein Assay (Bio-Rad Protein Assay). 30 [mu] g of the sample was isolated on two 12% SDS-polyacrylamide gels (30% acrylamide, 1% bis, 1 M Tris, 10% SDS, 10% APS, TEMED) in the same manner. One gel was stained with Coomassieblue, and the other gel was nitrocellulose for 1 hour at 90 V in transfer buffer containing 48 mM Tris, 39 mM glycine, 20% methanol and 0.037% SDS for electrophoretic protein. Transferred to the membrane. The nitrocellulose membrane to which the protein was transferred was then treated with blocking solution (TRIS buffer containing 0.1% (v / v) Tween 20 and 6% (w / v) skim milk powder) to reduce nonspecific binding. . Protein Extracted Immune Responses: Rabbit Polyclonal IgG Bax, Bcl-2, Cytochrome C, Caspase-3 and Caspase-9 Antibodies (1: 1000, 24 h) , 4 ° C, Santa Cruz, Cell signaling) or rabbit-derived anti-mouse Bax, Bcl-2, cytochrome C, kephas-3 and kephas-9 antibodies (1: 1000, Abcam Limited, UK) It was. After washing, Horseradish peroxidase binding goat anti-mouse and goat anti-rabbit IgG HRP (1: 10000, Bio-Rad) were added and incubated for 1 hour at room temperature. Proteins were measured by chemiluminescence using ECL-sensing reagent (Western blotting detection reagent, Amersham Pharmacia Biotech). Western blots were analyzed by densitometry using a computer-based Sigma Gel (SPSS Inc. Chicago, USA) system. Concentrations are expressed as ± standard error. One-way ANOVA analysis according to the Tukey-Kramer multiple-comparisons test was performed to determine the significant differences between the relevant groups.

Example 4. MTT Assay

Primary neurons in exponential growth phase were taken for growth analysis using MTT [3- (4,5-dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide]. MTT experiments were performed to determine the toxicity of ethanol and PTZ on cortex and hippocampus following ethanol and PTZ exposure. Hippocampal derived neurons (1 × 10 ⁵ cells / well) were cultured in 96-well plates, ethanol; PTZ; Vitamin C; Ethanol and vitamin C; PTZ and vitamin C; Treated with ethanol, PTZ and vitamin C. MTT solution was added to each well so that the final MTT concentration was 0.5 mg / ml and incubated at 37 ° C. for 4 hours. Formazan dissolved in an organic solvent was added and stirred for 10 to 20 minutes. Plates were measured by scanning at 550-570 nm (L1) and 620-650 nm (L2) with a microplate reader spectropolarizer: 620-650 nm absorbance results are cell suspension and well defects measured. Final Optical Density (OD) was obtained using the formula OD = L1-L2 and used to calculate cell viability, expressed as (absorbance of treated wells / absorbance of control wells) x 100.

Example 5. Mitochondrial Membrane Potential Measurement Using JC-1

In order to measure mitochondrial membrane potential, JC-1 mitochondrial membrane potential detection kit (Biotium Inc., Hayward, CA, USA) was used according to the manufacturer's protocol. JC-1 emits green or red fluorescence depending on the mitochondrial membrane potential: the green signal represents depolarized mitochondria and the red signal represents polarized mitochondria (Reers et al., 1991).

As such, fluorescence changes from red to green indicate a decrease in mitochondrial membrane potential. Neurons were placed in cell culture plates precoated with poly-lysine (0.02 g / l) and chamber slides (1 × 10 ⁶ cells / ml). Medium contains Dulbecco's modified Eagle medium (DMEM), 10% heat-inactivated fetal bovine serum, 1 mM pyruvate, 4.2 mM sodium hydrocarbon, 20 mM HEPES, 0.3 g / L bovine serum albumin, 50 U / ml penicillin and 50 mg / l streptomycin. The culture was carried out at a temperature of 5% CO ₂ , 95% humidity, 37 ℃. After 3 days, cortical and hippocampal neurons were normal medium (control C); Ethanol 100 mM (E) containing medium; Medium containing PTZ 20 mM (PTZ); Medium containing Vitamin C 1 mM (Vit-C); Medium containing 100 mM ethanol and 1 mM vitamin C (E + Vit-C); PTZ 20 mM and vitamin C 1 mM (PTZ + Vit-C) containing medium. All drug treatment groups were incubated at 37 ° C. for 24 hours in in vitro culture. Drug-treated cells were collected from three culture plates, reacted for 15 minutes at 37 ° C using JC-1, and resuspended twice with 1X assay buffer. Changes in mitochondrial membrane potential were measured at the single cell level by the FACS Calibur flow cytometer (Becton Dickinson, San Jose, Calif., USA) under the following conditions (FIG. 6B).

FL1, 511 volts; FL2, 389 volts; FL1-10.5% FL2; FL2-25.9% FL1; 488 nm argon electron laser and 585 nm band pass filter. A total of 10,000 cells were analyzed by CellQuest software, version 3.0 (Becton Dickinson, San Jose, Calif., USA), and the quantification of cells with low mitochondrial membrane potential, such as total cell rate.

Example 6 Determination of Mitochondrial Cytochrome C Release and Kephase-3 Activity

Intracellular analysis of free and caspase-3 expression of cytochrome C was performed by immunofluorescence technique. Primary cultured hippocampal neurons were fixed with 4% paraformaldehyde (PFA), then treated with ethanol, PTZ and combinations thereof and washed with PBS. Cytochrome C was reacted overnight at 4 ° C. with an anticytochrome C antibody (1: 250, Santa Cruz Biotechnology, CA, USA) and rabbit anti-mouse FITC labeled antibody (1: 100, Santa Cruz Biotechnology, CA, USA) It was confirmed by reacting for 90 minutes at room temperature. Expression of Kespase-3 was reacted overnight at 4 ° C. with a rabbit anti-Cespase-3 antibody (1: 250, Cell signaling) and goat anti-rabbit TRITC labeled antibody (1: 100, Santa Cruz Biotechnology, CA, USA) and reacted for 90 minutes under dark conditions at room temperature. Glass cover slips were fixed on glass slides with an encapsulant (Molecular Probes, Eugene, OR, USA). Staining patterns of cytochrome C (green) and caspase-3 (red) were shown using confocal microscopy (Fluoview FV 1000, Olympus, Japan).

Example 7. Data Analysis and Statistics

Western blot bands were analyzed by scanning with a concentration meter using a computer based on the Sigma Gel system (SPSS Inc, Chicago, IL). Concentrations are expressed as ± standard error. One-way ANOVA according to the Tukey-Kramer multi-comparison test was performed to determine the significant differences between the groups. In all cases, the acceptance value for statistical significance is when * p is 0.05 and ** p is less than 0.01 compared to the control.

result

Effect of Vitamin C on the Survival Rate of Hippocampal Neurons

MTT analysis was performed to investigate the effect of treatment of 100 mM ethanol and 20 mM PTZ on the survival of hippocampal neurons, as shown in FIG. 1. As a result, as shown in Fig. 1, strong detrimental effects were confirmed at a concentration of 100 mM or more of ethanol, and the hippocampal neurons were reduced in survival after 24 hours of culture by PTZ and ethanol.

In addition, 1 mM of vitamin C, 20 mM of PTZ, and 100 mM of ethanol, respectively, were treated with the cells for 24 hours, respectively. As a result, 1 mM of vitamins compared to 40.5 ± 2.0% of cell survival when only ethanol was treated. Treatment with C alone showed a significantly higher survival rate of 90.2 ± 2.5%, and treatment with 100 mM ethanol and vitamin C showed 87.8 ± 2.2% survival (FIG. 1). These results indicate that vitamin C has an effect of protecting neurons from ethanol-induced neuronal toxicity.

In addition, compared to 44.5 ± 2.5% cell survival rate when only PTZ treatment, 1 mM of vitamin C treatment alone showed a significantly higher survival rate, when combined with 20 mM PTZ and vitamin C 88.8 ± 1.7 Survival of% is shown (FIG. 1). These results indicate that vitamin C has the effect of protecting neurons from neuronal toxicity induced by PTZ.

Effect of Vitamin C on Bcl-2 Protein Expression

In order to confirm the effect of vitamin C on Bcl-2 protein expression associated with apoptosis and programmed cell death, the amount of Bcl-2 after ethanol and PTZ treatment in neurons was shown in FIG. 2A. . As a result, the expression level of Bcl-2 in cells treated with ethanol and PTZ was significantly reduced compared to cells treated with vitamin C.

In addition, when vitamin C was treated with ethanol and PTZ, the decreased expression level of Bcl-2 was significantly increased. In addition, when treated with vitamin C, ethanol and PTZ, the expression level of Bcl-2 was significantly increased compared to the cells treated with ethanol and PTZ (Fig. 2a). These results indicate that vitamin C increases the expression of Bcl-2, which inhibits apoptosis, thereby preventing apoptosis of neurons induced by ethanol or PTZ.

Effect of Vitamin C on Bax Protein Expression

In order to confirm the effect of vitamin C on the expression of Bax protein that induces apoptosis, the amount of Bax after the treatment of ethanol and PTZ in neurons is shown in FIG. 2b. As a result, Bax expression in cells treated with ethanol and PTZ was significantly increased compared to cells treated with vitamin C.

In addition, when vitamin C was treated with ethanol and PTZ, it was confirmed that the expression of increased Bax significantly decreased. In addition, when treated with vitamin C, ethanol and PTZ, Bax expression was significantly reduced compared to the cells treated with ethanol and PTZ (Fig. 2b). These results indicate that vitamin C reduces the expression of Bax, which induces apoptosis, thereby preventing apoptosis of neurons induced by ethanol or PTZ.

Effect of Vitamin C on Cytochrome C Protein Expression

To determine the effect of vitamin C on cytochrome C protein expression, which activates the cephase-9, which is known to be involved early in the apoptotic process associated with mitochondria, the cytochrome C activity of ethanol and PTZ in neurons The amount was confirmed and shown in FIG. 3.

As a result, as shown in Figure 3, the expression of cytochrome C significantly increased in the cells treated with ethanol and PTZ compared to the cells treated with vitamin C. However, when vitamin C was treated with ethanol or PTZ, the expression level of cytochrome C was significantly decreased. In addition, when the vitamin C, ethanol and PTZ were all treated, the expression level of cytochrome C was significantly decreased compared to the cells treated with ethanol and PTZ.

These results indicate that vitamin C can prevent the apoptosis of neurons induced by ethanol or PTZ by reducing the expression of cytochrome C induced in the process of apoptosis.

Effect of Vitamin C on Keasephase-9 Protein Expression

In order to confirm the effect of vitamin C on the expression of the kease phase-9 protein, which is a mediator of apoptosis in mammalian cells, the amount of the kease phase-9 when ethanol and PTZ were treated to neurons was shown in FIG. 4. It was.

As a result, as shown in Figure 4, in the ethanol and PTZ treated cells compared with the vitamin C-treated cells, the expression level of kephase-9 significantly increased. However, when vitamin C was treated with ethanol and PTZ, the expression level of kephas-9 was significantly decreased. In addition, when all the vitamin C, ethanol and PTZ were treated, the expression level of kephase-9 was significantly reduced compared to the cells treated with ethanol and PTZ.

These results indicate that vitamin C can reduce the expression of kephase-9 induced by apoptosis, thereby preventing apoptosis of neurons caused by ethanol or PTZ.

Effect of Vitamin C on Kease Phase-3 Protein Expression

To determine the effect of vitamin C on the expression of the kease phase-3 protein, which acts as a whole during the early stage of apoptosis, induces cell death. The amount was confirmed and shown in FIG. 5.

As a result, as shown in Figure 5, in the ethanol and PTZ treated cells compared to the vitamin C-treated cells, the expression level of kephase-3 significantly increased. However, when vitamin C was treated with ethanol and PTZ, it was confirmed that the expression level of kephase-3 was significantly decreased. In addition, when all of vitamin C, ethanol and PTZ were treated, the expression level of kephase-3 was significantly decreased compared to the cells treated with ethanol and PTZ.

These results indicate that vitamin C can reduce the expression of kephase-3 induced in apoptosis, thereby preventing apoptosis of neurons induced by ethanol or PTZ.

Effect of Vitamin C on the Release of Cytochrome C and the Activity of Kephas-3

The present invention was carried out by confirming the mode of mitochondrial membrane potential and the mode of sitechrome C released into the cytoplasm from the inside of the mitochondria by the activity of kepha phase-3 associated with ethanol and PTZ-induced apoptosis by vitamin C. The neuronal protective effect of vitamin C was confirmed. To this end, after treatment with ethanol and PTZ to the neuron and immunofluorescence staining, it was confirmed by confocal microscopy and flow cytometry (shown in Figure 6).

As a result, as shown in Figure 6a, when the ethanol and PTZ treated neurons, the amount of cytoplasmic cytochrome C (FITC, green) and Keze phase-3 (TRITC, red) increased compared to the control, The addition of vitamin C significantly reduced the expression of cytoplasmic cytochrome C and kephas-3.

In addition, as shown in FIG. 6B, the mitochondrial membrane potential was measured when the ethanol and PTZ were treated using the JC-1 fluorescent dye. As a result, the mitochondrial membrane potential was decreased to increase the FL1 fluorescence inside the mitochondria. When treated together, the mitochondrial membrane potential was repaired again, showing similar results as the control group.

These results suggest that vitamin C exhibits neuronal protective effects against neuronal toxicity induced by ethanol or PTZ.

Claims (8)

1. A composition for protecting neurons in the fetus in a mother exposed to alcohol or pentylenetetrazol (PTZ) containing vitamin C.
2. The composition of claim 1, wherein the mother is an early pregnancy.
3. The composition of claim 1, wherein the composition is used for the prevention or treatment of alcohol related neurodevelopmental disorders (ARNDS) disease or fetal alcohol syndrome (FAS).
4. The composition of claim 1, wherein the neuron is a hippocampal neuronal cell.
5. The composition of claim 1, wherein the composition protects fetal neurons by performing any one or more of the following actions.

   (a) increased expression of Bcl-2 protein reduced by alcohol or PTZ;

   (b) decreased expression of Bax protein increased by alcohol or PTZ;

   (c) reduced release of cytochrome-c released into the cytoplasm by alcohol or PTZ;

   (d) decreased expression of caspase-9 increased by alcohol or PTZ;

   (e) decreased expression of caspase-3 increased by alcohol or PTZ; And

   (f) recovery of mitochondrial membrane potential increased by alcohol or PTZ.
6. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
7. Health functional food comprising the composition of any one of claims 1 to 5.
8. A method of protecting neurons of a fetus in a mother, comprising administering the composition of any one of claims 1 to 6 to a mother or a fetus exposed to alcohol or PTZ.

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