KR101974717B1 - Composition comprising Connexin 43 as an effective ingredient for preventing or treating of neurological disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cranial nerve disease using Connexin 43 as an active ingredient, and a health functional food for improvement.

Description

[0001] The present invention relates to a composition for preventing or treating cranial nerve disease, which comprises connexin 43 as an active ingredient,

TECHNICAL FIELD The present invention relates to a composition for preventing or treating cranial nerve disease using cornexin 43 as an active ingredient, and a health functional food for improvement.

Apoptosis, a type of cell death, plays an important role in early development and growth of normal adult tissues. In addition, the apoptosis is a common phenomenon during the normal development of the central nervous system, and plays an important role in organ formation and synapse formation of the central nervous system (CNS). However, survival of neurons in the adult brain is essential for the normal function of the central nervous system during a lifetime. Excessive nerve damage or neuronal cell death occurs in many disease states or in an injured patient. In particular, nerve cell loss or death is most common in central nervous system related diseases such as stroke, Alzheimer's disease, Parkinson's disease and Huntington's disease.

Parkinson's disease is one of the second most common neurodegenerative diseases following Alzheimer's disease. Parkinson's disease is characterized by instability, stiffness, tremors during rest, and postural instability, and progresses slowly without definite pathogenesis. The pathological features of Parkinson's disease include the death of the dopaminergic neurons (SN) of the substantia nigra in viable neurons and the presence of Lewy bodies (ubiquitin positive, eosinophilic and β-synuclein including cytoplasmic inclusions). A recent etiological study of twins strongly suggests that environmental factors play an important role in typical non-familial Parkinson's patients aged 50 years and older. Environmental factors such as life in rural areas, well water intake and exposure to chemicals (pesticides, herbicides, industrial chemicals and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) the risk is increased. in order to study the neurological degeneration in Parkinson's disease, active metabolite neurotoxin, 1-methyl-4-phenylpyridinium (MPP +) in the MPTP has been widely used. the MPP ⁺ is cellular and animal models All selectively inhibit the complex I complex of mitochondrial electron chains, leading to severe Parkinson-like syndrome with loss of dopaminergic cells.

Gap junctional intercellular communication (GJIC) modulation changes can affect cell growth and viability of neurons. The homeostasis of various organs, including the brain, is influenced by GJIC, which can effectively control the pH, glutamate and K ⁺ of neurons in the brain. Connexin 43 is an important member of the gap-junction protein family that is widely expressed in various tissues and organs of vertebrate animals including humans. It is an important member of the gap-junction protein family including various cell functions such as cell growth and differentiation, .

The present invention is intended to provide a pharmaceutical composition for preventing or treating cranial nerve diseases containing Connexin 43 (Cx43) as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for the prophylaxis or treatment of cranial nerve diseases containing Connexin 43 as an active ingredient.

The composition can prevent neuronal cell death from neuroblastoma induced by MPP ⁺ (1-Methyl-4-Phenylpyridine).

The death of the neuronal cells may be due to a mitochondria-dependent pathway.

The above composition shows that the death of the neuronal cells in the mitochondria results in the release of Cytochrome C, activity of caspase-3, or activity of PARP protein hydrolysis (poly ADP ribose polymerase proteolysis) in the mitochondria .

The composition may increase the expression of Bax or Bcl-2 (B cell lymphoma 2) mRNA.

The cranial nerve diseases can be selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt Jakob disease, stroke, multiple sclerosis, learning disorder, cognitive disorder and memory impairment.

In one embodiment of the present invention, there is provided a health functional food for preventing or ameliorating a brain disease containing Connexin 43 as an active ingredient.

The present invention relates to a composition for the prophylaxis or treatment of cranial nerve diseases, which comprises connexin 43 as an active ingredient, wherein said connexin 43 is a neuroblastoma derived from MPP ⁺ And can be used for preventing or treating cranial nerve diseases.

FIG. 1 (a) shows the results of RT-PCR of the level of connexin 43 in SH-SY5Y cells treated with MPP ⁺ for 0 to 24 hours. 1 (b), 1 (C) and 1 (d) are the results of immunoblot analysis of SH-SY5Y cells treated with MPP ⁺ 1 mM, H ₂ O ₂ 100 μM and 6-OHDA 25 μM, (e) is a graph showing the ratio of connexin 43 protein /? -actin. 1 (f) shows cell viability in SH-SY5Y cells treated with toxin for 0 to 48 hours.
Figure 2 shows the effect of connexin 43 on apoptosis in human SH-SY5Y cells. FIG. 2 (a) is a Western blot graph showing Cx43 protein expression levels in shRNA-control, shRNA-Cx43 control-vector or overexpression-Cx43 SH-SY5Y cells. FIG. 2 (b) shows cell viability after shRNA-control, shRNA-Cx43 control-vector or overexpression-Cx43 SH-SY5Y cells treated with 0.5 mM to 1 mM of MPP + for 48 hours. FIG. 3 (c) shows FACs analysis results of PI-staining of SH-SY5Y cells expressing shRNA-control, shRNA-Cx43 control-vector or overexpression-Cx43 after MPP ⁺ 1 mM for 60 hours, 2 (d) is a graph that quantifies the sub-G0-G1 apoptotic population (a: control cells, b: MPP ⁺ 1 mM treated cells, c: MPP ⁺ 1 mM overexpression-Cx43).
Figure 3 shows the effect of overexpression of conexin 43 on MMP loss inhibition in MPP ⁺ induced cell death.
Figure 4 shows the effect of connexin 43 on the overexpression of the Bcl-2 family in SH-SY5Y cells. FIG. 4 (a) is a graph showing the results of treatment of Bax, Bcl-2 and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) after MPP ⁺ 1 mM for 24 hours in Cx43-knockdown (shRNA) and Cx43- 4 (b) shows the results of RT-PCR. After the cells were treated with MPP ⁺ 1 mM for 48 hours, Cx43-knockdown (shRNA) and Cx43-expressing (Cx43) SH- And GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). 4 (c) shows the ratio of Bax / Bcl-2 mRNA after treating MPX ⁺ 1 mM for 24 hours with Cx43-knockdown (shRNA) and Cx43-expressing (Cx43) SH- d) represents the ratio of Bax / Bcl-2 protein.
Figure 5 shows the effect of connexin 43 on mitochondrial regulation of the cell death pathway in SH-SY5Y cells. 5 (a) shows Western blot results of cytosolic cytochrome C after treatment of Cx43-knockdown (shRNA) and Cx43-expressing (Cx43) SH-SY5Y cells with MPP ⁺ 1 mM for 24 hours, ) Shows Western blot results of PARP level. FIG. 5 (c) shows Casoase-3 activity measured by the colorimetric assay kit (Sigma, CASP3P), and FIG. 5 (d) is a graph quantifying the PARP level.

The present inventors have completed the present invention by confirming whether there is a change in the level of expression of connexin 43 during apoptosis in MPP ⁺ induced neuroblasts and the change in the level of connexin 43 expression in mitochondria-dependent apoptosis.

Hereinafter, the present invention will be described in detail.

A pharmaceutical composition for the prevention or treatment of neurological diseases

The present invention provides a pharmaceutical composition for the prophylaxis or treatment of cranial nerve diseases containing Connexin 43 as an active ingredient.

The composition can inhibit neuronal cell death in neuroblastomas induced by MPP ⁺ (1-Methyl-4-Phenylpyridine). According to one embodiment of the present invention, when MPP ⁺ is treated with the overexpressed transgenic cells of connexin 43, it can be confirmed that the cell viability is significantly increased as compared with the connexin 43 knockdown transgenic cells (see FIG. 2 b)).

The death of the neuronal cells may be due to a mitochondria-dependent pathway.

Specifically, the destruction of the transmembrane potential is one of the early changes in mitochondrial-mediated cell death, and the destruction of the mitochondrial membrane potential implies the death of MPP ⁺ -induced neuronal cells. In one embodiment of the present invention, mitochondrial membrane potential, which is an index of mitochondrial activity, was measured in order to confirm MPP ⁺ -induced neuronal apoptosis of mitochondrial function of conexin 43. As a result, as shown in FIG. 3, when MPP ⁺ 1mM was treated with connexin 43 knockdown transfected cells, the membrane potential of mitochondria decreased, but when MPP ⁺ 1mM was treated to transgenic cells overexpressing connexin 43, And the mitochondrial membrane potential was significantly increased.

The composition may inhibit release of cytochrome C, activity of caspase-3, or PARP protein hydrolysis in the mitochondria.

Mitochondria play an important role in the regulation of apoptosis. Specifically, cytochrome C released from mitochondria activates caspases in the cytoplasm and causes changes in the membrane potential loss of mitochondria. Caspase-3 is a crucial biomarker for neuronal cell death and plays a role as an apoptosis index. Connexin 43 according to the present invention is a cell- By controlling mitochondria-dependent apoptosis pathway in MPP ⁺ -induced neuroblastoma, it can be used for prevention or treatment of neurological diseases by preventing the death of neuronal cells.

The composition may inhibit the expression of Bax mRNA or increase the expression of Bcl-2 (B cell lymphoma 2) mRNA.

Specifically, Bax and Bcl-2 are proteins that play an important role in the membrane permeability of mitochondria. Bax, a pore forming cytoplasmic protein, releases cytochrome c from the intermembrane space where mitochondrial cell apoptosis occurs to the cell matrix Bcl-2, which stabilizes the permeability of the membrane, inhibits cell death by inhibiting the release of cytochrome C, so that mitochondrial cells can be maintained. According to one embodiment of the present invention, the expression ratio of Bax / Bcl-2 mRNA (Fig. 4 (c)) in comparison with connexin 43 knockdown transformed cells when MPP ⁺ was treated with overexpressed transgenic cells of connexin 43, And the expression ratio of Bax / Bcl-2 protein (Fig. 4 (d)) were significantly increased.

Cranial nerve diseases that can be prevented or treated by the compositions of the present invention are selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt Jakob disease, stroke, multiple sclerosis, learning disorders, cognitive disorders, And is preferably, but not limited to, Parkinson's disease.

The dose of connexin 43 that may be contained in the composition of the present invention is preferably from 0.1 mM to 100 mM, but is not limited thereto. When the concentration of the connexin 43 is less than the above range, there is a problem that cell death is not prevented and it is difficult to exert the effect of preventing or treating a brain nerve disease. When the concentration exceeds the above range, toxicity There may be concerns.

As described above, the connexin 43 according to the present invention is a mitochondrial-dependent method such as reducing the membrane potential of the mitochondria, inhibiting the release of cytochrome C in the mitochondria, the activity of caspase-3 or the hydrolysis of the PARP protein Inhibits apoptosis of neuroblastoma, and can be used for the prevention or treatment of neurological diseases.

The pharmaceutical compositions for the prevention or treatment of cerebral diseases according to the present invention may be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

The solid preparation for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in cornexin 43. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for preventing or treating a neurological disease according to the present invention varies depending on the condition of the patient, the body weight, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for preventing or treating brain diseases according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Health functional food for prevention or improvement of cerebral nerve disease

The present invention provides a health functional food for preventing or ameliorating a brain disease containing Connexin 43 as an active ingredient. The details of the conexin 43 are as described above.

In the health functional food for preventing or improving cranial nerves according to the present invention, when the above-mentioned connexin 43 is used as an additive for a health functional food, it can be added as it is or can be used together with other food or food ingredients, It can be used properly. The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

Formulations of health functional foods may be in the form of powders, granules, pills, tablets, capsules, as well as in the form of ordinary foods or beverages.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

Generally, the connexin 43 may be added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight based on 100 parts by weight of the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range. Further, since the present invention uses fractions from natural products, there is no problem in terms of safety, Or more.

The beverage in the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food for preventing or ameliorating a brain disease according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, , Stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. In addition, the composition for improving sleep of the present invention may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Preparation Example ]

Culture plates (six and 96 wells) and culture dishes (100 mm) were incubated in Nunc Inc. (North Aurora Road, Naperville, IL) with Sigma Aldrich (St. Louis, Mo., USA) for MPP + and 5-diphenyl-tetrazolium bromide , IL, USA). Fetal bovine serum (FBS) was purchased from Gibco-BRL Technologies (Rockville, MD, USA) and Propidium iodide (PI) was purchased from BD Clontech (Terra Bella Ave, CA, USA). Antibodies to Connexin 43 (# 3512), Bax (# 2772), Bcl-2 (# 2872), cleaved PARP (# 5625) and β-actin (# 3700) (Boston, MA, USA).

[ Example ]

Example  One. SH - SY5Y  Toxin treatment of cells

1-1) Connexin  43 Protein expression changes

Hydrogen peroxide (H ₂ O ₂ ) and 6-OHDA have strong oxidative properties and can lead to oxidative stress and apoptosis, leading to the production of reactive oxygen species (ROS). Oxidative stress due to an imbalance of aerobic metabolism can pose a serious threat to cellular homeostasis, and high levels of ROS oxidize lipids, proteins and DNA to induce tissue damage and apoptosis. According to recent studies, ROS is deeply involved in the pathophysiology of various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease (PD). In addition, ROS can alter mitochondrial function and mitochondrial permeability transition pore.

SH-SY5Y cells were treated with MPP + 1 mM, H 2 O 2 100 μM and 6-OHDA 25 μM, respectively, and cultured at 5 × 10 ⁴ cells / 96-well pate for 1 to 48 hours. Cells were then washed twice with cold PBS, and 50 or 100 mL of RIPA buffer (fresh protease inhibitor cocktail containing PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) To a SH-SY5Y cell (1 x ¹⁰⁶ cells / mL) cultured in the same manner as in Example 1, thereby obtaining a total cell lysate. The cell lysate (15 μg / lane) was applied to 10% SDS-PAGE. Proteins were transferred to a PVDF membrane and fixed using an electroblotting apparatus (Biorad, Hercules, CA, USA). The membranes were blocked (room temperature) in TBS containing 1% Tween-20 and 5% dry milk for 1 hour, incubated with primary antibody (1: 1000) and horseradish peroxidase (1: 10,000) Conjugated secondary antibody was incubated overnight. The optical density of the antibody-specific band was then analyzed using an Image Analyzer LAS-3000 (Fuji, Japan).

As a result, as shown in Fig. 1, it was confirmed that the expression pattern of connexin 43 protein in MPP ⁺ -treated cells was similar to that of cells treated with H ₂ O ₂ and 6-OHDA (time-dependent decrease) . That is, the decrease in expression of connexin 43 protein is sensitive to MPP ⁺ treatment in the concentration range of 0.5 mM to 1 mM, indicating that connexin 43 plays an important role in MPP ⁺ resistance in SH-SY5Y cells .

1-2) Evaluation of cell viability

Cell viability was measured using MTT assay as is well known. First, SH-SY5Y cells treated with the respective toxins in Example 1-1 were cultured at 5 × 10 ⁴ cells / 96-well pate, and MTT (0.5 mg / mL) dissolved in PBS (phosphate-buffered saline) / Ml) was added. Crystals formed by viable cells were measured at a wavelength of 550 nm using a microplate reader (Molecular device, Sunnyvale, Calif., USA).

As a result, as shown in Fig. 1 (f), it was confirmed that the viability of cells treated with each toxin was reduced by about 50% as compared with the control.

Example  2. Connexin  43 Production of expression plasmids

In order to construct a recombinant vector of pcDNA3.1-conexin43, the human consexin 43 cDNA open reading frame (ORF) was amplified using the forward primer 5'-TGGCTAGCACATGGGTGACTGGA-3 'and the reverse primer 5'-ACCTGGAGATCTAGATGGATCC-3' . Two restriction sites NheI and BamHI were added at both ends of the gene, respectively. Expression vector Connexin 43 gene was cloned into the EcoRI and BamHI restriction enzyme sites of pcDNA3.1.

Example  3. Cell culture and transfection ( transfection )

To determine whether MPP ⁺ resistance is mediated by connexin 43, edogenous connexin 43 in SH-SY5Y was knocked down to connexin 43-shRNA and shRNA vector was used as a control

(V / v) FBS and 100 U / mL penicillin / streptomycin were inactivated in DMEM-cultured human dopaminergic SH-SY5Y cells and cultured in a 37 ° C incubator with 5% CO ₂ and 95% humidified air Lt; / RTI > Connexin 43 shRNA plasmids and control shRNA plasmids were obtained from Santa Cruz Biotechnology. The cells were diluted in nuclease free water to a final concentration of 10 μM and the plasmid (pcDNA 3.1-connexin 43 recombinant vector, connexin 43 shRNA plasmid) was transfected using a Transfection Reagent (Lipofectamine 2000, Invirogen, Carlsbad, CA, USA) And transfected into SH-SY5Y cells at a concentration of 15 nM. After 48 hours of transfection, subsequent measurements were performed.

Example  4. Evaluation of viability of transformed cells

The cells were treated with MTT assay in the same manner as in Example 1-2 except that SH-SY5Y cells were treated at 5 × 10 ⁴ cells / 96-well pate for 48 hours with MPP ⁺ 0.5 mM to 1 mM Respectively.

As a result, as shown in Fig. 2 (b), in the case of SH-SY5Y in which connexin 43 was overexpressed, it was confirmed that the survival rate was significantly increased as compared with that in 43-knockdown cells of connexin. In addition, the protective role of connexin 43 in MPP ⁺ inducible apoptosis was confirmed by PI staining. As shown in Fig. 2 (c), when a cell was incubated in a medium, a typical single peak having diploid DNA content 2 ~ 3%, and a characteristic low-level epileptic DNA content peak indicating sub-G0-G1 apoptotic populations was detected. As shown in FIG. 2 (d), it was confirmed that the sub G0-G1 population ratio was significantly reduced in the case of the overexpressed cell (c) treated with MPP ⁺ 1 mM compared to the positive control (b).

Example  5. Total RNA isolation and expression analysis

Total RNA was extracted from SH-SY5Y cells (1 × 10 ⁶ cells / well) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) To perform RT-PCR, 2.5 μg of total RNA was reverse-transcribed using First-Strand cDNA synthesis kit (Invitrogen). PCR was performed using the cDNA prepared above as a template, and the primers used were shown in Table 1 below.

sense Anti-sense Cx43 5'-CGCTCCCCTCTCGCCTATGT-3 ' 5'-CTGGCACGACTGCTGGCTCT-3 ' Bcl-2 5'-ACGACTTCTCCCGCCGCTAC-3 ' 5'-CCCAGCCTCCGTTATCCTGG-3 ' Bax 5'-CACCAAGGTGCCGGAACTGA-3 ' 5'-AATGCCCATGTCCCCCAATC-3 ' GAPDH 5'-GCAGTGGCAAAGTGGAGATTG-3 ' 5'-TGCAGGATGCATTGCTGACA-3 '

Internal control GAPDH was used to assess the relative expression levels of Bcl-2 and Bax. PCR product was analyzed on 1% agarose gel.

To measure the expression of connexin 43 in MPP ⁺ -induced apoptotic SH-SY5Y cells, expression of connexin 43 during MPP ⁺ treatment time was analyzed. As a result, it was confirmed that the expression of connexin 43 was decreased in SH-SY5Y cells induced by MPP + as shown in Fig. 1 (a). Further, as shown in Fig. 4 (a)

As a result, as shown in Figs. 1 (e) and 1 (f), H ₂ O ₂ And SH-SY5Y cells induced by MPP ⁺ similar to 6-OHDA (Fig. 1 (f)), and the expression of connexin 43 protein was decreased in a time-dependent manner (Fig. 1 (e)).

Example  6. Immunoblot  분석

Cells were washed twice with cold PBS and cultured in a 6-well plate in 50 or 100 mL RIPA buffer (fresh protease inhibitor cocktail containing PBS, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) (1 x 10 < ⁶ > cells / mL), thereby obtaining total cell lysate. The cell lysate (15 μg / lane) was applied to 10% SDS-PAGE. Proteins were transferred to a PVDF membrane and fixed using an electroblotting apparatus (Biorad, Hercules, CA, USA). The membranes were blocked (room temperature) in TBS containing 1% Tween-20 and 5% dry milk for 1 hour, incubated with primary antibody (1: 1000) and horseradish peroxidase (1: 10,000) Conjugated secondary antibody was incubated overnight. The optical density of the antibody-specific band was then analyzed using an Image Analyzer LAS-3000 (Fuji, Japan).

As a result, as shown in Fig. 4, the expression of Bcl-2 was high in connexin 43 overexpressed cells treated with MPP ⁺ , and the expression of Bax was low. The ratio of Bax / Bcl-2 to Bax / Bcl-2 was investigated, and as a result, the Bax / Bcl-2 ratio was found to be higher than that of connexin 43 overexpressed transformed cells Respectively. That is, connexin 43 can be used for the prevention or treatment of neurological diseases by inhibiting apoptosis through a mitochondria-dependent pathway.

Example  7. Caspase -3 Activity Assay

To better understand the effect of connexin 43 on caspase-3 activity as well as MPP ⁺ inducible apoptosis and cytochrome C release, connexin 43 knockdown cells and connexin 43 over-expressing cells were incubated with MPP ⁺ 1 mM for 48 hours Respectively. Caspase-3 activity was measured using a Colorimetric Caspase-3 Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. Capase-3 activity assay was measured as known. The reaction mixture (total volume, 200 μl) was run on a 96-well plate. After incubation of the mixture at 37 캜 for 90 minutes, release and absorbance values were measured at wavelengths of 360 and 460 nm, respectively.

As a result, as shown in FIG. 5, it was confirmed that the cytochrome C loss and the caspase-3 activity were significantly increased in the mitochondria in the case of the connexin 43 knockdown transformed cells. On the other hand, the level of cytochrome c in the cytoplasm was lower in the case of the transgenic cells expressing connexin 43. That is, the expression of connexin 43 in SH-SY5Y cells inhibits apoptotic MPP ⁺ induced cell death by regulating the mitochondrial apoptotic pathway by inhibiting the loss of cytochrome C from the mitochondria to the cytoplasm and inhibiting caspase-3 activity .

Example  8. Apoptotic  Flow of cell Cytometric  Detection

SH-SY5Y cells (1 × 10 ⁶ cells / well) were washed twice with cold BPS, exposed to MPP + for 60 hours, and then centrifuged. Cell pellets were resuspended in cold 70% ethanol, fixed at 4 ° C for 24-48 hours, and then washed with PBS. The cells were then resuspensioned with DNA staining reagent containing 50 μg / mL RNase, 0.1% Triton X-100, 0.1 mM EDTA (pH 7.4), and 50 μg / mL PI. DNA staining was carried out at 4 ° C for 30 minutes. Red fluorescence (DNA) was detected with a 563-607 nm band pass filter using a FACS Caliber flow cytometer (Becton Dickinson, San Jose, Calif., USA). Ten thousand cells were analyzed in each sample. The percentage of apoptotic cells accumulating at the sub-G1 peak was calculated using the Cell Quest software (Becton Dickinson, San Jose, Calif., USA).

Example  9. Mitochondrial  Membrane Potential ( DYm ) Measurement

JC-1 was used to analyze the mitochondrial transmembrane potential. The retention capacity of JC-1 dye correlates with the mitochondrial transmembrane potential. After MPP ⁺ treatment, the cells were incubated with DMEM containing 5 μl of JC-1 for 15 min at 37 ° C. Then, it was washed with 200 μl of assay buffer, and added to 100 μl of assay buffer. OD values were measured in a Microplate Reader (Tecan, Meilen, Zurich, Switzerland) at wavelengths of 485 and 535 nm, respectively, and then fluorescent densities were calculated.

As a result, it was confirmed that mitochondrial membrane potential was remarkably decreased when MPP ⁺ 1 mM was treated with control cell and knockdown cells of connexin 43. However, when the cells overexpressing connexin 43 were treated with MPP ⁺ , it was confirmed that the mitochondrial membrane potential was remarkably increased.

Example  10. Statistical analysis

All measurements were performed three times and repeated three times using different samples, the results being expressed as mean ± SEM. Statistical significance was determined by Dunnett's t-test for variance analysis (ANOVA) and multiple comparisons. P values less than 0.05 were considered statistically significant.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (12)

A pharmaceutical composition for preventing or treating Parkinson's disease containing Connexin 43 as an active ingredient.
The method according to claim 1,
The pharmaceutical composition for preventing or treating Parkinson's disease, wherein the connexin 43 prevents neuronal cell death from MPP ⁺ (1-Methyl-4-phenylpyridine) -mediated neuroblastoma.
3. The method of claim 2,
Wherein the neuronal cell death is caused by a mitochondria-dependent pathway.
delete The method according to claim 1,
Wherein the connexin 43 increases the expression of Bax or Bcl-2 (B cell lymphoma 2) mRNA.
delete A health functional food for preventing or improving Parkinson's disease containing Connexin 43 as an active ingredient.
8. The method of claim 7,
Wherein said connexin 43 prevents neuronal cell death from neuroblastoma induced by MPP ⁺ (1-Methyl-4-phenylpyridine).
9. The method of claim 8,
Wherein said nerve cell death is caused by a mitochondria-dependent pathway.
8. The method of claim 7,
Wherein said connexin 43 inhibits the release of cytochrome C, activity of caspase-3, or PARP protein hydrolysis in mitochondria. Health functional foods for prevention or improvement.
8. The method of claim 7,
Wherein the connexin 43 increases the expression of Bax or Bcl-2 (B cell lymphoma 2) mRNA. delete

Images