KR20090107607A - Pharmaceutical Composition For Prevention Or Treatment Of Neurodegenerative Diseases Comprising Fisetin - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing a fisetin is provided to prevent or treat neurodegenerative diseases such as Alzheimer disease, Parkison's disease, and multiple sclerosis. CONSTITUTION: A pharmaceutical composition for preventing and treating neurodegenerative diseases contains a fisetin and pharmaceutically acceptable carrier. The pharmaceutical composition for treating neurodegenerative diseases suppresses the activation of microglia. The carrier is selected from vaseline, lanoline, polyethylene glycol, alcohol and their combination.

Description

Pharmaceutical Composition For Prevention Or Treatment Of Neurodegenerative Diseases Comprising Fisetin}

The present invention relates to a pharmaceutical composition for the prevention and treatment of degenerative neurological diseases containing picetin and a pharmaceutically acceptable carrier to inhibit the activity of microglia.

Degenerative neuropathy is a disease caused by the degeneration of neuronal cells, including Alzheimer's disease (AD), Parkinson's disease (PD), HIV dementia (HIV-associated dementia), stroke and multiple sclerosis. Etc. In order to treat such neurological diseases, pharmaceutical compositions using polyphenol-based compounds have been developed as therapeutic agents. Flavonoids are a kind of the above polyphenols and are used in various fields because they have biologically useful effects including antioxidant activity, anti-inflammatory action and anticancer action. Fisetin, a type of flavonoid, also has anticancer, anti-angiogenic, neuroprotective, neurotrophic, and antioxidant properties, found in strawberries and other fruits and vegetables. Recently, another effect of cetinine has been shown to be anti-inflammatory in macrophages and mast cells. However, until now, there has been no research on the fact that phycetin can prevent and treat neurodegenerative diseases by inhibiting the activity of microglia.

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating neurodegenerative diseases which contain cecetin and a pharmaceutically acceptable carrier and inhibit the activity of microglia.

The pharmaceutical composition according to the present invention for achieving the above object is characterized in that it contains a picetin and a pharmaceutically acceptable carrier, to inhibit the activity of microglia to prevent and treat neurodegenerative diseases.

Preferably, the carrier is selected from the group consisting of petrolatum, lanolin, polyethylene glycol, alcohol, and combinations thereof.

Preferably, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, HIV dementia, stroke and multiple sclerosis.

Preferably, the above-mentioned phycetin inhibits the secretion of nitric oxide (NO), TNF-α (TNF-alpha) and PGE ₂ (prostaglandin 2) in microglia to prevent and treat neurodegenerative diseases. .

Preferably, the above-mentioned phycetin inhibits gene expression of iNOS to prevent and treat neurodegenerative diseases.

Preferably, the above-mentioned phycetin inhibits the gene expression of TNF-alpha (TNF-α) to prevent and treat neurodegenerative diseases.

Preferably, the above-mentioned phycetin inhibits the gene expression of COX-2 to prevent and treat neurodegenerative diseases.

Preferably, the above-mentioned phycetin inhibits the activities of P38 MAPK and NF-κB to prevent and treat neurodegenerative diseases.

Preferably, the above-mentioned phycetin inhibits the degradation of IκB to prevent and treat neurodegenerative diseases.

Preferably, the above-mentioned phycetin inhibits the migration of NF-κB p65 into the nucleus, thereby preventing and treating neurodegenerative diseases.

Preferably, the above-mentioned phycetin inhibits neuronal cell death due to activation of microglia to prevent and treat neurodegenerative diseases.

As described below below, the pharmaceutical composition according to the present invention contains picetin and a pharmaceutically acceptable carrier, thereby preventing and treating neurodegenerative diseases that inhibit the activity of microglia.

Microglial activity plays an important role in the development and development of degenerative brain diseases. When microglial cells are activated, they secrete proinflammatory inflammatory factors NO (nitrogen monoxide), TNF-α (TNF-alpha) or PGE ₂ (prostaglandin 2), which damage brain cells. Microglial activation activates the expression of iNOS, TNF-α and COX-2 (cyclooxygenase-2) genes to secrete proinflammatory inflammatory factors. To express the iNOS and TNF-α genes, NF-κB and p38 MAPK activities are required.In order to activate NF-κB, the degradation of IκB and NF-κB p65 transfer into the nucleus (nuclear translocation). You have to go through the process.

Since fisetin inhibits the secretion of NO, TNF-α and PGE ₂ due to activation of microglia, it also significantly inhibits the expression of iNOS, TNF-α and COX-2 genes. Compounds containing pacetin can be used to prevent or treat Alzheimer's disease (AD), Parkinson's disease (PD), HIV dementia (HIV-associated dementia), stroke and multiple sclerosis neurodegenerative diseases. Will be. At this time, it may further contain a pharmaceutically acceptable carrier to prepare a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, the carrier includes petrolatum, lanolin, polyethylene glycol, alcohol, and combinations of two or more thereof. do.

Justice

The term iNOS, as used herein, is an abbreviation for inducible Nitric Oxide Synthase, which is an immunogenic nitric oxide synthase.

As used herein, the term COX-2 is an abbreviation for cyclooxygenase-2 and is a prostaglandin E2 synthase.

As used herein, the term TNF-α (TNF-alpha) is an abbreviation for Tumor Necrosis Factor-alpha and is a type of tumor necrosis factor.

The term PGE ₂ as used herein is an abbreviation for prostaglandin E2 and is a type of pro inflammatory factor.

The term NF-κB, as used herein, stands for Nuclear Factor-kappa B. It is composed of p65 and p50 proteins, which are the subunits of NF-κB. Hereinafter, p65 and p50 of NF-κB are represented as NF-κB p65 and NF-κB p50, respectively. NF-κB is a transcription factor of COX-2.

As used herein, the term p38 MAPK is an abbreviation for p38 Mitogen-Activated Protein Kinase and is an enzyme that blocks cell division.

The term IκB, as used herein, stands for Inhibitor-kappa B and is an inhibitor of NF-κB.

Sample and Cell Culture

The bacterial lipopolysaccharide (LPS) (E. coli serotype 055: B5) and fisetin (fisetin) were purchased from Sigma-Aldrich (St. Louis, MO) and prepared. The BV-2 microglia of mice were treated at 37 ° C. and 5% CO ₂ in DMEM (Dulbecco's modified Eagle's medium) medium containing 50 μg / ml of fetal bovine serum (FBS) and gentamicin (10% inactivated). Incubated. Neuroblastoma cells from B35 rats were DMEM (Dulbecco's) with 10% inactivated fetal bovine serum (FBS), penicillin (10 U / ml) and streptomycin (10 µg / ml). cultured in modified Eagle's medium) at 37 ° C., 5% CO ₂ .

For co-culture experiments, BV-2 microglia were cultured in 96-well plates at three times the 1.5 × 10 ⁴ cell count / well concentration. BV-2 microglia were pretreated with 2 μg / ml phycetin for 30 minutes. Discard the cultured supernatant and add 0.5 μg / ml of lipopolysaccharide (LPS) to B35 rat neuroblastoma cells stably displaying EGFP (3 × 10 ⁴ cell counts / well) and co-administer for 24 hours. Incubated.

Experimental Example 1 (Inhibition of NO, TNF-α and PGE ₂ Production)

In order to confirm the anti-inflammatory action of picetin, experiments were conducted to measure nitric oxide (NO), TNF-α and PGE ₂ , which are derived from lipopolysaccharide (LPS) secreted by BV-2 microglia. It was.

Take 50 µl of the sample sample, mix 50 µl of Griess reagent (1% sulfanilamide / 0.1% naphthylethylene diamine dihydrochloride / 2% phosphoric acid) in 96-well plates, and then at 25 ° C. Incubate for 10 minutes. Absorbance was measured with a microplate reader at 550 nm.

To determine the increased NO product in BV-2 microglia, the concentration of nitrogen dioxide ions in the culture supernatant was calculated. The concentration of nitrogen dioxide ions was calculated using the sodium nitrate standard curve. Enzyme-linked immunosorbent assay (ELISA) was used in the culture supernatant to measure the increased TNF-α and PGE ₂ products in BV-2 microglia.

Nitrogen monoxide (NO), TNF-α, and PGE ₂ were significantly increased in the culture medium in which lipopolysaccharide (LPS) (0.1 μg / ml) and BV-2 microglia were reacted for one hour. However, in the case of BV-2 microglia treated with various concentrations of picetin, the concentrations of nitric oxide (NO), TNF-α, and PGE ₂ were decreased while showing the concentration-dependent aspect of the picetin (FIGS. 1A and 1B). , 1c)

Experimental Example 2 (Inhibition of Gene Expression of iNOS, TNF-α and COX-2)

Experiments were conducted to determine whether or not picetin inhibits the expression of iNOS and TNF-α genes. The mRNA levels of iNOS, TNF-α and COX-2 in BV-2 microglia treated with picetin were investigated using a reverse transcriptase polymerase chain reaction (RT-PCR) analyzer. Whole RNA was isolated using the Trizol reagent (Molecular Research Center Inc., Cincinnati, OH) following the instructions in the reagents. Reverse transcription was performed using Moloney murine leukemia virus (M-MLV) and oligo (dT) primers.

P CR amplification was performed for 30 seconds at 94 ° C, 30 seconds at 55 ° C, and 1 minute at 72 ° C. And 23 cycles were repeated and incubated for 7 minutes at 72 ℃. The nucleotide sequences of the primers are based on sequences of published cDNAs of iNOS, TNF-α, COX-2 and β-actin in mice. The sequence in which the iNOS replication proceeds is CCC TTC CGA AGT TTC TGG CAG CAG C and the reverse sequence is GGC TGT CAG AGC CTC GTG GCT TTG G. The sequence in which the TNF-α replication proceeds is CAT CTT CTC AAA ATT CGA GTG ACA A, and the reverse sequence is ACT TGG GCA GAT TGA CCT CAG. The sequence in the direction in which COX-2 replication proceeds is GAA GGG ACA CCC TTT CAC AT, and the reverse sequence is ACA CTC TAT CAC TGG CAT CC. The sequence in which the β-actin replication proceeds is ATC CTG AAA GAC CTC TAT GC, and the reverse sequence is AAC GCA GCT CAG TAA CAG TC. β-actin was used as an internal control to assess the dependence of the expression of iNOS gene and TNF-α gene.

2 shows that iNOS, TNF-α and COX-2 genes were suppressed from BV-2 microglia treated with picetin.

Experimental Example 3 (degradation of IκB and reduced migration of NF-κB p65 into the nucleus)

Immunofluorescence assay was performed to determine if phycetin reduced NF-κB and p38 MAPK activation in BV-2 microglia stimulated with lipopolysaccharide (LPS). In order for NF-κB to be activated, the degradation of IκB and the nuclear translocation of NF-κB p65 must be performed.

Cultured on sterile cover glass of 24-well plates for localization of NF-κB p65 cells in BV-2 microglia (1 × 10 ⁵ cell number / well in 24-wells) It was treated with phycetin and lipopolysaccharide (LPS). After 60 minutes after treatment with the lipopolysaccharide (LPS), the cells were fixed for 20 minutes with methanol (methanol) at -20 ℃, and washed for 5 minutes with PBS (phosphate buffered saline). Fixed cells were infiltrated with 0.5% of Triton X-100 at room temperature in PBS for 1 hour and then washed with 0.05% Tween-20 (Polysorbate-20 Twin-20) in PBS for 10 minutes. Then, washed with PBS 0.05% Tween-20 / 1% BSA (Bovine Serum Albumin) for 5 minutes. Cells infiltrated with the reagent were treated with 1 μg / ml NF-κB p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 60 minutes at room temperature and 5 minutes with 0.05% Tween-20 / 1% BSA in PBS. Washed off. The cells were then diluted 1: 2000 with Alexa Fluor 488-labeled goat anti-mouse antibody (Molecular Probes Inc., Eugene, OR), incubated at room temperature for 60 minutes, followed by 0.05% Tween-20 in PBS. Rinse for 5 minutes and wash again with PBS for 5 minutes. And the cells were washed after staining for 20 minutes at 37 ℃ temperature with 0.5μg / ml Hoechst dye solution. Finally, cover slips containing cells were placed in an oven and dried at 37 ° C. for 45 minutes. After drying, the cover glass is attached to a 1: 1 mixture of xylene and malinol. Fluorescence microscopy showed more than 50 cells with NF-κB p65 entering the cell nucleus.

In the above experiments, α-tubulin was used as an internal control to assess the dependence of IκB degradation and p38 MAPK phosphorylation.

Stimulation with lipopolysaccharide (LPS) induces NF-κB p65 to move into the nucleus within 60 minutes, and treatment with pacetin significantly inhibits this migration (FIGS. 3A and 3B).

In addition, when treated with picetin, it can be seen that degradation of IκB is inhibited and phosphorylation of p38 MAPK is inhibited (FIG. 3C). The portion labeled with phospho-p38 in Figure 3c refers to the phosphorylated p38 MAPK.

Experimental Example 4 (Preventing Neurotoxicity of Microglial Cells)

The method of co-culture of microglia and neurons was used to determine whether or not phycetin prevents neurotoxicity of microglia. Experiments were carried out by co-culture of BV-2 microglia and B35 neuroblastoma cells activated with lipopolysaccharide (LPS). B35 neuroblastoma cells induced by BV-2 microglia treated with nothing were used as controls.

The diversity of B35 neuroblastoma cells induced by the activated BV-2 microglia can be measured by estimating the number of cells represented by the EGFP (fluorescence gene) among the cells in the coculture. Because B35 neuroblastoma cells appear stable in EGFP.

Referring to Figure 4, it can be seen that the inhibition of the death of B35 neuroblastoma cells induced by picetin activated BV-2 microglia.

FIG. 1A is a diagram showing that picetin inhibits the production of nitric oxide (NO) of BV-2 microglia stimulated by lipopolysaccharide (LPS).

FIG. 1B is a diagram showing that cecetin inhibits TNF-α production of BV-2 microglia stimulated by lipopolysaccharide (LPS).

FIG. 1C is a diagram showing that picetin inhibits the production of PGE ₂ in BV-2 microglia stimulated by lipopolysaccharide (LPS).

FIG. 2 is a diagram showing that cecetin inhibits gene expression of iNOS, TNF-α and COX-2 in BV-2 microglia stimulated by lipopolysaccharide (LPS).

3A and 3B show that cecetin inhibits migration of NF-κB p65 into the nucleus of BV-2 microglia.

FIG. 3C is a diagram showing that picetin inhibits degradation of IκB and phosphorylation of p38 MAPK. FIG.

FIG. 4 shows that cecetin inhibits B35 neuroblastoma cell death induced by activated BV-2 microglia. FIG.

Claims (11)

A pharmaceutical composition for preventing and treating degenerative neurological diseases, containing cecetin and a pharmaceutically acceptable carrier to inhibit the activity of microglia. The pharmaceutical composition of claim 1, wherein the carrier is selected from the group consisting of petrolatum, lanolin, polyethylene glycol, alcohol, and combinations thereof. The pharmaceutical composition of claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, HIV dementia, stroke, and multiple sclerosis. 2. The method of claim 1, wherein the picetin inhibits the secretion of nitric oxide (NO), TNF-α (TNF-alpha) and PGE ₂ (prostaglandin 2) in microglia to prevent and treat neurodegenerative diseases. Pharmaceutical compositions. The pharmaceutical composition of claim 1, wherein the phycetin inhibits gene expression of iNOS to prevent and treat neurodegenerative diseases. The pharmaceutical composition according to claim 1, wherein the picetin inhibits gene expression of TNF-alpha (TNF-α) to prevent and treat neurodegenerative diseases. The pharmaceutical composition of claim 1, wherein the phycetin inhibits gene expression of COX-2 to prevent and treat neurodegenerative diseases. The pharmaceutical composition according to claim 1, wherein the picetin inhibits the activities of P38 MAPK and NF-κB to prevent and treat neurodegenerative diseases. The pharmaceutical composition according to claim 1, wherein the picetin inhibits the degradation of IκB to prevent and treat neurodegenerative diseases. The pharmaceutical composition according to claim 1, wherein the picetin inhibits the migration of NF-κB p65 into the nucleus to prevent and treat neurodegenerative diseases. The pharmaceutical composition of claim 1, wherein the phycetin inhibits neuronal cell death due to activation of microglia to prevent and treat neurodegenerative diseases.

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