KR20130037029A - 1-(5-bromo-2-hydroxy-4-methoxyphenyl)ethanone having anti-inflammatory effects and pharmaceutical composition containing the same - Google Patents

Abstract

PURPOSE: A 1-(5-bromo-2-hydroxy-4-methoxyphenyl)ethanone compound with anti-inflammatory effect and a pharmaceutical composition containing the same are provided to prevent or treat inflammation-mediated neurodegenerative diseases. CONSTITUTION: A 1-(5-bromo-2-hydroxy-4-methoxyphenyl)ethanone compound has inflammatory effect and is denoted by chemical formula 1. The compound blocks NF-kappaB and MAPK signaling pathway. The compound prevents or treats Alzheimer's disease, Parkinson's disease, myasthenia gravis, multiple sclerosis, Huntington's chorea, or progressive muscle dystrophy. The compound is derived from Paeonia suffruticosa ANER or hippocampus. A pharmaceutical composition for anti-inflammation contains the compound.

Description

1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone having an anti-inflammatory effect and a pharmaceutical composition containing the same {1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone having anti-inflammatory effects and pharmaceutical composition containing the same}

The present invention relates to a 1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone compound (SE1) having an anti-inflammatory effect and a pharmaceutical composition containing the same, more specifically NF-κB (SE1) compound having the prophylactic or therapeutic effect of inflammation-related diseases that can be exemplified by neurodegenerative diseases such as Alzheimer's by blocking the nuclear factor kappa B and miogen-activated protein kinases (MAPK) signaling pathways, and containing the same It relates to a pharmaceutical composition.

In recent years, inflammation has been a fundamental factor in various neurodegenerative diseases and several cell types have been identified as a causative agent of central nervous system (CNS) inflammation, among which microglia are important cell components. Microglial cells are resident innate immune cells in the brain and are responsible for immune surveillance (Block and Hong, 2005). Microglia are readily activated by changing their morphology and phenotype in response to brain injury or immunological stimulation (Davalos et al., 2005; Nimmerjahn et al., 2005; Perry et al., 2007). Long-term deregulated activation of microglia results in radical neurotoxicity outcomes, which in most cases are related to disease progression rather than onset (Block et al., 2007; Ekdahl et al., 2009). Activated microglial cells include reactive oxygen species, pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) It produces many of the same neurotoxic factors, which damage neurons and eventually kill them. Thus, microglial activation must be tightly regulated to avoid these inflammatory side effects.

Stimulations associated with microglia activation and deregulation can range from environmental toxins such as the insecticide rotenone, to brain damage or disease in which neuronal death or damage occurs (Nimmerjahn et al., 2005 ). Microglial activation at the experimental level was most effectively demonstrated in models using bacterial lipopolysaccharide (LPS), which caused acute overactivation (Hanisch and Kettenmann, 2007). Therefore, LPS-activated microglia have been widely used as a model for studying the results of microglia activation and candidates capable of inhibiting it (Hanisch and Kettenmann, 2007).

The fundamental signaling mechanism of microglia deregulated activation is of primary concern since it can be understood in terms of noxious activation regulation by understanding this. The NF-κB signaling pathway has been found to be the major signaling mechanism involved in inflammation. NF-κB is a transcription factor that mediates immune and inflammatory responses by promoting expression of pro-inflammatory mediators, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and cytokines. The MAPK signaling pathway is also involved in modulating the inflammatory response of microglia. Thus, there is a need to provide potential candidates that can inhibit the inflammatory response.

Seahorses are marine tibial fish and are widely known for their special medicinal components. China hippocampus of Hebrews Pocatello Moose CUDA block rilreo (Hippocampus kuda Bleeler) is used as one of the most popular ingredients in traditional Chinese TCM appetite, antioxidant, anti-tumor, anti-aging, anti-fatigue and Ca ^{2 +} a variety of biological activities, including the channel block Has been studied for several years (Li et al., 2008; Ryu et al., 2010; Zhang et al., 2003).

For example, Korean Patent Application Publication No. 10-2009-0043656 (published: 2009.05.07) 'Antioxidant composition containing hippocampus extract' describes that the hippocampus extract has an antioxidant activity, Korean Patent Application Publication No. 10-2009-0063302 (published: June 18, 2009) The hippocampus-derived bis (2-ethylheptyl) phthalate compound and pharmaceutical use thereof describes that the compound derived from hippocampus has antioxidant and anti-inflammatory effects And, Korean Patent Application Publication No. 10-2011-0036415 (published: 2011.04.07) 'peptide derived from hippocampus hydrolyzate for treating arthritis' describes a peptide for preventing or treating arthritis having the amino acid sequence LEDPFDKDDWDNWK.

However, anywhere in the prior art 1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone (1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone: hereafter referred to as 'SE1' Anti-inflammatory effect, molecular morphological properties and its benefits as a pharmaceutical active ingredient have not been studied.

1. Korean Patent Application Publication No. 10-2009-0043656 (published: 2009.05.07) 2. Korean Patent Application Publication No. 10-2009-0063302 (published: 2009.06.18) 3. Republic of Korea Patent Application Publication No. 10-2009-0063302 (published: 2009.06.18)

Hippokamus in the present invention Compound 1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone (1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone which is a bioactive metabolite from CUDA Bleeder: , Is referred to as 'SE1' to provide the bioactive characteristics of the compound, more specifically for use in the prevention or treatment of inflammation. On the other hand, since the SE1 is also a major component of moutan cortex, a herbal material used as a therapeutic drug for atherosclerosis and infection, it can also be used to separate and extract from the neck.

In addition, the present invention reveals the anti-inflammatory mechanism of the SE1, more specifically the MAPK and NF-κB signaling pathway blocking effect of SE1.

In addition, the present invention is to examine the basis of the SE1 can be effectively used in neurodegenerative diseases of the brain, such as Alzheimer's from the molecular form of SE1.

The present invention provides an anti-inflammatory effect of 1- (5-bromo-2-hydroxy-4-methoxyphenyl) ethanone ('SE1') of Formula 1 below:

The SE1 compounds, but are not particularly limited as long as its origin, preferably the hippocampus or mokdanpi, more preferably Hi myrtle mousse It is derived from CUDA bliller.

In accordance with the present invention, SE1 mediates anti-inflammatory responses in activated microglia by blocking two important pathways MAPK and NF-κB leading to inflammatory marker expression.

The MAPK signaling pathway molecules, ERK, JNK, and P38, are involved in the regulation of inflammatory responses, and phosphorylation of these molecules finally signals the expression of pre-inflammatory mediators, particularly cytokines, and unregulated phosphorylation of MAPKs causes excessive inflammatory molecules. To the contrary, which leads to adverse effects.

According to the present invention, SE1 blocks the phosphorylation of JNK and p38 MAPK, which is highly involved in the regulation of inflammatory molecules for ERK.

The NF-κB pathway is the major signaling pathway involved in inflammation, where the inducer of inflammation causes the inactive transcription factor NF-κB to be activated and translocated into the nucleus to express a wide range of inflammatory molecules. NF-κB consists of five subunits, p50 (NF-κB1), p52 (NF-κB2), p65 (RelA), RelB and c-Rel. The most abundant are p50 and p65. In microglia, under normal conditions, NF-κB is bound to IκB and is in the resting state in the cytoplasm. Upon immune stimulation, the upstream signaling molecule IKK induces phosphorylation and degradation of IκB, which releases free NF-κB. Free NF-κB translocates into the nucleus and promotes expression of proinflammatory proteins, iNOS, COX-2 and cytokines. These inflammatory mediators then stimulate the release of NO from each downstream molecule, such as iNOS, from the cell. NO and PGE ₂ are major inflammatory mediators and play an important role in inflammatory diseases. Long-term unregulated production of these molecules from activated microglia leads to damage and death of cells in the CNS, especially neurons.

The SE1 compound according to the present invention regulates this process in an activated microglial model by inhibiting NF-κB activation and translocation, which decreases the expression of iNOS, COX-2 and cytokines. Due to the low expression of these inflammatory genes, their respective downstream toxic products NO, PGE ₂ and cytokine production are lowered, resulting in inhibition of the inflammatory response.

Inflammatory diseases of the present invention include inflammatory diseases that cause neurodegenerative diseases, and examples of specific diseases to which the compounds of the present invention are applicable include Alzheimer's disease, Parkinson's disease, myasthenia gravis, multiple sclerosis, Huntington's chorea or progressive muscular dystrophy Although these etc. are mentioned, It is not limited to these.

According to the invention, SE1 has an anti-inflammatory effect in the 10-50 μM dosage range.

The methoxy groups and Br molecules present in the SE1 compounds of the invention contribute to anti-inflammatory efficacy.

Since the SE1 compound of the present invention has a small molecule size, it is highly bioavailable and likely to reach the microglia of the brain through the blood-brain barrier.

As used herein, the term "active ingredient" includes a substance or a group of substances (a pharmacologically active ingredient or the like, which is expected to express directly or indirectly the efficacy and effect of the drug by inherent pharmacological action). Means a main component).

The pharmaceutical composition comprising the SE1 compound of the present invention as an active ingredient may be provided in a form obvious to those skilled in the art such as tablets, pills, granules, liquids, capsules, etc., containing other carriers or excipients. In addition, when the pharmaceutical composition of the present invention contains the SE1 compound as an active ingredient, the daily dosage may be determined according to the prescription of the doctor in charge, may be administered once or divided into several times a day.

The pharmaceutical composition according to the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. That is, the pharmaceutical composition may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used may be used. Are prepared using. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and the like, which may be added to the glycoprotein extracts at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin every day. The mixture is prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

The SE1 compound of the present invention has an anti-inflammatory effect and a prophylactic or therapeutic effect of inflammation-mediated neurodegenerative diseases by a mechanism that blocks phosphorylation of MAPK molecules and nuclear translocation of NF-κB, and in particular, the molecular size of SE1 compound is small. Therefore, it can easily cross the blood-brain barrier and has a remarkably good effect of preventing or treating brain injury diseases such as Alzheimer's disease.

1 is a graph showing the effect of SE1 on the viability of BV-2 and PC-12 cells.
FIG. 2A is a graph showing the effect of SE1 on LPS-induced NO production in BV-2 cells, and FIG. 2B is a diagram showing that SE1 inhibits LPS-induced iNOS protein and mRNA expression in BV-2 cells. It is shown.
FIG. 3A is a graph showing the effect of SE1 on LPS-induced PGE ₂ production in BV-2 cells, and FIG. 3B is LPS-induced COX-2 protein and mRNA expression in SE1 in BV-2 cells It is to suppress the.
4 is a graph showing the effect of SE1 on the production of LPS-induced TNF-α (A), IL-1β (C) and IL-6 (E) and LPS-induced TNF-α (B), IL Is a graph showing the effect of SE1 on gene expression of −1β (D) and IL-6 (F).
5 shows the SE1 effect on LPS-induced MAP kinase activity in BV-2 cells.
6 shows SE1 effects on LPS-induced IKKα / β expression, IKBα degradation and NF-κB activation in BV-2 cells.

Hereinafter, the present invention will be described in more detail in the Examples. Since the following examples are intended to illustrate the invention, it will be apparent to those skilled in the art that the scope of the invention is not limited thereto.

Materials and methods

¹ H NMR (400 MHz) and ¹³ CNMR (100 MHz) spectra were run on a JEOL JNM-ECP 400 NMR spectrometer (JEOL, Japan) using CDCl ₃ (1D, 12.75 ppm at ¹ H, 113.9 ppm at ¹³ C NMR). Recorded. MS spectra were acquired on a JEOL JMS-700 spectrometer (JEOL, Japan). Silica gel 60 (230-400 mesh, Merck, Germany) and thin layer chromatography (TLC) plates (Kieselgel 60F254, 0.25 mm, Merck) were used as column chromatography and analytical TLC, respectively. BV-2 mouse microglial cells were provided by Professor Il-Whan Choi of Inje University, Korea. Cell culture media (Dulbecco's modified Eagle's minimal essential medium (DMEM)), penicillin / streptomycin, fetal bovine serum, equine serum and other materials required for cell culture include Gibco Bial, Life Technologies Gibco BRL, Life Technologies, Grand Island, NY, USA. The LPS of the MTT reagent (3- (4,5-dimethyl-2-yl) -2,5-diphenyl tetrazolium bromide), the Griess reagent and E. coli 026: B6 was determined by Sigma Chemical Company (Sigma). Chemical Co.). Primary and secondary antibodies used in Western blot analysis were Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and Amersham Pharmacia Biosciences (Piscataway, NJ). Purchased from Other chemicals and reagents were commercially available grades for analysis.

The live hippocampus, Hippokamus Kuda Blyler, was collected in October 2005 on Zhoushan Island, Zhejiang, China, and freeze-dried after removing internal organs. Prof. Ming-Lu Deng (Changchun University of Chinese Medicine, zoologist) confirmed that he is a Hippokamus kuda bliller.

SE1  Extract and check

Lyophilized Hippocamus Cuda bliller (3 kg) was triturated to a powdered state, refluxed with MeOH (3 × 5 L) for 3 hours, and the solvent was evaporated in vacuo to yield a crude MeOH extract (217 g). First, after receiving 3 ml of 50 ml each of 50 ml of n-hexane, which is a non-polar solvent, and checking whether there is a main substance, the same operation is repeated by continuously flowing 3 L of EtOAc. The extract was subjected to silica gel flash chromatography, eluting with n-hexane / EtOAc / MeOH (gradient) in a manner of flowing 3 L of MeOH, a polar solvent, in order to check whether there was a significant substance, and providing 10 main fractions. . Fraction 6 (502 mg, EtOAc), a suitable material for the experiment, was repeatedly purified on silica gel through toxicity testing and separation purification. Successful purification gave compound (16.38 mg). Based on comprehensive spectral analysis and previously published data (Qing et al., 2009), the compound obtained is 1- (5-bromo-2-hydroxy-4-methoxyphenyl) having the formula It was found to be ethanone (SE1):

Formula 1

In addition, the obtained compound was determined as C ₉ H ₉ O ₃ Br from LREIMS, ¹ H, ¹³ C and ¹³ C DEPT spectral data.

SE1 compound: ¹ H NMR (400 MHz, CDCl ₃ ): δ _H ppm 12.70 (1H, s, HO-2), 7.86 (1H, s, H-6), 6.46 (1H, s, H-3), 3.92 (3H, s, H-9), 2.56 (3H, s, H-8); ¹³ C NMR (100 MHz, CDCl ₃ ): δ _c ppm 114.6 (C-1), 164.4 (C-2), 100.7 (C-3), 161.8 (C-4), 101.1 (C-5), 134.7 (C-6), 201.9 (C-7), 26.3 (C-8), 56.6 (C-9) (see Table 1); LREIMS m / z 246.10 (41) [M] ⁺ , 244.15 (42), 231.05 (100), 229.05 (97), 216.00 (2), 188.00 (2), 173.05 (5), 149.10 (2), 121.15 ( 3), 107.10 (22), 93.10 (4), 79.05 (24), 69.05 (13), 53.00 (21).

¹ H and ¹³ C NMR data for SE1 location SE1 δ _H (mult, J) δ _C (mult) One 114.6 (s) 2 164.4 (s) 3 6.46 (1 H, s) 100.7 (d) 4 161.8 (s) 5 101.1 (s) 6 7.86 (1 H, s) 134.7 (d) 7 201.9 (s) 8 2.56 (3H, s) 26.3 (q) 9 3.92 (3H, s) 56.6 (q) 10 11 2-OH 12.70 (1H, s) CDCl ₃ at 400 Hz for ¹ H and 100 Hz for ¹³ C Recorded from

Cell Culture and Cell Viability Assays assay )

BV-2 cells were 95% air, 5% CO in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% heat-inactivated fetal bovine serum (FBS), 100 μg / mL streptomycin and 100 μg / mL penicillin ₂ was stored in the atmosphere. BV-2 cells were maintained passaged twice a week and cells were used for experiments at 70-80% confluence. PC-12 cells were stored in the same atmosphere in high glucose DMEM supplemented with 10% horse serum and 5% FBS.

The cell compatibility of SE1 compounds was tested at three concentrations of SE1 (5, 10 and 50 μg / mL) using MTT cell viability assays in cultured BV-2 and PC-12 cells. Cell viability was determined by 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) reduction assay as described in Hansen et al. (1989). . This is based on the theory that living cells convert MTT into visible formazan crystals during incubation and the absorbance of the dissolved formazan crystals in dimethylsulfoxide is proportional to cell viability.

MTT is used as an indicator of cell viability through mitochondrial reduction to formazan (Rajapakse et al., 2008). Cells were preincubated overnight in 96-well plates at 1 × 10 ⁴ cell density per well, then washed with phosphate-buffer (PBS). Cells were treated for 24 hours with SE1 at various concentrations (5, 10 and 50 μM), 100 μL MTT was added and incubated for 4 hours at 37 ° C. After removing the culture supernatant, the obtained dark blue crystals were dissolved in DMSO (Dimethylsulfoxide). The absorbance was read at 540 nm with an Enzyme Linked Immunosorbent Assay (ELISA) microplate reader (Tecan Austria GmbH, Tecan Austria GmbH). Relative cell viability was calculated relative to untreated control. As shown in FIG. 1, SE1 did not show significant (P <0.05) cytotoxicity against cells at all concentrations tested 24 hours after treatment. These data suggest that SE1 does not affect cell proliferation in PC-12 cells or BV-2 cells and can therefore be used safely in future experiments. Therefore, nontoxic concentrations ranging from 5 to 50 μM / mL were selected for later analysis.

Nitric Oxide Production Assay

LPS stimulation of microglial cells increases iNOS expression, which mediates NO production and ultimately damages adjacent neuronal cells by nitrite peroxide (ONOO ⁻ ), which is NO or a more toxic metabolite. NO is a short-lived molecule because it is easily converted to nitrates (NO ₂ ⁻ ). Thus, nitrate concentration is an indicator of the amount of NO produced, and nitrate is also easily detected spectroscopically when analyzed by grease reaction.

Nitric oxide (NO) concentrations in the culture supernatants were measured by a Greries reaction as described in Cocker and Laurent, 1998. That is, BV-cells were preincubated overnight in 96-well plates using DMEM without phenol red at a density of 1 × 10 ⁴ cells per well and then treated with SE1 (5, 10 and 50 μM) for 1 hour. Thereafter, NO production was stimulated by incubating with LPS (1 μg / mL final concentration) for 48 hours. 50 μL of the culture supernatant from each sample was then mixed with the same amount of grease reagent (1% sulfanylamide / 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride / 2.5% phosphoric acid), 15 Incubate for minutes. Absorbance was read at 540 nm with an ELISA microplate reader (Tecan, Geembeha, Austria). The numerical values obtained were compared with the numerical values of the standard concentrations of sodium nitrite dissolved in DMEM and the nitrite concentrations in the conditioned medium of the sampled cells were calculated.

As a result, LPS induced unregulated production of nitrite compared to the non-stimulated blank group (FIG. 2A). Pretreatment with SE1 effectively reduced LPS-stimulated NO production in BV-2 cells at all tested concentrations, and inhibition was throughput dependent.

To study the effect of SE1 on LPS-induced expression of iNOS enzyme and mRNA, BV-2 cells were treated with LPS (1 μg) after 1 hour treatment with SE1 at different concentrations (5, 10 and 50 μM) Treated for 18 hours and tested by Western blot and RT-PCR, respectively. Unstimulated cells showed very low levels of iNOS expression compared to LPS-induced iNOS overexpression at both protein and mRNA levels (FIG. 2B). This overexpression was significantly inhibited by SE1 treatment in a concentration dependent manner. This result is in good agreement with SE1-mediated NO inhibition, suggesting that SE1 inhibits NO production by inhibiting unregulated transcription and translation of iNOS.

PGE ₂ Enzyme immunity Assay

Inducible cyclooxygenase COX-2 produces proinflammatory prostaglandin E ₂ (PGE ₂ ) to develop inflammation. To determine SE1 efficacy on inhibition of PGE ₂ production, PGE ₂ release is measured using anti-PGE ₂ -coated ELISA plates using conditioned media.

PGE ₂ synthesis measurements were performed using an enzymatic immunoassay without prior extraction or purification using a commercially available PGE ₂ enzyme immuno-metric assay kit (Amersham Pharmacia Biosciences, NJ). Was carried out. BV-2 cells were treated with SE1 at different concentrations (5, 10 and 50 μM) for 1 hour and stimulated with LPS (1 μg / mL) for 18 hours. The conditioned media was harvested and subjected to a PGE ₂ enzyme immunoassay assay (PGE ₂ -EIA) according to the manufacturer's instructions. PGE ₂ concentration was calculated according to the formula obtained from the standard curve preparation using the PGE ₂ standard solution provided with the EIA kit.

As a result, PEG ₂ production was significantly increased in BV-2 cells as a result of LPS stimulation compared to the non-irritating group. However, the SE1 treatment group significantly inhibited the increase in concentration of PGE ₂ at all tested concentrations (5, 10 and 50 μg / mL) in a throughput dependent manner (FIG. 3A).

To study the underlying mechanism of SE1-mediated inhibition of PGE ₂ production, expression of COX-2 protein and COX-2 mRNA levels were treated with SE1 (5, 10 and 50 μM) for 1 hour after LPS (1 μg). Western blot and RT-PCR analysis were performed using protein and RNA collected from BV-2 cells stimulated for 18 hours.

As a result, LPS-induced overexpression of COX-2 enzyme and mRNA was clearly inhibited SE1 throughput dependent at both protein and gene levels (FIG. 3B), which is an inhibitory effect similar to PGE ₂ .

TNF -α, IL -1β and IL -6 Enzyme Immunity Assay

In the CNS, proinflammatory cytokines are produced primarily from activated microglia and are involved in the development and / or exacerbation of brain inflammation.

The inhibitory effect of SE1 on proinflammatory cytokine TNF-α, IL-1β and IL-6 production was determined by enzyme-linked immunosorbent assay (ELISA). Cells were treated for 1 hour with SE1 at different concentrations (5, 10 and 50 μM) followed by 18 hours with LPS (1 μg / mL). Conditioned media were analyzed using the mouse cytokine-specific Biotral ™ ELISA kit (Amersham Pharmacia Biosciences, New Jersey, USA) according to manufacturer's instructions. The concentrations of TNF-α, IL-1β and IL-6 were calculated by standard curve using each of the recombinant cytokines provided with the ELISA kit.

As a result, compared to unstimulated cells, cytokine concentrations were increased in the culture medium of LPS-stimulated BV-2 cells and decreased throughput-dependently by SE1 treatment (A, C, E in FIG. 4).

Reverse transcription  ( RT ) - PCR  analysis

To determine if SE1 affects cytokines at the transcription level, RNA isolated from BV-2 pretreated with SE1 (5, 10 and 50 μM) for 1 hour and stimulated for 18 hours by LPS (1 μg) was isolated. RT-PCR was performed.

RT-PCR was performed to detect iNOS, COX-2 and cytokine (TNF-α, IL-1β and IL-6) mRNA expression. Total RNA was extracted from BV-2 cells after treatment for 1 hour with SE1 at different concentrations (5, 10 and 50 μM) followed by 18 hours with LPS (1 μg / mL). Cells were lysed with Trizol® and centrifuged at 25 ° C. for 15 minutes at 12,000 rpm before adding chloroform. The supernatant was separated and isopropanol was added in a 1: 1 ratio. RNA pellets were obtained by centrifugation at 10,000 rpm for 10 minutes. After washing with ethanol, the extracted RNA was dissolved in diethyl pyrocarbonate-treated RNase-free water, and the absorbance was measured at 260 nm using a GENios® microplate reader (Tecan Austria GmbH). RNA amount was quantified. The resulting RNA (1 μg) contained 1 × reverse transcriptase (RT) buffer, 1 mM dNTPs, oligo (dT) 15 primers 500 ng, rodent molony leukemia virus (MMLV) reverse transcriptase 140 U and RNase inhibitor 40 U Reverse transcription was carried out at 42 ° C. for 45 min in the cDNA in the master-mix. Polymerase Chain Reaction (PCR) was carried out in an automatic Whatman thermocycler (Biometra, Kent, UK) to provide iNOS, COX-2, TNF-α, IL-1β and glyceraldehyde 3-Phosphate dehydrogenase (GAPDH) mRNA was amplified. Each transcription was identified using specific forward and reverse primers according to the manufacturer's instructions (Promega, Madison, WI). GAPDH expression was included as an internal housekeeping gene control. Primer sequences used to amplify the desired cDNA were as follows.

iNOS forward and reverse primers: 5′-CCTTCCGAAGTTTCTGGCAGCAGC-3 ′ and 5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3 ′;

COX-2 forward and reverse primers: 5′-GGGGTACCTTC CAGCTGTCAAAATCTC-3 ′ and 5′-GAAGATCTCGCCAGGTACTCACCTG-3 ′;

TNF-α forward and reverse primers: 5′-ATGAGCACAGAAAGCATGATC-3 ′ and 5′-TACA GGCTTGTCACTCGAATT-3 ′;

IL-1β forward and reverse primers: 5′-ATGGCAACTGTTCCTGAACTCAACT-3 ′ and 5′-TTTCCTTTCTTAGATATGGACAGGAC-3 ′;

IL-6 forward and reverse primers: 5′-AGTTGCCTTCTTGGGACTGA-3 ′ and 5′-CAGAATTGCCATTGCACAAC-3 ′; And

G3PDH forward and reverse primers: 5'-TGAAGGTCGGTGTGAACGGATTTGGC-3 'and 5'-CA TGTAGGCCATGAGGTCCACCAC-3'.

Amplification cycle conditions were 35 cycles of 95 ° C. 30 sec, 55 ° C. 30 sec and 72 ° C. 1 minute. PCR reaction products electrophoresed on 1.5% agarose gel in 1 × TAE buffer for 30 minutes at 100 V were then subjected to AlphaEase® gel image-analysis software (Alpha Innotech, San Lindo, CA, USA). Material) to visualize with ethidium bromide.

As a result, cytokine expression was significantly regulated by LPS treatment and significantly inhibited by SE1 treatment in all three cytokines tested (B, D, F of FIG. 4). This means that SE1 downregulates mRNA expression, inhibiting TNF-α, IL-1β and IL-6.

Western Blot  analysis

To further study the SE1 inhibition mechanism of LPS-induced microglia activation leading to unregulated inflammation, the effect of SE1 on the MAPK signaling pathway was studied. BV-2 cells were treated with SE1 for 1 hour and LPS for 30 minutes. Western blot analysis was performed to study the effect of SE1 on the phosphorylation of extracellular signal-regulated kinase (ERK), c-jun-N-terminal kinase (JNK) and P38 MAPK.

Western blots used standard procedures. That is, BV-2 cells treated with SE1 at different concentrations (5, 10 and 50 μM) and then 18 hours with LPS (1 μg / mL) were treated with 50 mM Tris-HCl (pH 7.5), 0.4% Nonidet P Lysis for 30 minutes at 4 ° C. in lysis buffer containing −40, 120 mM NaCl, 1.5 mM MgCl ₂ , 2 mM phenylmethylsulfonyl fluoride, 80 μg / mL leupetin, 3 mM NaF and 1 mM DTT I was. Cell debris was removed by centrifugation and the supernatant containing the extracted protein was kept at -80 ° C until use. CelLytic ™ NuCLEAR ™ extraction kit (Sigma-Aldrich Co., Sigma-Aldrich Co., Missouri, USA) was used to obtain nuclear extracts of BV-2 cells according to the manufacturer's instructions. Protein concentration of cell lysates was determined using the Lowry method. Cell lysates containing the same amount of protein (about 20 μg of total protein) were separated by SDS-PAGE gel electrophoresis and electro-blotting on nitrocellulose membranes. The membrane was blocked with 5% BSA and then incubated with the desired antibody (Santa Cruz Biotechnology Ink .: Santa Cruz Biotechnology Inc.) to detect each protein according to the manufacturer's instructions using a chemiluminescent ECL assay kit. It was. Western blots were visualized using a LAS3000® Luminescent image analyzer and protein expression was quantified using Multi Gauge V3.0 software (Fujifilm Life Science, Tokyo, Japan). .

As a result, as shown in Figure 5, LPS treatment induced the phosphorylation of all three MAPK molecules compared to the LPS untreated group. While both JNK and P38 phosphorylation were inhibited SE1 throughput dependently, there was no significant effect on ERK phosphorylation.

SE1 of NF -κB nuclear transcription inhibition

Activation and nuclear translocation of the transcription factor NF-κB promotes expression of inflammatory genes such as iNOS, COX-2, TNF-α, IL-1β and IL-6. This phenomenon is tightly regulated by upstream signaling molecules such as IKK (IB kinase) and IKB (Inhibitor of B). Therefore, it was studied whether SE1-mediated inhibition of the inflammatory response is regulated through NF-κB activation and inhibition of the former in LPS treated BV-2 cells.

Cells were stimulated with LPS (1 μg / mL) after treatment for 1 hour with SE1 (5, 10 and 50 μM). The cells were harvested after 1 hour to prepare cytoplasm and nuclear extracts to detect IKK α / β, IκBα, NK-κB p65 and NF-κB by Western blot. Protein expression levels were determined by density relative to the control treated with LPS alone.

As a result, the cytoplasmic concentration of IKK was decreased and the IκB concentration was increased in the SE1 treated group compared to the LPS alone treated group (FIG. 6). At the same time, the increased nuclear concentrations of NF-κB p65 and NF-κB p50 by LPS treatment decreased during SE1 treatment. This indicates that SE1 inhibits NF-κB nuclear translocation by regulating the upstream signaling molecules IKK and IκB.

Statistical analysis

All data are reported as mean ± SD of 3 independent experiments, unless indicated otherwise. Statistical comparisons between different treatments were made by one way ANOVA with a post hoc test of Student Newman Keul using the SPSS program (version 12.0). Differences with P <0.05 values were considered statistically significant.

Claims (6)

1- (5-Bromo-2-hydroxy-4-methoxyphenyl) ethanone compound (SE1) of Formula 1, characterized by having an anti-inflammatory effect:
Formula 1

SE1 compound characterized by having an anti-inflammatory effect by blocking NF-κB (nuclear factor kappa B) and MAPK (Mitogen-activated protein kinases) signaling pathways. The method according to claim 1 or 2,
SE1 compound for the prevention or treatment of Alzheimer's disease, Parkinson's disease, myasthenia gravis, multiple sclerosis, Huntington's chorea or progressive muscular dystrophy. The method according to claim 1 or 2,
SE1 compound, characterized in that it is derived from neck skin or hippocampus. A pharmaceutical composition for anti-inflammatory, comprising the SE1 compound of claim 1 or 2 as an active ingredient. The method of claim 5,
A pharmaceutical composition for preventing or treating Alzheimer's disease, Parkinson's disease, myasthenia gravis, multiple sclerosis, Huntington's chorea or progressive muscular dystrophy.

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