KR20120052011A - Pharmaceutical composition for preventing and treating inflammation comprising ethyl linoleate - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing ethyl linoleate is provided to suppress expression of inflammation expression marker genes and to treat inflammation. CONSTITUTION: A pharmaceutical composition for preventing and treating inflammation contains ethyl linoleate as an active ingredient. The composition suppresses expression of inflammation expression marker genes including COX-2(cyclooxygenase-2), IL-8beta(interleukin 1-beta), iNOS(inducible NO synthase), IL-6(interleukin-6), TNF-alpha(tumor necrosis factor-alpha), NF-kappaB(nuclear factor-kappaB), or IL-12(interleukin-12).

Description

Pharmaceutical composition for preventing and treating inflammation comprising ethyl linoleate}

The present invention relates to a pharmaceutical composition for preventing and treating inflammation comprising ethyl linoleate as an active ingredient, and more particularly, to prevent inflammatory diseases by using the anti-inflammatory activity of ethyl linoleate extracted from garlic. And to pharmaceutical compositions for treatment.

Inflammation is in response to various stimuli such as cytokine (lipopolysaccharide, LPS), IL-1β, TNF-α, reactive oxygen species (ROS), radiation, etc. It is an essential defense mechanism of the host being induced.

Nitric oxide (NO) is produced from L-arginine by nitric oxide synthetase (NOS) in human macrophages. INOS in the NOS of the human body can be activated by immune stimulants such as lipopolysaccharide (LPS), cytokine such as IL-1, TNF-α, radiation, etc. Only when large amounts are expressed in several cells. When iNOS expression is induced by external stimuli such as LPS, inflammation factor, and irradiation, a large amount of NO is continuously produced for 4-6 hours, which is known to cause an inflammatory response in the human body. In this respect, compounds that inhibit NO production by iNOS can be used as a therapeutic agent for various inflammatory diseases in the human body.

Prostaglandins, such as PGE 2, PGF 2a, and PGI 2, are other hormones derived from arachidonic acid and are regulated by cyclooxygenase (COX). COX-1 is present in most tissues and maintains homeostasis in the body, while COX-2 is involved in the production of prostaglandins at sites of inflammation. Inhibition of this COX gene can be used for effective regulation and control of immune-related cells or cancer cells.

Heme oxygenase-1 is a key factor in the defense of oxidative stress responses and is a rate-limiting enzyme that triggers heme metabolism. When heme is decomposed by heme oxygeanse-1, biliverdin and carbon monoxide free iron are produced. Biliverdin continues to be bilirubin by biliverdin reductase. The biliverdin, carbon monoxide and free iron produced by heme oxygenase-1 have been reported to be an important role in the regulation of anti-inflammatory mechanisms.

Thus, iNOS and COX-2 specific inhibitors are essential for inflammation treatment purposes, and drugs that can specifically inhibit iNOS and COX-2 would be useful in treating diseases mediated by NO and PGE 2 overproduction. will be.

On the other hand, garlic ( Allium sativum ) is a perennial plant belonging to the family Liliaceae and has been used as a medicinal food in the folklore since ancient times. Many scientific characteristics of garlic have been proved through modern scientific research. Especially, it contains antibiotics, allium, which has the effect of reducing preservatives, expectorants, and intestinal spasms. It is known.

In this regard, the present inventors investigated the anti-inflammatory activity of garlic extract, revealed that ethyl linoleate is the active ingredient, the ethyl linoleate inhibits the activity of iNOS and COX-2, and inhibits the production of proinflammatory cytokines. By blocking the activation pathway of NF-κB and further revealing that ethyl linoleate induces the expression of hemeoxygenase-1 in macrophages, ethyl linoleate exhibits good anti-inflammatory activity in macrophages. It confirmed and completed this invention.

An object of the present invention is to provide a pharmaceutical composition for preventing and treating inflammation comprising ethyl linoleate as an active ingredient.

Another object of the present invention is to provide a method of screening an ethyl linoleate concomitant for preventing and treating inflammation.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating inflammation comprising ethyl linoleate as an active ingredient.

The term "ethyl linorate" in the present invention refers to one of the essential fatty acids that act to inhibit the activity of reactive oxygen species by stimulation of bacteria and to inhibit hyperkeratinization induced by the lack of linoleic acid. The ethyl linoleate of the present invention was extracted from garlic. In the present invention, it is confirmed that the anti-inflammatory active ingredient of garlic extract is ethyl linoleate, and the anti-inflammatory activity of the ethyl linoleate and its mechanism are to be clarified.

When inflammatory stimuli are given to macrophages, expression of various inflammation-related genes is enhanced, including the production of iNOS (inducible NO synthase) and prostaglandin E2 (enzymes) involved in NO production. COX-2 (cyclooxygenase-2), an enzyme that mediates inflammatory reactions, and various inflammatory cytokines, TNF-α (tumor necrosis factor-alpha), IL-1β (interleukin 1-beta), IL-6, IL- 12 and others control the inflammatory response.

In the present invention, in order to investigate the effect of ethyl linoleate on the molecular and cellular changes at the cellular level by the inflammatory response, it is rapidly induced by inflammatory stimulation induced by the lipopolysacchride (LPS), a bacterial cell wall component in the macrophages of murine. Cyclooxygenase-2 (COX-2), interleukin 1-beta (IL-1β), inducible NO synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) ), Whether mRNA expression and protein expression of NF-κB (nuclear factor-kappaB), IL-12 (Interleukin-12), etc. are inhibited by the treatment of ethyl linoleate.

As a result, in specific embodiments of the present invention, ethyl linoleate inhibits NO and PGE 2 release by affecting iNOS and COX-2 expression levels without affecting cell viability. It was confirmed to inhibit the activity of 2. In addition, the mRNA levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 were examined by real-time RT-PCR, and the expression of TNF-α, IL-1β and IL-6 mRNA was reduced. It was.

On the other hand, NF-κB (nuclear factor-kappaB) is present in the cytoplasm in the form of inhibitory kappaB (IκB) if it is not activated. NF-κB, which is cleaved and released at this time, moves to the nucleus and activates various target genes, iNOS and COX-2.

The present inventors confirmed the effect of ethyl linoleate on macrophages on the activation pathway of NF-κB, which is the most representative pathway in inflammatory reaction, to examine the mechanism of inflammatory response of ethyl linoleate. LPS-induced ERK1 / 2, JNK and p38 MAPK activity was significantly inhibited by inhibiting the activity of NF-kB was confirmed (Fig. 4A). This strongly suggests that ELA inhibits inflammatory activation of macrophages by LPS.

In the present invention, the term "hemeoxygenase-1 (hereinafter referred to as HO-1)" is an inflammation inhibitory enzyme that metabolizes heme into free iron, bilirubin and carbon monoxide, and refers to an important regulator of inflammation. In addition, HO-1 protein is a multifunctional enzyme that effectively regulates hyperimmune responses, and it has been observed that severe inflammatory diseases are found in humans with defects in the gene of this enzyme. In stimulated macrophages, HO-1 expression and CO treatment reduce proinflammatory mediators such as NO, TNF-α, IL-1β and IL-6.

In a specific embodiment of the present invention, ELA can induce mRNA and protein expression of HO-1 in murine macrophages RAW264.7 (FIG. 8A), and LPS-induced proinflammatory cytokines due to induction of HO-1 By revealing reduced release, it was confirmed that the anti-inflammatory effect of ELA could be mediated by the induction of HO-1. In addition, treatment with SnPP, a HO-1 inhibitor, revealed that ELA significantly inhibited the inhibitory effects on NO, TNF-α, IL-1β and IL-6 secretion in LPS-stimulated macrophages (FIG. 6) These results suggest that HO-1 induction mediates the inhibitory effect of ELA on LPS-induced inflammatory responses.

As described above, the ethyl linoleate of the present invention has the effect of preventing and treating inflammation by inhibiting the expression of an inflammatory expression marker gene for an inflammatory response. Therefore, the ethyl linoleate of the present invention can be usefully used as a composition for preventing and treating anti-inflammatory.

The throughput of the pharmaceutical composition for preventing and improving inflammation used in the present invention should be a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level may refer to an individual type and severity, age, sex, It can be determined according to the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the concurrent drug and other factors well known in the medical field. Effective amounts may vary depending on the route of treatment, the use of excipients, and the possibility of use with other agents, as will be appreciated by those skilled in the art.

Pharmaceutically treated forms of the pharmaceutical compositions for preventing and improving inflammation of the present invention can be used in the form of their pharmaceutically acceptable salts, and can also be used alone or in combination with other pharmaceutically active compounds, as well as in suitable collections. have.

In addition, the pharmaceutical composition for preventing and improving inflammation of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations and patches according to a conventional method. It may be used, and may further include suitable carriers, excipients and diluents commonly used in the preparation of the composition.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

In another aspect, the present invention provides a method for treating hemeoxygenase-1 expression in a subject with inflammation; (b) administering ethyl linoleate alone and co-administering ethyl linoleate and a candidate to the subject; And (c) comparing the increase in hemeoxygenase-1 expression in the single administration and co-administration of the present invention relates to a method for screening ethyl linoleate co-administration for inflammation prevention and treatment.

In order to screen the ethyl linoleate concomitant of the present invention, the expression pattern of hemeoxygenase-1, which is increased by ethyl linoleate, is compared with the administration of ethyl linoleate alone and the combination of ethyl linoleate and a candidate substance. In addition, it may be determined whether the candidate substance can effectively treat inflammation.

Preferably the expression level can be observed at the protein level as well as at the gene (mRNA) level, and the identification of the expression level can be confirmed by techniques known in the art.

In the present invention, "mRNA expression level measurement" is a process of confirming the presence and expression of mRNA of the inflammatory marker gene in a biological sample in order to diagnose inflammation, the analysis method for this RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assays, northern blotting, DNA chips, and the like.

In the present invention, "protein expression level measurement" refers to a process of confirming the presence and expression level of a protein expressed in an inflammatory marker gene in a biological sample in order to diagnose inflammation. An antibody that specifically binds to a protein of the gene. Check the amount of protein using. Analytical methods for this purpose include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, immunoprecipitation assay.

As compared to the expression pattern of hemeoxygenase-1 in the combination group of ethyl linoleate alone and ethyl linoleate and the candidate substance as described above, the expression of the hemeoxygenase-1 gene or protein was administered alone in the combination administration group. If identified at lower levels, the candidate can be selected as a combination dose in the treatment of inflammation.

As described in detail above, ethyl linoleate according to the present invention has an effect of inhibiting the expression of inflammatory expression marker genes rapidly increased by inflammatory stimulation induced by LPS in macrophages which are immune cells, and ethyl linoleate is HO By revealing a novel mechanism for reducing LPS-induced proinflammatory cytokine release by inducing -1, a composition comprising the same can be usefully used as a medicament effective in the treatment of inflammatory diseases.

Figure 1 shows the results of the identification of ethyl linoleate ((9E, 12E) -ethyl octadeca-9,12-dienoate) as the anti-inflammatory stimulating molecule of the present invention (Structure Formula (A), HMBC correlation and GC profile of ELA) (C)).
Figure 2 shows the effect of ethyl linoleate on NO and PGE 2 production in RAW264.7 cells activated by LPS. Cells were pretreated for 2 hours at various ELA concentrations (1 μM, 5 μM, 10 μM) and stimulated with LPS (1 μg / ml) for 24 hours. Cell viability was then assessed using the MTT assay (A). In addition, the cells were treated with LPS (1 μg / ml) for 16 hours in the presence and absence of ELA, and culture medium was collected to measure NO and PGE2 production (B, C).
Figure 3 shows the effect of ethyl linoleate on iNOS and COX-2 expression in RAW264.7 cells activated by LPS. Data are presented as mean ± SD of three independent experiments per group, ** P <0.05 and *** P <0.01 vs 1 μg / ml LPS-treated group; Significant differences between groups were determined by the Student-Newman-Cowls test.
Figure 4 shows the effect of ethyl linoleate on LPS-induced proinflammatory cytokine mRNA expression.
Figure 5 shows the results of inhibition of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IP-12) production by ethyl linorate.
Figure 6 shows the effect of ethyl linoleate on PI-3K / Akt and MAPK signaling in LPS-stimulated macrophages.
Figure 7 shows the inhibitory effect of ethyl linoleate on LPS-induced translocation of NF-kB in RAW 264.7 cells.
8 shows the effect of ethyl linoleate on induction of HO-1 expression in RAW 264.7 cells.
Figure 9 shows that the inhibitory effect of ethyl linoleate on NO, TNF-α, IL-1β and IL-6 production in LPS induced-RAW 264.7 cells is inhibited due to inhibition of HO-1 activity.

It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Materials and Experiment Methods

material

Garlic ( Allium sativum ) was purchased from New Green Company (Changyoung, South Korea) in January 2008. Voucher specimens (Accession No. NGL-PDRL-80-1) were deposited in the Pusan National University Herbarium. The plant was identified by one of the inventors (YW Choi).

ethyl Linoleic  detach

Dried garlic (1309 g) was ground to a powder and extracted with n-hexane, EtOAc and MeOH at room temperature. Hexane extract (9.751 g) was evaporated in a rotary evaporator and chromatographed by silica gel (40 μm; Baker, Phillipsburg, NJ, USA) column (70 × 8.0 cm). 24 fractions of ethyl linorate (109 mg) were obtained from 100 fractions obtained using 10% methylene chloride in hexane, 5% and 25% acetone in methylene chloride, 25% acetone in chloroform and 25% methanol in chloroform.

reagent

Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco / BRL (Grand Island, NY, USA). Specific inhibitors of p38, ERK1 / 2 and JNK (SB203580, PD98059 and SP600125) were purchased from AG Scientific (sanDiego, CA, USA). 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT), LPS ( Salmonella enteritidis ) and other reagents were purchased from Sigma (St. Louis, MO, USA). Antibodies against Protoporphyrin IX (SnPP), iNOS, COX-2, JNK, ERK1 / 2, p38 and HO-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-IκBα, p-JNK, p-ERK1 / 2, p-p38, p-Akt and Akt polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA USA).

Cell culture

Murine macrophage RAW 264.7 cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD). Cells were DMEM supplemented with 10% FBS; It was grown in GIBCO and incubated at 37 ° C. in a humid atmosphere of 5% CO ₂ and 95% air. Separation every 2-3 days to maintain exponential growth stage. Cell numbers were counted using a hemacytometer.

Cell viability  Assay MTT Assay )

Cytotoxicity of ELA was measured using microculture tetrazolium (MTT) -based colorimetric assay. Cells were incubated in 24-well plates at ⁴ × 10 ⁵ cell density per well. About 5 μl of MTT solution (5 mg / ml) was added to each well (final concentration 62.5 μg / ml). After 4 hours of incubation at 37 ° C. for 5% CO ₂ , the supernatant was removed and the foamy crystals formed in the visible cells were dissolved in 150 μl of DMSO. Absorbance of each well was measured using a Vmax microplate reader (Molecular Devices) at 570 nm.

Measurement of nitric oxide concentration

NO synthesis in the medium was measured by microplate assay method as described in Schmidt and Kelm (1996). To determine nitric oxide, 100 μl alloquit was removed from the conditioned medium and with an equal volume of Griess reagent (1% sulfanilamide / 0.1% N- (1-naphthyl) -3thylenediamine dihydrochloride / 2.5% H ₃ PO ₄ ). Incubated at 37 ° C. for 10 minutes. Nitric oxide concentration was measured using a microplate reader (Wallac1420, Boston, MA USA) at an absorbance of 540 nm. On the other hand, NaNO ₂ was used as a standard.

PGE  Measurement of 2 concentration

RAW264.7 cells were plated in 24-well culture plates at a density of ⁵ × 10 ⁵ cells / ml. The cells were incubated for 1 hour at various ELA concentrations (1-10 μM) and then treated with LPS (1 μg / ml) for 18 hours. PGE 2 levels were then quantified in the culture medium using an ELISA kit (R & D, Minneapolis, MI) according to the manufacturer's instructions.

TNF -α, IL -1β, IL -6 and IL Measurement of -12 concentration

RAW264.7 cells ( ⁵ × 10 ⁵ cells / ml) were incubated for 1 hour at various ELA concentrations (1-10 μM) in 24-well culture plates and then treated with LPS (1 μg / ml) for 6 or 24 hours. . After 6 hours of incubation, TNF-α, IL-1β, IL-6 and IL-12 levels were quantified in the culture medium using the ELISA kit (Koma Biotech, Seoul, Korea) according to the manufacturer's instructions.

Reverse transcription ( RT ) real time PCR

Total RNA was isolated using RNA spin mini RNA isolation kits (GE Healthcare) according to the manufacturer's instructions. 1 μg of total RNA was reverse transcribed using Maxime RT PreMix (Intron Biotechnology) and an anchored oligo-dT ₁₅ -primer. Real-time PCR was performed on a Chromo4 ^™ instrument (BIO-RAD) with the SYBR Green Master Mix (Applied Biosystems, CA). Relative amounts of target mRNAs were determined by comparative threshold (Ct) method by standardizing the target mRNa Ct values for GAPDH (ΔCt). The primer sequence is as follows: TNF-α-sense (5'-ttctcattcctgcttgtggc--3 ', TNF-α-antisense (5'-gtttgctacgacgtgggcta-3', IL-1β-sense (5'-gttgacggaccccaaaagat-3 ', IL-1β-antisense (5'-cctcatcctggaaggtccac-3 ', IL-6-sense (5'-cacggccttccctacttcac-3', IL-6-antisense (5'-tgcaagtgcatcatcgttgt-3 ', iNOS-sense (5'-agatcccgaaacgcttcact -3 ', iNOS-antisense (5'-ctgctgagggctctgttgag-3', COX-2-sense (5'-tactacaccagggcccttcc-3 ', COX-2-antisense (5'-catatttgagccttgggggt-3', HO-1-sense ( 5′-cgggccagcaacaaagtg-3 ′, HO-1-antisense (5′-agtgtaaggacccatcggaga-3 ′, GAPDH-sense (5′-aggtggtctcctgacttc-3 ′, and GAPDH-antisense (5′-taccaggaaatgagcttgac-3 ′.

Immunofluorescence Confocal  Microscopic observation

RAW264.7 cells were cultured directly into glass cover-slip 35 mm dishes. The cells were fixed for 10 minutes at 37 ° C. with 3.5% paraformaldehyde PBS and permeated for 10 minutes with 100% MeOH. To examine the NF-κB position of the cells, the cells were treated with anti-NF-κB p65 polyclonal antibody (1: 100) for 1.5 hours. After washing with PBS, the cells were incubated for 1 h at 37 ° C. in a diluted 1: 100 PBS secondary FITC-conjugated donkey anti-rabbit IgG antibody. Nuclei were stained with 1 μg / ml DAPI and analyzed by confocal microscopy using a Zeiss LSM 510 Meta microscope.

Preparation of Nuclear Extracts

After washing RAW264.7 cells (3 × 10 ⁶ cells) three times with cold PBS, cell pellets were suspended in hypotonic buffer (Active Motif, California, USA) and incubated in ice for 15 minutes. Then, Detergent (Active Motif, California, USA) was added to the cell extracts, and then cultured for 1 minute and centrifuged at 4 ° C. for 1 minute at 13000 rpm. Supernatants containing cytoplasmic and nuclear proteins were extracted with complete lysis buffer B (Active Motif, California, USA) for 30 minutes at 4 ° C. After centrifugation at 4 ° C. for 5 minutes at 13,000 rpm, the supernatant was harvested and stored at −70 ° C.

Weston Blot  analysis

Cells were incubated in cold lysis buffer consisting of 1% Triton X-100, 1% deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). Protein amount of cell lysate was determined using Bradford reagent (Bio-Rad; Hercules, CA, USA). Proteins in each sample (50 μg total protein) were electrophoresed on 7.5% SDS-polyacrylamide gels, then transferred to PVDF membranes and exposed to appropriate antibodies. The protein was visualized by an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody. Images were acquired using an ImageQuant 350 analyzer (Amersham Biosciences), and density measurements were performed using ImageQuant TL software (Amersham Biosciences).

result

Determination of the structure of the active compound

Active molecules isolated from garlic powder were identified by ¹ H, ¹³ C, Dept, HSQC, HMBC NMR spectra in CDCl ₃ . From the molecular ion signal at m / z 308 of ethyl linoleate, the constitutive composition of C ₂₀ H ₃₆ O ₂ was confirmed. This was confirmed by the ¹ H, ¹³ C, and Dept NMR technique, two primary [δc 14.1 (C-2 ′, 14.3 (C-18)), 13 secondary [δc 22.6 (C-17), 31.5 (C-16), 29.4 (C-15), 27.2 (C-14), 25.6 (C-11), 27.2 (C-8), 29.1-29.6 (C-4-C-7), 34.4 ( C-2), 25.0 (C-3), 60.2 (c-1 '), four tertiary [δc 130.2 (C-13), 131.0 (C-12), 127.9 (C-10), 128.1 (C -9)], and one quaternary carbon [δc 173.9 (C-1)] From the above data, the presence of two double bonds was determined: C ₂₀ H ₃₆ O ₂ composition, together with the double bond Indicates that there were thirteen unsaturations in the ELA chain system The ¹ H NMR of the ELA has two methyl triplets (δ _H 0.89, 18-H; 1.25 2′-H) The absorption of the two olefin protons is δ Detected at _H = 5.39 (x2H, 11 and 12-H) and 5.35 (x2H, 9 and 11-H) These abnormal down-field shifted proton signals are methylene groups at δ _H = 4.12 (1'-H). 2.29 (2-H) showed the above amount This is due to the fact that the saints are each present in the esters of the ELA chain system.The two methylene protons are part of adjacent methylene groups, as can be seen by ¹ H ¹ H-COSY The other protons of each methylene group It appears in the aliphatic region of the spectrum: H-11 is located in the triplet with two methylene protons (5.39 11-H / 5.35 H-10), and 15-17-H is δ _H = 1.33-1.35 (4-H In 7-H).

Two protons (2H, 11-H) belonging to one methylene group appeared as triplets at δ _H = 2.27. The corresponding cross peak appeared in ¹ H ¹ H-COSY (data not shown). Association with other groups was confirmed by two-dimensional NMR experiments. ¹ H ¹ H-COSY was found to be the methylene group (H = 2.29, dd, J = 7.5 / 3.0) by the same cross peak of the double doublet of 3-Ha / 3-Hb. This was further confirmed by HMBC (1-C bound to both methylene double doublet 2-H and multiplet 3-H). The same applies to 11-C (also bound to both methine protons (10-H and 12-H)). The association of the chain was confirmed, as 14-C binds to methion protons 12-H and 13-H and methylene multilets 15-H and 16-H and 18-H binds to 16-C and 17-C. . Adjacent quaternary carbon 1-C reveals a connection to 2-H, 3-H, and 1′-H, which is consistent with the omega-6 fatty acid structure. From this information, the association of ELA was inferred. The conformational structure of the ELA compound was established as shown in FIG. 1.

ELA of NO  And PGE  2 generation suppression effect

To investigate cell viability as a response to ELA, the inventors first examined the potential cytotoxic effects on the viability of RAW264.7 cells for 24 hours in the presence or absence of LPS. As a result of the MTT assay, cell viability was not affected by ELA 1-10 mM because inhibition of inflammation was not due to the cytotoxicity of ELA (FIG. 2A). In addition, in LPS-induced RAW264.7 cells ELA showed a concentration dependent inhibitory effect on NO and PGE 2 production (FIGS. 2B-2C). Furthermore, the effect on ELA's iNOS and COX-2 mRNA and protein levels in LPS-stimulated macrophages was investigated. iNOS and COX-2 mRNA and protein levels were significantly up-regulated in response to LPS and ELAs strongly inhibited these mRNA and protein expressions (FIG. 2D-2F). These results indicated that ELA inhibited NO and PGE 2 release by affecting iNOS and COX-2 expression levels without affecting cell viability.

ELA of Proinflammatory  Inhibitory Effect on Cytokine Production

As a result of measuring the levels of TNF-α, IL-1β, IL-6 and IL-12 in medium of RAW 264.7 cells treated with varying amounts of ELA and LPS, ELA was determined to be TNF-α, IL-1β, IL-6 And was shown to significantly inhibit IL-12 production (FIG. 3C-3D). In addition, mRNA levels of TNF-α, IL-1β and IL-6 were investigated using real-time RT-PCR. Changes in TNF-α, IL-1β and IL-6 production were also shown to be reduced in mRNA expression.

ELA On by PI -3K / Akt , MAPK  And NF -κB pathway inhibitory effect

MAPK is known to be associated with LPS mediated induction of human and murine macrophages and plays an important role in the activity of NF-κB. We investigated the effect of ELA on p38, ERK1 / 2, and JNK activity in LPS-stimulated RAW264.7 cells. Since the activity of MAPK requires phosphorylation of threonine and tyrosine residues, Western blot analysis was used as anti-phosphate-MAPK antibody. The activity of MAPK appeared 15 minutes after treatment with LPS (1 μg / ml). Cells were pretreated for 1 hour with ELA or PD98059, SB203580 and SP600125 (positive control) and stimulated for 15 minutes with 1 μg / ml LPS. Phosphorylation of ERK1 / 2, JNK and p38 was increased in cells treated with LPS alone, but ELA concentration-dependently inhibited ERK1 / 2, JNK and p38 phosphorylation. The LPS-induced ERK1 / 2, JNK and p38 phosphorylation was also significantly inhibited by PD98059, SB203580 and SP600125 (positive control). These results suggest that ELA significantly inhibits LPS-induced ERK1 / 2, JNK and p38 MAPK activities in RAW264.7 cells (FIG. 4A).

In addition, the inventors examined the NF-κB p65 subunit levels in the cytoplasm and nucleus after treatment with LPS by Western blot analysis in the presence or absence of ELA. As a result, LPS treatment reduced cytoplasmic NF-κB p65 subunits and increased nuclear NF-κB p65 subunits. However, ELA inhibited all nuclear translocations of LPS-induced NF-κB p65 subunits. Immunofluorescence assay with anti-p65 antibody showed that p65, a component of NF-κB, has transcriptional activity. Western blot analysis also showed weaker nuclear p65 expression while unstimulated cells showed nuclear accumulation of p65, whereas LPS-stimulated cells decreased due to treatment with ELA (FIGS. 4B and 4C).

RAW264 .7 in the cell HO -1 expression inducing effect

To identify possible factors responsible for differential inhibition of LPS-mediated iNOS and COX-2 induction, we examined whether ELA induces HO-1 mRNA and protein expression in RAW264.7 cells. After treatment with 20 μM ELA in RAW264.7 cells and examining the mRNA and protein expression of LPS mediated proinflammatory factor HO-1, ELA increased the mRNA and protein expression of HO-1 in a time-dependent manner (FIG. 8A ). Cells were treated with ELA 1-20 μM for 24 hours, showing significant HO-1 mRNA and protein expression levels.

HO -1 activity inhibition ELA Inhibits anti-inflammatory activity

To investigate the potential for HO-1 gene expression induced by ELA, RAW264.7 cells were treated with ELA (20 μM) after 1 hour pretreatment with a specific HO-1 inhibitor, Protoporphyrin IX (SnPP). The cells were then stimulated with LPS. As a result, as shown in Figure 6, SnPP significantly neutralized the inhibitory effect on NO, TNF-α, IL-1β and IL-6 secretion in LPS-stimulated macrophages of ELA. These results suggest that HO-1 induction mediates the inhibitory effect of ELA on LPS-induced inflammatory responses.

Claims (4)

Inflammation prevention and treatment pharmaceutical composition comprising ethyl linoleate as an active ingredient. The composition of claim 1, wherein the composition inhibits expression of an inflammatory expression marker gene. The method of claim 2, wherein the inflammatory expression marker gene is COX-2 (cyclooxygenase-2), IL-1β (interleukin 1-beta), iNOS (inducible NO synthase), IL-6 (Interleukin-6), TNF-α (tumor necrosis factor-alpha), nuclear factor-kappaB (NF-κB), and IL-12 (Interleukin-12). (a) identifying hemeoxygenase-1 expression in an individual with inflammation; (b) administering ethyl linoleate alone and co-administering ethyl linoleate and a candidate to the subject; And (c) comparing the increase in hemeoxygenase-1 expression in the single administration and co-administration of the ethyl linoleate concomitant for inflammation prevention and treatment.

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