KR20190117394A - Composition comprising 15-keto prostaglandin e2 for anti-oxidation - Google Patents

Abstract

The present invention relates to an antioxidant pharmaceutical composition, a cosmetic composition, and a health functional food composition comprising 15-keto prostaglandin E2 as an active component. In the case of the composition comprising 15-keto prostaglandin E2 according to the present invention increases the activity of an antioxidant enzyme HO-1 by increasing the expression of Nrf2, and also produces a low level of ROS to activate a protective mechanism in the cell, thereby exhibiting an excellent antioxidant effect.

Description

Antioxidant composition containing 15-keto prostaglandin E2 as an active ingredient {COMPOSITION COMPRISING 15-KETO PROSTAGLANDIN E2 FOR ANTI-OXIDATION}

The present invention relates to an antioxidant composition, and more particularly to an antioxidant pharmaceutical composition, cosmetic composition and health functional food composition comprising 15-keto prostaglandin E ₂ as an active ingredient.

Reactive Oxygen Species (ROS) are the oxygen produced by organisms that breathe organically by the enzymes in cells and the stimulation of various actions. Reactive oxygen species have a free radical, which means they are not stable, and thus have strong activity. Reactive oxygen species are known to produce toxic or oxidative stress in living organisms when production is excessive. It causes fatal damage in cell tissues by oxidizing huge molecules such as proteins or lipids in the cells, destroying the homeostasis of cells and killing the cells, and causing cancer, muscle dysplasia, Alzheimer's disease, Parkinson's disease, ischemic diseases, Causes of various degenerative diseases such as sclerosis or aging. Reactive oxygen species are representative of superoxide anion radicals (O2-), hydrogen peroxide (H ₂ O ₂ ), hydroxyl radicals (OH).

On the other hand, it is known through many studies that reactive oxygen species are involved in aging of the skin. Accordingly, many studies and studies on antioxidants that inhibit oxidation by active oxygen have been made. Antioxidants are widely distributed in copper and plant systems, and many phenolic compounds, flavonoids, tocopherols, vitamin C, and selenium are known in fruits and vegetables.

The damage caused by free radicals is defended by the ability of the living body, but the defense is not made of 100%, the body is subjected to some harmful effects by free radicals. This adverse action occurs slowly and, in some cases, for years or even throughout life, and accumulates, degrading the function of cells and tissues in the skin, which causes disease and aging.

Prostaglandin (PG) is produced by cyclooxygenase (COX) from arachidonic acid and regulates many biological functions, including homeostasis, reproduction and immune responses. Non-ideal overexpression of COX-2 has been frequently observed in several cancer tissues and transformed cells, leading to the accumulation of certain prostaglandins, in particular prostaglandin E ₂ (PGE ₂ ). Excessively increased PGE ₂ is known to play an important role in tumor formation and angiogenesis by stimulating the proliferation and metastasis of transformed cells.

Intracellular levels of PGE ₂ are regulated by synthesis and degradation. One of the major enzymes involved in the metabolism of prostaglandins is (NAD ⁺ ) -dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The enzyme 15 (S) of PGE ₂ - and greatly reduces the hydroxyl group is oxidized to the 15-keto-prostaglandin _{E 2 (15-keto prostaglandin E} 2, 15-Keto PGE 2) making the biological activity of PGE _2.

To date, little is known about the physiological role of 15-keto prostaglandin E ₂ . Recent studies on the mechanism by which 15-keto prostaglandin E ₂ inhibits the cancer process in pancreatic and liver cancers have begun to emphasize the unique role of 15-keto prostaglandin E ₂ .

HO-1, on the other hand, is an important enzyme that converts heme to biliverdin, carbon monoxide (CO) and iron. HO has three isomers. HO-2 and HO-3 are continuously expressed in cells, while HO-1 is a protein induced in stress, ischemia and inflammatory conditions, and is known to have mainly cytoprotective, anti-inflammatory, antioxidant and anti-proliferative effects. Induction of HO-1 in activated macrophages reduces the production of TNF-α, IL-6 and IL-1β, and overexpression of HO-1 results in COX-2 and inducible nitric oxide synthase. expression of oxide synthase (iNOS) is reduced. HO-1 has been recognized as an important molecule to protect cells and tissues from oxidative stress such as stimulation of free radicals through various experimental studies conducted on cells, animals and humans.

HO-1 gene expression is regulated by a variety of transcription factors, a representative of which is NF-E2-related factor-2 (NF-E2-related factor-2, Nrf2). Nrf2 is a redox-sensitive transcription factor and is known to regulate various antioxidant enzyme expressions. Nrf2 usually exists in the cytoplasm as an inactive complex with Keap1, but when activated it moves into the nucleus and binds to an antioxidant response element (ARE), which is NAD (P) H: quinone oxidation. Increases the expression of several detoxification / antioxidant enzymes such as quinone oxidoreductase (NQO1), glutathione S transferase (GST), and HO-1.

Nrf2's protective actions against oxidative damage have been suggested throughout the body, including the removal of carcinogens, the suppression of inflammation-causing agents, and the inhibition of the progression of degenerative brain diseases. Recently, a method for preventing various diseases related to oxidative stress in cells by activating Nrf2 has been proposed, and has been concentrating on the discovery and development of drugs that induce Nrf2 activity and expression of antioxidant enzymes. For example, Woongjin Pharmaceutical's Ursodeoxycholic acid (UDCA), a major component of bile, is known to be effective in improving liver function by protecting liver cells, which increases the expression of Nrf2. It has been shown to have this effect by increasing the expression of antioxidant enzymes.

As such, Nrf2 is known to play a role in detoxification of environmentally harmful substances in normal cells and to play a major role in protecting cells in oxidative stress conditions by leading to maintenance of antioxidant systems and increased Nrf2 expression in stress situations. have.

However, the effects of 15-keto prostaglandin E ₂ and Nrf2 on the antioxidant effect and antioxidant activity due to increased expression of Nrf2 and antioxidant enzymes via 15-keto PGE ₂ are not known.

Accordingly, the present inventors have diligently studied to overcome the problems of the prior art, in the case of a composition containing 15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ), increasing the expression of Nrf2 is an antioxidant enzyme The present invention was completed by increasing the activity of HO-1, generating low levels of ROS, and activating a defense mechanism in cells, thereby completing the present invention.

The main object of the present invention is to provide an antioxidant composition comprising 15-keto prostaglandin E ₂ as an active ingredient that can exhibit an antioxidant effect by increasing the expression of Nrf2 to increase the activity of the antioxidant enzyme HO-1. .

Another object of the present invention to provide a pharmaceutical composition, cosmetic composition and health functional food composition using the composition for antioxidant.

According to an aspect of the present invention, the present invention provides a pharmaceutical composition for antioxidant comprising 15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ) as an active ingredient.

Reactive Oxygen Species (ROS) can cause oxidative stress in living organisms when they are excessively produced, causing fatal damage in cell tissues, cancers, dysplasia, Alzheimer's disease, Parkinson's disease, ischemic diseases, and arteries. It is known to cause various degenerative diseases such as sclerosis and aging. Accordingly, the present inventors have searched for a substance capable of preventing or treating a disease caused by reactive oxygen species by reducing cellular damage caused by reactive oxygen species, and found that 15-keto prostaglandin E ₂ is suitable as an antioxidant, and the present invention. To complete.

In the antioxidant pharmaceutical composition of the present invention, the 15-keto prostaglandin E ₂ is characterized in that to activate the antioxidant enzyme.

In the antioxidant composition of the present invention, the antioxidant enzyme is hemeoxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (catalase), glutathione per Oxidase (glutathione peroxidase), reductase (glutathione reductase) and peroxyiredoxin (peroxiredoxin) may be at least one antioxidant enzyme selected from the group, preferably forceoxygenase-1 (hemeoxygenase-1; HO- It is characterized by being 1)

According to one embodiment of the present invention, as a result of confirming gene expression of HO-1 in cells treated with 15-keto prostaglandin E ₂ , the expression of HO-1 gene as the time of 15-keto prostaglandin E ₂ treatment increased It was confirmed that this braille increased. These results suggest that 15-keto prostaglandin E ₂ according to the present invention is suitable as a substance for exhibiting an antioxidant effect by activating an antioxidant enzyme (see Example 1 and FIGS. 1A to 1D).

In the pharmaceutical composition for antioxidant of the present invention, the 15-keto prostaglandin E ₂ is characterized by increasing the expression of Nuclear Factor-Erythroid derived 2-related factor-2 (Nrf2). Nrf2 is a transcription factor that acts when cell damage in the body occurs to produce various antioxidant enzymes. The HO-1 gene expression, which is an antioxidant enzyme, is known to be regulated by the Nrf2 transcription factor. Accordingly, the antioxidant effect of the activation of the Nrf2 transcription factor was confirmed.

According to one embodiment of the present invention, as a result of confirming the expression of Nrf2 in the cells treated with 15-keto prostaglandin E ₂ , it was confirmed that the expression of Nrf2 increases in a time-dependent manner, moreover, the activation of Nrf2 is HO-1 activity It was confirmed to increase the. These results suggest that these results suggest that 15-keto prostaglandin E ₂ according to the present invention is suitable as a substance for exerting an antioxidant effect by increasing the expression of Nrf2 and activating antioxidant enzymes (Example 2 and FIG. 2a to 2d).

In the antioxidant pharmaceutical composition of the present invention, the 15-keto prostaglandin E ₂ is characterized by promoting the expression of HO-1 by inducing phosphorylation of protein kinase B (AKT).

According to one embodiment of the invention, the result, 15-keto-prostaglandin E _2, make the HO-1 expression control Induced by the 15-keto-prostaglandin E _2, whereas to induce the phosphorylation of protein kinase B (AKT) protein Inhibitors of kinase B have been shown to reduce the expression of HO-1, an antioxidant enzyme induced by 15-keto prostaglandin E ₂ . These results suggest that 15-keto prostaglandin E ₂ activity is dependent on AKT, and 15-keto prostaglandin E ₂ promotes the expression of HO-1 by inducing phosphorylation of protein kinase B (AKT). It can be shown that (Example 4 and, Figures 4a to 4c).

In the antioxidant composition of the present invention, the 15-keto prostaglandin E ₂ is characterized in that it exhibits an antioxidant effect by inhibiting the phosphorylation of signal transducer and activator of transcription (STAT) 3. STAT3 activation is involved in the essential processes of many types of cell survival, growth and proliferation, and is a major target protein caused by immune diseases.

According to one embodiment of the invention, the result confirmed the expression of STAT3 To confirm the antioxidant effect of the 15-keto-prostaglandin E _2, 15-keto-prostaglandin E ₂ are STAT3 induced by Interleukin-6 (IL-6) It was confirmed that phosphorylation of was suppressed. These results suggest that 15-keto prostaglandin E ₂ may survive the cells by inhibiting the phosphorylation of STAT3 and have an antioxidant effect (see Example 5 and FIG. 5A). In addition, as a result of confirming the antioxidant effect by the active oxygen species hydrogen peroxide, the degree of phosphorylation of cleavage of PARP-1, a protein related to apoptosis induced by hydrogen peroxide, and BCl-2, an anti-apoptotic protein, was induced by 15-keto prostaglandin E ₂ . It was confirmed that the reduction phenomenon was protected by 15-keto prostaglandin E ₂ . These results suggest that 15-keto prostaglandin E ₂ protects against cell death by hydrogen peroxide, thereby exhibiting an antioxidant effect (see Example 5 and FIG. 5B).

In the antioxidant pharmaceutical composition of the present invention, the 15-keto prostaglandin E ₂ is characterized by showing an antioxidant effect by generating a low level of reactive oxygen species.

According to one embodiment of the present invention, it was confirmed that 15-keto prostaglandin E ₂ produced low levels of reactive oxygen species. These results indicate that 15-keto prostaglandin E ₂ can produce low levels of reactive oxygen species and promote expression of antioxidants such as HO-1, thereby preventing cell damage induced by potent reactive oxygen species such as hydrogen peroxide. (See Example 7 and Fig. 7).

In the antioxidant composition of the present invention, the pharmaceutical composition is one or more formulations selected from the group consisting of liquids, powders, aerosols, injections, infusions (ring gels), patches, capsules, pills, tablets and suppositories. It features.

In the antioxidant pharmaceutical composition of the present invention, the 15-keto prostaglandin E ₂ may be included in the concentration of 1 ~ 60μM compared to the pharmaceutical composition, preferably characterized in that it is included in a concentration of 10 ~ 50 μM. If the concentration is lower than the antioxidant effect may be insignificant, and if the concentration is higher than the concentration is not preferable in terms of economics.

According to one embodiment of the invention, 15-keto-prostaglandin E in order to determine the effects on expression of HO-1 by _2, 15-keto-prostaglandin E ₂ by processing the different concentrations result confirmed the expression of HO-1, 15 Keto prostaglandin E ₂ It was confirmed to increase HO-1 expression in a concentration dependent manner (see Example 3 and Figure 3a).

The pharmaceutical composition for antioxidant of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage is weight, age, sex of the patient. The range varies depending on health, diet, time of administration, method of administration, duration or interval of administration, excretion rate, constitution specificity, and the nature of the formulation.

The daily dosage of the composition comprising 15-keto prostaglandin E ₂ of the present invention is about 8 to 10 mg / kg, preferably about 9 to 9.5 mg / kg, but may be added or subtracted according to clinical trial results. It is preferable to divide and administer several times.

The pharmaceutical composition for antioxidant of the present invention may be formulated into various agents for administration. The pharmaceutical compositions can be formulated in various forms with the addition of excipients. The excipients are nontoxic and inert pharmaceutically suitable solid, semisolid or liquid formulation aids of all types, for example fillers, weights, binders, wetting agents, disintegrants, dispersants, surfactants or diluents and the like. Can be mentioned.

According to another aspect of the present invention, the present invention provides an antioxidant cosmetic composition comprising 15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ) as an active ingredient.

In the antioxidant cosmetic composition of the present invention, the cosmetic composition is characterized in that one formulation selected from the group consisting of supple cosmetics, nourishing cosmetics, nutrition lotion, nutrition cream, massage cream, nutrition serum, essence and pack.

According to another aspect of the present invention, it provides a health functional food composition for antioxidant containing 15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ) as an active ingredient.

In the antioxidant health functional food composition of the present invention, the health functional food includes the form of tablets, capsules, pills or liquids, and the health functional foods include dairy, including drinks, candy, snacks, noodles, ice cream Products, soups, beverages, including ionic beverages, alcoholic beverages and nutritional products including vitamin complexes may be included.

As it described above, in the case of a composition comprising a 15-keto-prostaglandin _{E 2 (15-keto prostaglandin E} 2) according to the invention, by increasing the expression of Nrf2 to increase the antioxidant effect of enzymatic activity of HO-1, low It produces a good level of ROS and activates the defense mechanisms within the cell, thus showing an excellent antioxidant effect.

1 is a result confirming HO-1 activation of 15-keto prostaglandin E2 (Fig. 1A: RT-PCR results, Figure 1B: Western blotting results, Figure 1C: Western blotting results according to the concentration of 15-keto PGE ₂ 1D: immunofluorescence results).
Figure 2 is a result confirming the Nrf2 activation of 5-keto prostaglandin E ₂ (Fig. 2A: Western blotting results, Figure 2B: immunofluorescence results, Figure 2C: Western blotting results, Figure 2D: ARE luciferase assay).
Figure 3 is a result of confirming the treatment effect of the concentration of the 15-keto prostaglandin E ₂ for each time, HO-1 and Nrf2 (Fig. 3A: HO-1 expression results according to the concentration, Figure 3B: HO-1 expression over time 3C: Nrf2 results over time).
4 shows the results of confirming the mechanism of HO-1 expression induced by 15-keto prostaglandin E ₂ .
5 is a result of confirming the antioxidant effect.
6 is a result confirming the antioxidant effect according to the prostaglandin E ₂ type.
7 shows the results of confirming ROS produced by prostaglandin E ₂ .

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Cell Culture and Drug Treatment

CCD841 cells, which are normal human colonic epithelial cells, and RAW 264.7 cells, which are mouse macrophage lines, were distributed by Prof. Seo Young-jun from Seoul National University College of Pharmacy. (Dulbecco Modified Eagle Medium), 10% FBS (HyClone Laboratories, Inc, Logan, Utah, USA), 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco, Grand Island, NY, USA) ) Was added and cultured. Cells were incubated at 37% incubator at 5% CO ₂ . 15-keto prostaglandin E ₂ (15-keto Prostaglandin E ₂ , 15-keto PGE ₂ ) was purchased from Cayman Chemical Inc, USA.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

CCD841 cells, human normal colonic epithelial cells, were treated with 15-keto prostaglandin E2 according to time and concentration, followed by lysis with trizol, followed by vortexing with 0.2 ml of chloroform. Then, the supernatant was taken by centrifugation for 15 minutes at 12,000 rcf. 0.5 ml isopropanol was added thereto and allowed to stand at room temperature for 10 minutes, followed by centrifugation at 4 ° C and 12,000 rcf for 10 minutes to precipitate RNA. After removing the supernatant, the RNA was washed with 75% ethanol, and the RNA was dried at room temperature after centrifugation at 7,600 rcf. After quantifying the isolated total RNA, according to the method provided by the manufacturer, reverse transcription reaction synthesizes cDNA using oligo dT primer and RT enzyme. PCR was performed using the synthesized cDNA as a template. GAPDH was used as an internal standard. PCR products were immersed in 1.5% agarose gel in 1 × TBE buffer (electrophoresis) and electrophoresed at 100 V for 20 minutes and stained with Red Safe (Intron Biotechnology Inc., USA).

Western blot analysis

CCD841 cells, which are normal human colonic epithelial cells, and RAW 264.7 cells, which are mouse macrophage lines, were cultured in a 60 mm culture vessel by 5 × 10 ⁵ , and proteins were extracted and Western blotting was performed. Two kinds of cultured cells were treated with 15-keto prostaglandin E ₂ at different times and concentrations, washed with PBS, scraped with a scraper and lysed with lysis buffer for 1 hour, and then 13,000 rpm at 4 ° C. The supernatant was extracted by centrifugation for 15 minutes. After protein quantification, 25 μg of protein was electrophoresed on SDS-polyacrylamide gel and transferred to PVDF membrane for 2 hours. Reactions are performed using relevant antibodies, including HO-1, and detection using a detection solution with enhanced chemiluminascence (ECL).

Immunocytochemistry

CCD841 cells, which are human normal colonic epithelial cells, were cultured in 8 chamber slides, and then treated with 15-keto prostaglandin E ₂ for 12 hours. After washing with PBS, fixed at room temperature for 30 minutes using 10% neutral buffered formalin, and then incubated with 1% bovine serum albumin (BSA) for 1 hour at room temperature for nonspecific binding. Blocking. Incubated overnight at 4 ° C. with the primary antibody, anti-Nrf2 antibody, washed with PBS and then incubated for 1 hour at room temperature with FITC-bound secondary antibody. Cells stained with propidium iodide (PI) for 10 minutes after termination are confirmed using a confocal laser microscope (Confocal Microscope).

Reactive oxygen species

CCD841 cells, which are normal human colonic epithelial cells, are cultured daily on a 4 chamber slide at 2.5 X 10 ⁴ . Cultured cells were treated with 40 μM 15-keto prostaglandin E ₂ and 5 mM antioxidant N-acetyl-L-cysteine (NAC) for 12 hours. After washing with PBS, Hank's Balanced Salt Solution (HBSS) (Gibco, Grand Island, NY, USA) was mixed with 10 μM of DCF-DA, a fluorescent substance that can detect free radicals, and added to the cells. Incubate for minutes. Fluorescently stained cells are identified using a confocal laser microscope.

ARE luciferase assay

CCD841 cells, which are normal human colonic epithelial cells, were transfected with pCMV-β-galactosidase and luciferase reporter gene fusion construct (pGL2) or ARE Luciferase reporter gene for 36 hours, followed by 15-keto prostaglandin E ₂ for 12 hours. Treated. Activity was measured using promega luciferase asssay systems and quantified after analysis using beta-galactosidase Enzyme assay system with Reporter lysis buffer.

Statistical processing

The results of the experimental analysis were expressed as mean ± SEM, and the significance between each experimental group was performed by the T-test test, and the comparison between the experimental groups was one-way ANOVA (Prime 7, Graph-pad, San Diego, CA, USA). variance) was used to test the significance at * P <0.05, ** P <0.01, *** P <0.001 after statistical processing.

Example 1: 15-keto prostaglandin E ₂ HO-1 activation

15 to determine the antioxidant effects of HO-1 activation of the keto-prostaglandin E _2, 15-keto-prostaglandin E ₂ 40 μM treatment of a human to normal colon cells and the cells CCD841 of reverse transcriptase-polymerase chain reaction (RT- PCR) confirmed that, as time passes, the expression of HO-1 gene gradually increased (FIG. 1A). As a result of confirming HO-1 protein expressed in human normal colon cell CCD841 cells through western blotting, HO-1 expression gradually increased over time as in RT-PCR results (FIG. 1B). In addition, after treatment with 15-keto prostaglandin E2 by concentration (control, 20 μM, 40 μM), the expression of HO-1 protein was confirmed by Western blotting. Also increased (** p <0.01) was confirmed (Fig. 1C). Expression of HO-1 was also increased when confirmed by immunofluorescence (Immunocytochemistry) 12 hours after 40-M treatment of 15-ketoprostaglandin E2 in colonic cells (Fig. 1D).

Example 2: 15-keto prostaglandin E ₂ Activation of Nrf2 on Windows

Human normal colon cell CCD841 cells were treated with 40 μM 15-keto prostaglandin E ₂ and confirmed by Western blotting, indicating that the protein level of Nrf2 increased time-dependently and the highest expression level appeared after 12 hours of treatment. It was confirmed (** p <0.01) (FIG. 2A). In addition, the state of the Nrf2 expressed in the nucleus was confirmed by immunofluorescence (Fig. 2B). In addition, it was confirmed that the Nrf2 was knocked down using the Lipofectamine RNAiMAX Transfection method and HO-1 expressed by 15-keto prostaglandin E ₂ was achieved through Nrf2 activation (FIG. 2C). In addition, CCD 841 cells were co-transfected with pCMV-β-galactosidase and luciferase reporter gene fusion construct (pGL2) or ARE Luciferase reporter gene for 36 hours by ARE luciferase assay, followed by DMSO or 15-ketoprostaglandin E ₂ . Time was processed. The Nrf2 activity of these cells was analyzed for ARE transcriptional activity and 15-keto prostaglandin E ₂ also increased the transcriptional activity of Nrf2 (2D).

Example 3: 15-keto prostaglandin E for HO-1 and Nrf2 ₂ Check the treatment effect by concentration and time

To determine the effect of 15-keto prostaglandin E ₂ on the expression of HO-1 and its transcription factor Nrf2, first, 15-keto prostaglandin E ₂ was expressed in concentrations (0, 10 μM) in RAW 264.7 cells, a mouse macrophage line. , 20 μM, 40 μM) was confirmed by Western blotting the expression of HO-1 protein, it was confirmed that the expression of HO-1 increased depending on the 15-keto prostaglandin E ₂ concentration (Fig. 3A). On the other hand, 15-keto prostaglandin E ₂ 20 μM treatment time was changed (0, 3, 6, 12, 24 hours) to confirm the expression level of HO-1 protein, HO-expression is increased in a time-dependent manner It was confirmed (Fig. 3B). In addition, as a result of confirming the expression level of Nrf2 protein at different treatment times (0, 3, 6, 12, 24 hours) of 15-keto prostaglandin E ₂ 20 μM, it was confirmed that the expression of Nrf2 was increased in a time-dependent manner as well. (Figure 3C).

Example 4: 15-keto prostaglandin E ₂ Identification of HO-1 expression regulators

15-keto-prostaglandin E ₂ treatment and 1.5 hours 40 μM in human normal colon cells CCD841 cells, 3 hours, 6 hours, 12 hours by Western blotting confirmed the various genes result, 15-keto-prostaglandin E ₂ and the AKT It was confirmed that upregulation of phosphorylation of ERK (FIG. 4A). In addition, the colonic normal cells were pretreated with 10 mM of LY294002 (AKT inhibitor), U0126 (MEK1 / 2 inhibitor, ERK upstream), SB203580 (p38 MAPK inhibitor) or SP600125 (JNK inhibitor), respectively, for 1 hour, followed by 15- for 12 hours. Keto prostaglandin E ₂ treatment. 15-keto prostaglandin E ₂ induced Akt phosphorylation, and it was confirmed that the expression of HO-1 induced by 15-keto prostaglandin E ₂ was reduced by the Akt pharmacological inhibitor LY294002 (FIG. 4B). In a further study, inhibition of AKT activity after 15 hours of 15-keto prostaglandin E ₂ treatment with LY294002 (AKT inhibitor) in colonic cells for one hour reduced Nrf2 expression and decreased HO-1 expression. It was confirmed. It was found that 15-keto prostaglandin E ₂ activity was dependent on AKT (FIG. 4C).

Example 5: Confirm the antioxidant effect

Inflammatory factor IL-6 was pretreated at 25 ng / ml for 12 hours in normal colorectal CCD841 cells, and then treated with 15-keto prostaglandin E ₂ 40 μM for 12 hours, resulting in 15-keto prostaglandin E2 (Interleukin-6 ( Inhibition of phosphorylation of STAT3 induced by IL-6) was confirmed via western blotting (FIG. 5A). In addition, 300 μM of hydrogen peroxide (H ₂ O ₂ ) was treated for 6 hours on colon cell CCD841 cells. Hydrogen peroxide, a representative reactive oxygen species, induces cleavaged apoptosis-related protein PARP-1 in normal colon cell CCD841 cells. Inhibition of phosphorylation of the -apoptotic protein BCl-2, 15-keto prostaglandin E ₂ inhibited this phenomenon. That is, 15-keto prostaglandin E2 protected apoptosis by hydrogen peroxide (FIG. 5B).

Example 6: Prostaglandin E ₂  Check the effect by type

Prostaglandin E was doing in order to determine the antioxidant activity according to the type of Figure _2, determine the 15-keto-prostaglandin E ₂ and 3, 14-dihydro-HO-1 expression and activation of Nrf2 of prostaglandin E _2. As a result, the 13,14-dihydro prostaglandin E ₂ did not induce HO-1 expression and migration into the nucleus of Nrf2, whereas the 15-keto prostaglandin E ₂ induced HO-1 expression and Nrf2 into the nucleus. It was confirmed that (Fig. 6B).

Example 7: 15-keto prostaglandin E ₂ ROS generation on

The antioxidant effect according to ROS produced by 15-keto prostaglandin E ₂ was confirmed. ROS production by 15-keto prostaglandin E ₂ was confirmed by DCF-DA staining (FIG. 7A), and expression of Nrf2 and HO-1 was measured by western blotting (FIG. 7B). As a result, ROS produced by 15-keto prostaglandin E ₂ by the antioxidant N-acetylcysteine was eliminated (lower left of FIG. 7A), the migration of Nrf2 into the nucleus and the expression of HO-1 were also reduced (FIG. 7B). ). It was confirmed that blocking ROS through Luciferase assay reduced ARE binding activity of Nrf2 (FIG. 7C). Normal colon cell CCD841 cells were incubated with 15-keto prostaglandin E ₂ (40 μM) for 12 hours with or without DDT (500 mM) as a reducing agent. As a result, 15-keto prostaglandin E ₂ was reduced with Dithiothreitol (DTT) as a reducing agent. Intranuclear migration of Nrf2 was reduced and HO-1 expression was also reduced (FIG. 7D).

Claims (11)

15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ) comprising an active pharmaceutical composition for antioxidant.
The method of claim 1,
The 15-keto prostaglandin E ₂ is an antioxidant pharmaceutical composition, characterized in that to activate the antioxidant enzyme.
The method of claim 2,
The antioxidant enzyme is HO-1 (hemeoxygenase-1) characterized in that the pharmaceutical composition for antioxidant.
The method of claim 1,
The 15-keto prostaglandin E ₂ is characterized in that to increase the expression of Nrf2 (Nuclear Factor-Erythroid derived 2-related factor-2).
The method of claim 1,
The 15-keto prostaglandin E ₂ is an antioxidant pharmaceutical composition, characterized in that to promote the expression of HO-1 by inducing phosphorylation of protein kinase B (AKT).
The method of claim 1,
The 15-keto prostaglandin E ₂ is characterized in that the antioxidant effect by inhibiting the phosphorylation of signal transducer and activator of transcription (STAT) 3, characterized in that the antioxidant composition.
The method of claim 1,
The 15-keto prostaglandin E ₂ is an antioxidant pharmaceutical composition characterized in that it exhibits an antioxidant effect by generating reactive oxygen species.
The method of claim 1,
The pharmaceutical composition is an antioxidant pharmaceutical composition, characterized in that it is formulated in one formulation selected from the group consisting of liquids, powders, aerosols, injections, fluids (ring gels), patches, capsules, pills, tablets and suppositories. .
The method of claim 1,
The 15-keto prostaglandin E ₂ is an antioxidant pharmaceutical composition, characterized in that contained in a concentration of 10 to 50 μM compared to the pharmaceutical composition.
15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ) comprising an antioxidant cosmetic composition as an active ingredient.
Health functional food composition for antioxidant containing 15-keto prostaglandin E _{2 (} 15-keto prostaglandin E ₂ ) as an active ingredient.

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