KR101881722B1 - Pharmaceutical composition for preventing or treating of psoriasis comprising expressing or activity inhibitors of inflammatory mediator of eosinophil - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating psoriasis comprising an expression or an activity inhibitor of an eosinophil inflammatory regulator as an active ingredient. According to the present invention, the inflammatory regulator of an eosinophil increases the expression in the EoL-1 cell line, a cell line of an eosinophil, by TLR7 and inhibits an increase by imiquimod causing psoriasis in an animal model from which eosinophil cells are removed. The inhibition of the inflammatory regulator reduces eosinophil cell counts. Also, cytotoxic granulocytes of the eosinophil cell are significantly increased in tissue acquired from a lesion of a psoriasis patient to effectively use a material inhibiting expression or activity of the inflammatory regulator of the eosinophil in the treatment of psoriasis.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for the prevention or treatment of psoriasis, which comprises as an active ingredient an expression or activity inhibitor of an inflammatory modulator of eosinophil. [0002] The present invention relates to a pharmaceutical composition for the prevention or treatment of psoriasis,

The present invention relates to a pharmaceutical composition for the prophylaxis or treatment of psoriasis comprising as an active ingredient an expression or activity inhibitor of an inflammatory modulator of eosinophils.

Psoriasis is a chronic inflammatory skin disorder caused by abnormal proliferation and differentiation of keratinocytes due to increased infiltration of immune cells. Although the exact etiology of psoriasis is not known, antigen stimulation induces the activation of innate immune cells, resulting in proinflammatory cytokines in the skin resulting in the differentiation of Th1 and Th17 cells. IL-17 and IL-22 secreted by T cell-activated keratinocytes increase the production of proinflammatory cytokines and chemokines, which promote the inflammatory portion of psoriasis. The important role of the innate immune response in the early and developmental stages of psoriasis is emphasized by the efficacy of anti-tumor necrosis factor-α (TNF-α) used in the treatment of psoriasis.

The congenital immune system is a Toll-like receptor expressed in a variety of immune cells, including monocytes, macrophages, dendritic cells (DCs) and granulocytes. Lt; / RTI > receptor (TLR), which is known to be involved in pathogenesis. Signals through TLRs stimulate the development of an inflammatory environment in the skin, thereby forming congenital and acquired immune responses. At this time, expression of TLR1 and TLR2 is increased in keratinocytes of psoriatic skin, and skin-damage-associated antimicrobial peptide (TLR9), which is activated by its own DNA, induces the production of congenital cytokines. ≪ / RTI > The TLR7 agonist imiquimod (IMQ) aggravates psoriasis by increasing the production of cytokines associated with congenital immune responses and causes psoriatic dermatitis in mouse models. On the other hand, increased expression of TLRs 3, 7, 8 and 9 was observed in peripheral blood mononuclear cells of patients with psoriasis, indicating that activation of the immune system and activation of intracellular ligands released from damaged tissues .

The treatment methods of psoriasis in general are divided into local treatment, systemic treatment or light therapy. Recently, various immunological biological agents based on the etiology of psoriasis have been developed. In addition, multiple therapies are used in which the therapeutic methods are appropriately mixed to increase the psoriasis treatment effect and reduce side effects. Topical treatments are the most commonly used to treat mild psoriasis, and many of these treatments have beneficial effects on psoriasis alone. In particular, when psoriasis patients have systemic diseases such as digestive disorders, liver or kidney disorders, local treatment is safer and more effective than systemic treatment. In this regard, Korean Patent Registration No. 10-1492465 discloses a pharmaceutical composition containing an alcoholic organic acid copper compound, an iodine solution and an alcohol, and using polyethylene glycol or vaseline to treat psoriasis.

Eosinophils are proinflammatory granulocytes, are involved in the pathogenesis of allergic diseases, and defense responses to parasitic and viral infections. Mature eosinophils have cytotoxic granules containing major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Eosinophils also release a wide variety of cytokines, chemokines, and lipid mediators, which indicate that eosinophils are involved in multiple proinflammatory functions. TLR-mediated eosinophil activation leads to the secretion of several proinflammatory regulators by increasing the production of EDN and IL-8 from eosinophils stimulated by cognate ligands such as TLR7 and TLR9. The effect of eosinophils on inflammatory responses has been reported by several studies, but the role of eosinophils in the pathogenesis of psoriasis has not been clearly elucidated.

Accordingly, the present inventors have found that when TLR7 ligand is administered to an eosinophilic granule-containing cell, the expression of an inflammatory regulator secreted from eosinophils increases The psoriasis animal model prepared by using eosinophil-deprived mouse was significantly inhibited in the expression level of inflammatory regulator of eosinophil compared to the psoriasis animal model prepared by wild-type mouse, Neutropenia increased in each tissue and eosinophil granulocyte differentiation and activity were increased by the factors secreted from eosinophils and cytotoxic granulocytes of eosinophils were significantly , Thereby completing the present invention.

Korean Patent No. 10-1492465

It is an object of the present invention to provide a use for the treatment of psoriasis of an inflammatory regulator of eosinophil expression or an activity inhibitor.

It is another object of the present invention to provide a method for screening candidate substances for psoriasis by measuring the level of expression or activity of inflammatory modulators of eosinophils in differentiated eosinophils.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of psoriasis comprising as an active ingredient an expression or activity inhibitor of an inflammatory mediator of eosinophils.

The present invention also provides a health functional food for preventing or ameliorating psoriasis, which comprises as an active ingredient an expression or activity inhibitor of an inflammatory modulator of eosinophils.

The present invention also provides a cosmetic composition for preventing or ameliorating psoriasis, which comprises as an active ingredient an agent for inhibiting the expression or activity of an inflammatory modulator of eosinophils.

Furthermore, the present invention provides a method of treating a subject, comprising the steps of: treating a differentiated eosinophil cell with a toll-like receptor 7 ligand and a test substance; Measuring the level of expression or activity of the inflammatory regulator of the differentiated eosinophil cells treated with the test substance; And screening a test substance having a reduced level of expression or activity level of an inflammatory modulator of eosinophil cells compared to a control group not treated with the test substance.

The inflammatory regulator of eosinophils according to the present invention is expressed by TLR7 in the eosinophil cell line EoL-1 cell line, and the increase by the imiquimod that induces psoriasis in the animal model in which the eosinophil cell is removed is suppressed. Inhibition of inflammatory regulators reduces neutrophil cells. In addition, the cytotoxic granulocyte of the eosinophils is significantly increased in tissues obtained from lesions of psoriasis patients, and substances that inhibit the expression or activity of inflammatory modulators of the eosinophils can be useful for the treatment of psoriasis.

FIG. 1 is a result of confirming the expression of TLR7 gene by real-time PCR (A) and RT-PCR (B) in order to confirm whether EoL-1 cell line as a human eosinophil cell line can be used as a model cell of eosinophils.
FIG. 2 is a graph showing the change in the expression of CCR3 and TLR7 genes in the EoL-1 cell line differentiated with butyrate by real-time PCR.
FIG. 3 is a graph showing cell survival rate changes of EoL-1 cell line differentiated with butyrate. FIG.
FIG. 4 is a graph showing the change in expression of a gene encoding an inflammatory cytokine by real-time PCR after addition of a TLR7 ligand to EoL-1 cell line differentiated with nitrate. FIG.
FIG. 5 is a diagram showing the expression of inflammatory cytokines in the EoL-1 cell line (A) and R837-treated differentiated EoL-1 cells (B) by protein array analysis.
FIG. 6 is a result of observing whether or not psoriasis has occurred after application of vaseline (Control) or IMQ cream (IMQ) to wild-type mouse (WT) and eosinophil-deprived mouse (ΔdblGATA).
FIG. 7 is a graph showing the results of evaluating ear thickness, erythema and scarring after application of IMQ cream (IMQ) to a wild-type mouse (WT) and a mouse (ΔdblGATA) from which eosinophil cells have been removed.
Figure 8 shows the results of H & E staining of back skin and ear skin tissues after application of IMQ cream (IMQ) to wild-type mouse (WT) and eosinophil cell-free mouse (ΔdblGATA) It is the result of checking the degree of onset.
FIG. 9 shows the results of real-time PCR analysis of changes in expression of genes encoding inflammatory cytokines after application of vaseline (control) or IMQ cream (IMQ) to wild-type mouse (WT) Graph.
Figure 10 shows the composition of cell populations present in the skin, axillary lymph nodes and spleen of the mouse after application of Vaseline (Control) or IMQ cream (IMQ) to wild-type mouse (WT) and eosinophil- Flow cytometry analysis.
FIG. 11 shows the results of culturing the HL-60 cell line in a culture medium (HL-60 + control medium), a culture medium of the EoL-1 cell line (HL-60 + EoL-1 UCM) 60 + EoL-1 DRCM), and a change in the expression of the gene encoding the inflammatory cytokine (B).
12 is a graph showing a change in the expression of cytotoxic granulocyte genes of eosinophils in tissues obtained from lesions of psoriasis patients by real-time PCR.
FIG. 13 is a chart showing the infiltration of neutrophils by H & E staining of lesions of psoriasis patients.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of psoriasis comprising as an active ingredient an expression or activity inhibitor of an inflammatory mediator of eosinophils.

As used herein, the term "inflammatory mediator" refers to factors such as cytokines, chemokines, etc., which are known in the art and are associated with an inflammatory response. The inflammatory regulator may be an inflammatory regulator secreted by the activation of eosinophils and specifically an inflammatory regulator secreted by TLR-mediated eosinophil activation. Inflammatory regulators secreted through the TLR-mediated pathway in activated eosinophils are well known in the art.

The inflammatory regulator of the eosinophils may increase the expression or activity of neutrophils. Psoriasis can be caused or exacerbated by increased neutrophil expression or activity by inflammatory regulators of eosinophils. The inflammatory regulators of the eosinophils are IL1B, IL23, interferon-γ, interleukin-6, TNF, IL22, IL17 (interleukin-17), interleukin-8, interleukin-1 receptor antagonist, macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1), CXCL12 12), CCL5 (CC chemokine ligand 5), PRG2 (proteoglycan 2), and EDN (eosinophil-derived neurotoxin).

The pharmaceutical composition according to the present invention may contain an inhibitor of the expression of an inflammatory modulator of eosinophils as an active ingredient. The expression inhibitor may include any substance known in the art as long as it inhibits the mRNA of a gene encoding an inflammatory regulator of eosinophils. The expression inhibitor may be any one selected from the group consisting of antisense nucleotides, siRNA, and shRNA complementarily binding to the mRNA of the inflammatory regulator of eosinophils.

Said antisense nucleotide, siRNA and shRNA can be suitably prepared by a person skilled in the art if the mRNA sequence of the target gene for suppressing its expression is known. That is, the sequences of cytokines, chemokines, etc., which are inflammatory regulators of eosinophils according to the present invention, are apparent to those skilled in the art, and antisense nucleotides, siRNAs and shRNAs can be prepared from the known sequences.

The expression inhibitor may include one or more substitutions, insertions, deletions, and combinations thereof that do not degrade its activity. Such variants may have 80%, 90%, 95%, 98% or more homology with the expression inhibitor sequences of the present invention. The expression inhibitor may be prepared by various methods known in the art, such as chemically synthesized or synthesized by in vitro transcription.

 The antisense nucleotide can be complementarily (hybridized) to the base sequence of DNA, immature mRNA or mature mRNA, as defined in the base pair of Watson-click, thereby inhibiting the expression of an inflammatory regulator.

The siRNA refers to short double stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA. In addition, the siRNA is not limited to a complete pair of double-stranded RNA portions paired with each other in RNA, but also includes a mismatch (corresponding base is not complementary), a bulge (no base corresponding to one side chain) It can also include non-paired parts. The siRNA end structure can be either a smooth end or a protruding end if the target gene can be repressed by RNA interference, and the 3 'end protruding structure and the 5' end protruding structure are both possible.

The shRNA may be a double-stranded RNA of a hairpin structure having a loop region of a base constituting the shRNA. The base in such a loop region may be those known in the art, and the double stranded portion of the RNA may be constructed in the same manner as an siRNA that may have the characteristics as described above.

In addition, the pharmaceutical composition according to the present invention may contain, as an active ingredient, an inhibitor of the activity of an inflammatory regulator of eosinophils. The activity inhibitor may include any substance commonly known as a substance that inhibits the activity of an inflammatory regulator of eosinophils. The activity inhibitor may be any one or more selected from the group consisting of peptides, peptide mimetics, substrate analogs, aptamers, and antibodies that complementarily bind to inflammatory modulators of eosinophils.

The polypeptide sequence of the inflammatory regulator of the eosinophil may be a sequence well known in the art. The polypeptide may be a variant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. Amino acid exchanges in proteins or peptides that do not globally alter the activity of the molecule are known in the art. In some cases, it may be modified by phosphoric acid, sulfidation, acrylation, saccharification, methylation, phaneticylation, and the like. Thus, the polypeptide may comprise a known polypeptide sequence, variants or fragments thereof.

Such peptides and peptide mimetics may inhibit the activity of inflammatory regulators of eosinophils by inhibiting the binding of eosinophilic inflammatory modulators to other proteins. The peptide may be a mutant or fragment of an amino acid having a different sequence by deletion, insertion, substitution, or a combination of amino acid residues within a range that does not affect the function of the protein. The non-hydrolyzable peptide mimetics can be prepared by using as main residues a β-turn dipeptide core, keto-methylene pseudopeptides, azepine, benzodiazepine, β-amino alcohol or substituted γ-lactam ring.

The antibody may be a monoclonal antibody, a polyclonal antibody or a recombinant antibody. Antibodies to a particular protein can be readily prepared using techniques well known in the art if the sequence of the protein is known. Such monoclonal antibodies can be prepared using hybridoma methods or phage antibody library techniques known in the art. Generally, hybridoma cells that secrete monoclonal antibodies can be made by fusing immune cells and cancer cell lines isolated from immunologically appropriate host animals, such as mice injected with antigen protein. The cell fusion of these two groups is fused using a method known in the art such as polyethylene glycol, and the antibody producing cells are proliferated by a standard culture method. Subcloning is performed using a limiting dilution method to obtain a uniform cell population, and hybridoma cells capable of producing an antigen-specific antibody can be produced in vitro or in vivo. The antibodies prepared by the above methods can be separated and purified by methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.

The polyclonal antibody can be prepared by injecting an immunogen-causing biomarker protein or fragment thereof into an external host according to methods known in the art. The external host may be a mammal such as a mouse, rat, sheep, or rabbit. When the immunogen is injected intra-muscularly, intraperitoneally or subcutaneously, it may be administered with an adjuvant to increase antigenicity. Thereafter, blood can be routinely taken from an external host to obtain serum showing improved titer and specificity for the antigen, and separating and purifying the antibody therefrom.

In a specific example of the present invention, the present inventors treated the EoL-1 cell line, which is a human eosinophilic cell line, with a butyrate to differentiate the EoL-1 cell line into differentiated EoL-1 cells, i.e., eosinophilic granule- , Indicating that the expression level of the TLR7 gene was increased (see FIGS. 1 and 2). In addition, the expression of eosinophil-like inflammatory regulators such as IL1B, IL23, and IFNG is increased in the differentiated EoL-1 cells and the inflammatory regulation of eosinophils such as IL-1ra, CXCL12, MIF, PAI-1 and IL- And the activity of the factor was increased (see FIGS. 4 and 5).

On the other hand, when IMQ cream was applied to the DELTA dblGATA mouse from which eosinophil was removed, psoriasis was induced by application of IMQ cream to the wild type mice. The degree of psoriasis was less than that of psoriasis caused by ear thickness, erythema (See Figs. 6 to 8). In addition, the expression of various cytokines such as IL6, TNF, IL23, CCL11 and the like was significantly increased when psoriasis was induced in the wild type mouse, but the increase was suppressed in the eosinophilized mouse (see FIG. 9). Furthermore, the composition of cell populations existing in the skin, axillary lymph nodes and spleen of the mice was confirmed, and it was confirmed that the number of neutrophils increased in the wild-type psoriasis model mice was inhibited in the ΔdblGATA psoriasis model mouse (see FIG. 10).

In addition, the present inventors cultured HL-60 cell line using the culture medium of EoL-1 cell line or differentiated EoL-1 cell to confirm neutrophil differentiation and activity change by eosinophil. As a result, The differentiation and activity of the HL-60 cell line cultured using the culture medium of differentiated eosinophils was increased (see FIG. 11).

In addition, the expression of PRG2 and EDN, the cytotoxic granulocyte of eosinophils, was significantly increased in the tissues obtained from psoriatic lesions (see Fig. 12), and infiltration of neutrophils was observed in the tissues where eosinophils were present 13).

Therefore, it has been found from the above that the activation of eosinophils, specifically the inflammatory regulator secreted from eosinophils, significantly increases the activity of neutrophils, which are known to induce or exacerbate psoriasis, And it can be used effectively.

The pharmaceutical composition may contain 10 to 95% by weight of an effective amount of an inhibitor of the expression or activity of an inflammatory modulator of eosinophils according to the present invention, which is an active ingredient, based on the total weight of the pharmaceutical composition. In addition, the pharmaceutical composition of the present invention may further contain at least one active ingredient which exhibits the same or similar functions in addition to the above-mentioned effective ingredients.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof commonly used in biological formulations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo. Examples include Merck Index, 13th ed., Merck & Inc. Sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of at least one of these components. At this time, other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added as necessary.

When the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

The composition of the present invention may be formulated as an oral preparation or a parenteral preparation. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose, Lactose, gelatin, and the like. Lubricants such as magnesium stearate and talc may also be added. On the other hand, liquid formulations include suspensions, solutions, emulsions or syrups, which may contain excipients such as wetting agents, sweeteners, fragrances, preservatives and the like.

Formulations for parenteral administration may include injections such as sterile aqueous solutions, non-aqueous solutions, suspensions, and emulsions.

Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like.

The composition of the present invention can be administered orally or parenterally according to the desired method, and parenteral administration can be carried out by external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, Can be selected.

The composition according to the invention is administered in a pharmaceutically effective amount. It may vary depending on the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, The composition of the present invention may be administered alone or in combination with other therapeutic agents. When administered concomitantly, the administration can be sequential or simultaneous.

However, for the desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.001 to 10,000 mg / kg, specifically 0.1 g to 5 g / kg. The administration may be one time per day and may be divided into several times.

The present invention also provides a health functional food for preventing or ameliorating psoriasis, which comprises as an active ingredient an expression or activity inhibitor of an inflammatory modulator of eosinophils.

The expression or activity inhibitor of the inflammatory regulator of eosinophils may have the same characteristics as described above. For example, the inflammatory modulator of the eosinophils may be a factor that increases the expression or activity of neutrophils, and specifically IL1B, IL23, IFNG, IL6, TNF, IL22, IL17, IL-8, IL- PAI-1, CXCL12, CCL5, PRG2 and EDN.

The expression inhibitor may be any one selected from the group consisting of an antisense nucleotide complementary to the mRNA of the gene encoding the inflammatory regulator of eosinophils, siRNA and shRNA. Meanwhile, the activity inhibitor may be any one selected from the group consisting of peptides, peptide mimetics, substrate analogues, aptamers, and antibodies that complementarily bind to the inflammatory regulator of eosinophils.

Specifically, in an embodiment of the present invention, the present inventors have found that expression of the TLR7 gene in the differentiated EoL-1 cell line is increased, gene expression of an inflammatory regulator of eosinophils such as IL1B, IL23, and IFNG is increased, (See FIGS. 1, 2, 4, and 5), the activity of inflammatory regulators of eosinophils such as CXCL12, MIF, PAI-1, and IL-8 was increased.

In addition, when IMQ cream was applied to the DELTA dblGATA mouse from which eosinophil was removed, psoriasis was induced, the occurrence of psoriasis was weaker than that of IMQ cream applied to the wild type mouse, and it was increased by the onset of psoriasis And the number of neutrophils contained in the tissues of the mice was also decreased (see Figs. 6 to 10).

In addition, when HL-60 cells were cultured using the medium in which the differentiated eosinophils were cultured, differentiation and activity of HL-60 cells were promoted (see FIG. 11).

Furthermore, expression of PRG2 and EDN, the cytotoxic granulocyte of eosinophils, was significantly increased in the tissues obtained from psoriatic lesions (see FIG. 12), and infiltration of neutrophils was observed in the tissues where eosinophils were present 13).

Thus, it has been found from the above that the activation of eosinophils, specifically the inflammatory regulator secreted from eosinophils, significantly increases the activity of neutrophils, which are known to induce or exacerbate psoriasis, And it can be used effectively.

The expression or activity inhibitor of the inflammatory regulator of the eosinophils of the present invention may be added intact to the food or may be used with other food or food ingredients. At this time, the content of the active ingredient to be added may be determined depending on the purpose. Generally, the content in the health functional food may be 0.01 to 90 parts by weight of the total food weight.

There is no particular limitation on the form and kind of the health functional food. The form of the health functional food to which the substance is added may be a tablet, a capsule, a powder, a granule, a liquid, a ring and the like.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as an additional ingredient such as a normal health functional food. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohol, and the like. These components may be used independently or in combination. The proportion of such additives may be selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides a cosmetic composition for preventing or ameliorating psoriasis, which comprises as an active ingredient an agent for inhibiting the expression or activity of an inflammatory modulator of eosinophils.

The expression or activity inhibitor of the inflammatory regulator of eosinophils may have the same characteristics as described above. For example, the inflammatory modulator of the eosinophils may be a factor that increases the expression or activity of neutrophils, and specifically IL1B, IL23, IFNG, IL6, TNF, IL22, IL17, IL-8, IL- PAI-1, CXCL12, CCL5, PRG2 and EDN.

The expression inhibitor may be any one selected from the group consisting of an antisense nucleotide complementary to the mRNA of the gene encoding the inflammatory regulator of eosinophils, siRNA and shRNA. Meanwhile, the activity inhibitor may be any one selected from the group consisting of peptides, peptide mimetics, substrate analogues, aptamers, and antibodies that complementarily bind to the inflammatory regulator of eosinophils.

Specifically, in an embodiment of the present invention, the present inventors have found that expression of the TLR7 gene in the differentiated EoL-1 cell line is increased, gene expression of an inflammatory regulator of eosinophils such as IL1B, IL23, and IFNG is increased, (See FIGS. 1, 2, 4, and 5), the activity of inflammatory regulators of eosinophils such as CXCL12, MIF, PAI-1, and IL-8 was increased.

In addition, when IMQ cream was applied to the DELTA dblGATA mouse from which eosinophil was removed, psoriasis was induced, the occurrence of psoriasis was weaker than that of IMQ cream applied to the wild type mouse, and it was increased by the onset of psoriasis And the number of neutrophils contained in the tissues of the mice was also decreased (see Figs. 6 to 10).

In addition, when HL-60 cells were cultured using the medium in which the differentiated eosinophils were cultured, differentiation and activity of HL-60 cells were promoted (see FIG. 11).

Furthermore, expression of PRG2 and EDN, the cytotoxic granulocyte of eosinophils, was significantly increased in the tissues obtained from psoriatic lesions (see FIG. 12), and infiltration of neutrophils was observed in the tissues where eosinophils were present 13).

Thus, it has been found from the above that the activation of eosinophils, specifically the inflammatory regulator secreted from eosinophils, significantly increases the activity of neutrophils, which are known to induce or exacerbate psoriasis, And it can be used effectively.

The cosmetic composition of the present invention may contain 0.1 to 50% by weight, specifically 1 to 10% by weight, of an agent for inhibiting the expression or activity of an inflammatory modulator of the eosinophil. The cosmetic composition can be applied directly to the skin for the purpose of improving psoriasis.

The cosmetic composition may be formulated into cosmetic formulations conventionally prepared. The cosmetic composition may be formulated into a solution, suspension, emulsion, paste, gel, lotion, cream, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and spray. Specifically, it may be a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, the carrier may be an animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, And mixtures thereof. In addition, when the formulation of the cosmetic composition is a powder or a spray, it may contain lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or a mixture thereof. In particular, in the case of a spray, it may further include chlorofluorohydrocarbons, propane / butane, dimethyl ether, and the like.

When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, the carrier may include a solvent, a solvent, an emulsifier or a mixture thereof. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol and sorbitan fatty acid ester.

When the formulation of the cosmetic composition of the present invention is in the form of a suspension, a carrier, such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, or mixtures thereof. When the formulation of the cosmetic composition is an interfacial active agent-containing cleansing, the carrier may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, Aliphatic alcohols, fatty acid glycerides, fatty acid diethanol amides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, or mixtures thereof.

The cosmetic composition of the present invention may contain, in addition to the carrier component, an antioxidant, a stabilizer, a solubilizing agent, a moisturizer, a pigment, a fragrance, a sunscreen agent, a coloring agent, a surfactant or a mixture thereof as an adjuvant. The adjuvant may be any substance conventionally used in the production of a cosmetic composition.

Furthermore, the present invention provides a method of treating a subject, comprising the steps of: treating a differentiated eosinophil cell with a toll-like receptor 7 ligand and a test substance; Measuring the level of expression or activity of the inflammatory regulator of the differentiated eosinophil cells treated with the test substance; And screening a test substance having a reduced level of expression or activity level of an inflammatory modulator of eosinophil cells compared to a control group not treated with the test substance.

The differentiated eosinophil cell can be suitably produced by a person skilled in the art. Specifically, the differentiated eosinophil cell may be an eosinophilic granule-containing cell. The TLR7 ligand can be suitably selected and used by a person skilled in the art.

The expression or activity inhibitor of the inflammatory regulator of eosinophils may have the same characteristics as described above. For example, the inflammatory modulator of the eosinophils may be a factor that increases the expression or activity of neutrophils, and specifically IL1B, IL23, IFNG, IL6, TNF, IL22, IL17, IL-8, IL- PAI-1, CXCL12, CCL5, PRG2 and EDN.

The expression or activity level of the inflammatory regulator of the eosinophil cell can be measured by immunoprecipitation, radioimmunoassay, enzyme immunoassay, immunohistochemistry, RT-PCR, real-time PCR, Western blot or flow cytometry, Any method for measuring the amount of the transcript or the protein encoded therefrom can be used.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, but the present invention is not limited thereto.

Example  1. Model cells of eosinophils EoL -1 cell line is available

The EoL-1 cell line, a human eosinophilic cell line, is useful for the study of eosinophils, and is capable of differentiating into eosinophilic granule-containing cells by nascent salt stimulation. Therefore, it was confirmed by the following method whether the EoL-1 cell line can be used as a model cell of eosinophils.

First, EoL-1 (DSMZ, Germany) and HL-60 (ATCC, USA) cells is 10% FBS (fetal bovine serum) with RPMI 1640 culture medium containing the (Sigma-Aldrich, USA), 5% CO ₂ using a And 37 < 0 > C. Meanwhile, HaCaT cell line (ATCC, USA) was cultured in the same manner as above except that DMEM culture medium (Sigma-Aldrich, USA) was used. Total from the respective centrifugation the EoL-1, HL-60 and the HaCaT cell line cultured to give a cell, and obtained by using the QIAzol ^® cell lysis reagent (Qiagen, Germany) and RNeasy ^® mini kit (Qiagen, Germany) Cells RNA was extracted. 500 ng of extracted RNA was treated with DNase I enzyme (New England Biolabs, USA) and cDNA was synthesized using iScript ™ cDNA synthesis kit (Bio-Rad Laboratories, USA). Using a TLR7-F and TLR7-R primer, and SYBR ^® Green Super Mix iQ (Bio-Rad Laboratories, USA) according to the composite cDNA, Table 1 were carried out real-time PCR in the conventional manner. At this time, the instrument used the CFX Connect ™ real-time PCR detection system (Bio-Rad Laboratories, USA) and the control gene GAPDH gene.

Name of the primer The sequence (5 '- > 3') SEQ ID NO: TLR7-F GCT CTG TGG GAG TTC TGT CC SEQ ID NO: 1 TLR7-R ACC GTT TCC TTG AAC ACC TG SEQ ID NO: 2 CCR3-F GTC ATC ATG GCG GTG TTT TTC SEQ ID NO: 3 CCR3-R CAG TGG GAG TAG GCG ATC AC SEQ ID NO: 4 GAPDH-F CTG GTA TGA CAA TGA ATA CGG SEQ ID NO: 5 GAPDH-R GCA GCG AAC TTT ATT GAT GG SEQ ID NO: 6

At the same time performing a RT-PCR using a TLR7-F and TLR7-R primer, and AccuPower ^® PCR premix (Bioneer, Republic of Korea) as described in Table 1, the synthesized cDNA, and the result of 2% agarose gel . ≪ / RTI > At this time, GAPDH gene was used as a control.

As a result, unlike the HaCaT and HL-60 cell lines, the expression of the TLR7 gene was significantly higher in the EoL-1 cell line as shown in Figs. 1A and 1B (Fig. 1).

Example  2. To butyrate  Differentiated by EoL -1 cells TLR7  Confirmation of increased expression of the gene

Previous studies have shown that the expression of the mature eosinophil marker CCR3 (CC-chemokine receptor 3) gene is increased in the EoL-1 cell line treated with butyrate. Therefore, the expression of TLR7 gene in EoL-1 cells was confirmed by the following method by treatment with butyrate.

First, the EoL-1 cell line cultured in Example 1 was treated with 0.5 μM of butyrate, and cells were obtained after 2, 4 and 6 days of treatment. Real-time PCR was performed according to the conditions and method as described in Example 1 using the obtained cells and the primers for the CCR3 and TLR7 genes described in Table 1 above. At this time, GAPDH gene was used as a control group.

As a result, as shown in FIG. 2, expression of CCR3 and TLR7 genes was increased in the EoL-1 cell line treated with butyrate salt, and the expression level of the two genes was the highest at 2 days after culturing with butyrate 2).

Example  3. In the butyrate  Cell survival rate change

Flow cytometry analysis was carried out in the following manner to determine the effect of the butyrate on the cell survival rate of the EoL-1 cell line.

First, the EoL-1 cell line cultured in Example 1 was treated with 0.5 μM of butyrate, and cells were obtained after 2, 4 and 6 days of treatment. The obtained cells were treated with 7-amino-actinomycin D to stain the cells, and the survival rate of the differentiated EoL-1 cell line was confirmed.

As a result, as shown in Fig. 3, the cell survival rate was decreased in dependence on the time of the butyrate treatment (Fig. 3). Thus, from the above, it was decided to treat the butyrate salt, the highest expression of the TLR7 gene, and to use the cells two days later as the differentiated EoL-1 cells for future experiments.

Example  4. Differentiated EoL -1 cells TLR7  Identification of expression of inflammatory cytokine gene by ligand

After two days of treatment with the butyrate, the expression of IL1B, IL23, IL6, TNF, IFNG and IL13 gene, which are inflammatory cytokines, was examined by real-time PCR after treating the differentiated EoL-1 cells with TLR7 ligand.

First, EoL-1 cell line and differentiated EoL-1 cells were treated with 1 μg / ml R837 (Invivogen, USA) and cultured for 24 hours. EoL-1 cell line without R837 treatment and differentiated EoL-1 cells were prepared. Except for using the prepared four kinds of cells (EoL-1 cell line, EoL-1 cell line treated with R837, differentiated EoL-1 cells and differentiated EoL-1 cells treated with R837) and the primers shown in Table 2 below Was carried out under the same conditions and conditions as described in Example 1. [ At this time, GAPDH gene was used as a control group.

Name of the primer The sequence (5 '- > 3') SEQ ID NO: IL1B-F ATG ATG GCT TAT TAC AGT GGC AA SEQ ID NO: 7 IL1B-R CCC TTG CAC AGT TTG ACT CTC SEQ ID NO: 8 IL23-F GTC TCC TTC TCC GCT TCA SEQ ID NO: 9 IL23-R TTA GGG ACT CAG GGT TGC SEQ ID NO: 10 IL6-F GAC AGC CAC TCA CCT CTT CAGE SEQ ID NO: 11 IL6-R GTG CCT CTT TGC TGC TTT CAC SEQ ID NO: 12 TNF-F TCG GTA ACT GAC TTG AAT GTCA SEQ ID NO: 13 TNF-R GGT TGA GGG TGT CTG AAG GA SEQ ID NO: 14 IFNG-F TGT CAC ATT GGG CAA AGT T SEQ ID NO: 15 IFNG-R TCG CTT CCC TGT TTT AGC TGC SEQ ID NO: 16 IL13-F TGT TTG TCA CCG TTG GGG AT SEQ ID NO: 17 IL13-R TGA GTC TCT GAA CCC TTG GC SEQ ID NO: 18

As a result, as shown in FIG. 4, expression of IL1B, IL23 and IFNG gene was increased in differentiated EoL-1 cells compared to that of EoL-1 cell line. In addition, the expression of the cytokine gene induced by the butyrate was further increased in the differentiated EoL-1 cells treated with the TLR7 ligand. On the other hand, the IL6, TNF and IL13 genes were not significantly altered in expression by treatment with tricalcium phosphate or TLR7 ligand (Fig. 4).

Therefore, it was confirmed from the above that the TLR7 ligand had no significant effect on cytokine expression of the undifferentiated EoL-1 cell line.

Example  5. Differentiated EoL -1 cells TLR7  Identification of the expression of inflammatory cytokines by ligands

Protein array assays were performed to further identify inflammatory cytokines that increased expression by TLR7 ligand in differentiated EoL-1 cells.

First, cultures were obtained from the EoL-1 cell line and the differentiated EoL-1 cells treated with R837, respectively. Next, the expression of the inflammatory cytokine was confirmed according to the manufacturer's protocol using the obtained culture and a human cytokine array kit (R & D system).

As a result, as shown in FIG. 5, the CXC chemokine ligand 12 (CXCL12) involved in neutrophil activity, chemotaxis and survival in the differentiated EoL-1 cells (B) treated with R837 compared to the EoL-1 cell line ), IL-8, MIF (microphage migration inhibitory factor), and PAI-1 (plasminogen activator inhibitor type 1). In addition, the expression of IL-1ra (IL-1 receptor antagonist) was also increased in differentiated EoL-1 cells treated with R837 (FIG. 5).

In conclusion, from the above, it can be predicted that eosinophils may play an important role in the pathogenesis of psoriasis by confirming that the production of various inflammatory regulators is increased in differentiated EoL-1 cells treated with R837.

Example  6. Using eosinophil-free mouse model Psoriasis  Identification of the etiology of eosinophils

6-1. psoriasis  Identification of the production and occurrence of animal models

In order to more clearly confirm the pathogenesis of psoriasis and eosinophils, psoriasis animal models were prepared using? DblGATA mice, a mouse model of eosinophil-derived cells.

First, BALB / c mice (Oriental Bio, Korea) and? DblGATA mice (Jackson Laboratory, USA) were prepared by breeding under standard laboratory conditions. The? DblGATA mice at 8-10 weeks of age were topically applied IMQ cream (5%) (Aldara ™, 3M Pharmaceuticals, USA) once a day in an amount of 62.5 mg. At this time, the IMQ cream was applied daily for 6 to 8 days to the right ear with the mouse hair removed. On the other hand, control mice were coated with Vaseline (Uniever, Holland). All experiments were carried out in accordance with the guidelines for animal welfare.

As a result, as shown in Fig. 6, the phenotype of psoriasis was observed in both IMQ cream-coated BALB / c mice and? DblGATA mice, but the degree of psoriasis in the eosinophilized mice was weak (Fig. 6).

In addition, the ear thickness, erythema and scales of the mice were measured on the 2nd, 4th, 6th and 8th days after the IMQ cream was applied to the mice. The thickness of the ear of the mouse was expressed in mm, and erythema and scarring were evaluated based on the 5 point scale (0: none, 1: weak, 2: normal, 3: prominent, 4: very prominent).

As a result, as shown in Fig. 7, it was confirmed that the degree of psoriasis in the? DblGATA mouse was weak due to the thin ear thickness of? DblGATA mice and weak generation of erythema and scarring compared to BALB / c mice coated with IMQ cream 7).

6-2. Through tissue staining Psoriatic  Check the degree of occurrence

By confirming the epidermal thickness of the tissue obtained from the psoriasis animal model prepared in Example 6-1, the degree of occurrence of psoriasis was reaffirmed.

First, tissues of the ear and the like were taken from BALB / c mice and? DblGATA mice coated with the IMQ cream prepared in Example 6-1, fixed in 10% formalin and embedded in paraffin. The embedded tissue was sectioned to a thickness of 4 [mu] m, and the tissue was stained by treating with H & E dye. The stained tissue was observed with a CX41 microscope (Olympus, Japan).

As a result, as shown in FIG. 8, the skin of the IMQ-coated mouse was thicker than the mice not coated with the IMQ cream. On the other hand, when the IMQ cream was applied, the thickness of the skin of? DblGATA mice was less than that of BALB / c mice, and the degree of psoriasis in the? DblGATA mice was weak (Fig.

Thus, it was found that the degree of psoriasis was significantly lower in the mice from which eosinophils were removed.

6-3. Identification of the expression level of inflammatory cytokine gene

The expression level of various kinds of inflammatory cytokine genes was confirmed by real-time PCR in the psoriasis animal model prepared in Example 6-1. The experiment was carried out under the same conditions and method as in Example 1, except that the RNA obtained from the tissue of the psoriasis animal model prepared in Example 6-1 and the primer described in Table 3 below were used.

Name of the primer The sequence (5 '- > 3') SEQ ID NO: IL1B-2F GCA ACT GTT CCT GAA CTC AAC T SEQ ID NO: 19 IL1B-2R ATC TTT TGG GGT CCG TCA ACT SEQ ID NO: 20 IL6-2F TAG TCC TTC CTA CCC CAA TT SEQ ID NO: 21 IL6-2R TTG GTC CTT AGC CAC TCC TTC SEQ ID NO: 22 TNF-2F CCT GTA GCC CAC GTC GTA G SEQ ID NO: 23 TNF-2R GGG AGT AGA CAA GGT ACA ACC C SEQ ID NO: 24 IL22-F ACC AGA ACA TCC AGA AGA AT SEQ ID NO: 25 IL22-R CTC AGA CGC AAG CAT TTC SEQ ID NO: 26 IL23-2F CTA AGA GAA GAA GAG GAT GAA GAG SEQ ID NO: 27 IL23-2R CTG GCT GTT GTC CTT GAG SEQ ID NO: 28 IL17-F TCA GCG TGT CCA AAC ACT GAG SEQ ID NO: 29 IL17-R CGC CAA GGG AGT TAA AGA CTT SEQ ID NO: 30 CXCL2-F CCA ACC ACC AGG CTA CAG G SEQ ID NO: 31 CXCL2-R GCG TCA CAC TCA AGC TCT G SEQ ID NO: 32 CCL5-F AAG TGT GTG CCA ACC CAG AG SEQ ID NO: 33 CCL5-R CAA GCT GGC TAG GAC TAG AGC SEQ ID NO: 34 CCL11-F AGC TAG TCG GGA GAG CCT AC SEQ ID NO: 35 CCL11-R AAG GAA GTG ACC GTG AGC AG SEQ ID NO: 36

As a result, as shown in Fig. 9, IL1b, IL6, TNF, IL22, IL23 and IL17 genes, which are known to be important cytokines in psoriasis, and T cells Expression of the chemokines CXCL12 and CCL5, which promote leukocyte migration, increased. However, when IMQ cream was applied, the expression of most cytokines and chemokines in ΔdblGATA mice was significantly lower than that of BALB / c mice, but the expression of TNF and CCL11 genes was not significantly different. In addition, expression of IL6 and IL23 genes was significantly lower in [Delta] dblGATA mice without IMQ cream application (Fig. 9).

Thus, it can be seen from the above that eosinophils are intracellular sources of inflammatory regulators.

6-4. IMQ  Confirmation of infiltration of neutrophils into tissues by applying cream

The composition of cell populations present in the skin, axillary lymph nodes and spleen of psoriasis animal models was confirmed by flow cytometry.

First, skin tissues of BALB / c mouse and? DblGATA mouse coated with IMQ cream prepared in Example 6-1 were obtained and the obtained tissues were treated with 0.2% collagenase D, 10 μg / ml DNase I (Roche) and 10% FCS (fetal calf serum), and incubated at 37 ° C for 1 hour. After completion of the reaction, by using the washed tissue and washed cells ^{40/75% Percoll ® (Sigma} -Aldrich, USA) density differential centrifugation (density-gradient centrifugation) cells were obtained rule. On the other hand, axillary lymph nodes and spleen were obtained from the mice, gently crushed and filtered with a nylon mesh to obtain spleen cells from which the aggregates had been removed, respectively, from the axillary lymph nodes and the spleen. The obtained splenocytes were resuspended in an ammonium-chloride-potassium lysis buffer to remove red blood cells, and centrifuged to obtain cells.

Each of the cells obtained from the skin, axillary lymph nodes and spleen was treated with FACS buffer (PBS containing 10% FCS, 20 mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid and 10 mM EDTA ). CD4 (RM4-5, BD Biosciences, USA), SiglecF (E50-2440, BD) was prepared by adding anti-mouse CD16 / CD32 antibody (2.4G2, BD Biosciences, USA) (RB6-8C5, BioLegend) and F4 / 80 (BM8, BioLegend) were added and reacted, respectively. After reaction at 4 ° C for 30 minutes, the cells that had been reacted were measured using a FACSCalibur ™ flow cytometer (BD Biosciences, USA) and the results were analyzed using FlowJo software (Tree Star, USA).

As a result, as shown in Figure 10, Gr-1 ⁺ neutrophils only was penetration in all tissues analyzed for applying a IMQ cream BALB / c mice and ΔdblGATA mice, this time, Gr-1 ⁺ neutrophils BALB / c mice Compared with the control group. In addition, CD317 ⁺ dendritic cells and F4 / 80 ⁺ macrophages were smaller in the spleen of ΔdblGATA mice than in BALB / c mice when IMQ cream was applied. On the other hand, CD4 ⁺ T cells were increased after application of IMQ cream on skin of both BALB / c and ΔdblGATA mice, but no significant difference was observed between them. SiglecF ⁺ eosinophils were significantly increased in the skin of IMQ cream coated BALB / c mice, but not in the axillary lymphocytes and spleen. Furthermore, considering that the absolute number of eosinophils present in skin, axillary lymphocytes and spleen is much smaller than that of other types of cells, eosinophils promote infiltration of inflammatory cells, in particular neutrophils, based on IMQ stimulation 10).

Example  7. Correlation between eosinophils and neutrophils

The following experiment was conducted to determine the effect of eosinophilic factors on neutrophils.

Specifically, the HL-60 cell line was cultured as described in Example 1 and prepared. The prepared HL-60 cell line was cultured for 24 hours using a medium in which EoL-1 cells or differentiated EoL-1 cells were cultured. At this time, the control group was cultured using the culture medium described in Example 1. The degree of cell differentiation was confirmed by measuring the absorbance of the cultured cells at a wavelength of 450 nm. In addition, the expression of IL1B, IL23, IL6 and TNF genes was confirmed by real-time PCR using the cultured cells and the primers described in Table 3 above. The experiment was carried out under the same conditions and in the same manner as in Example 1 above.

As a result, as shown in FIG. 11A, the differentiation of HL-60 cell line cultured using the medium in which the differentiated EoL-1 cells were cultured was inhibited in the HL-60 cell line cultured in the control or EoL-1 cell line culture medium (Fig. 11A). In addition, the expression of IL1B, IL6, and TNF genes was significantly increased in the HL-60 cell line cultured using differentiated EoL-1 cells (FIG. 11B).

Thus, from the above, it was found that factors secreted from eosinophils under the inflammatory conditions stimulated by TLR7 promote the differentiation and activity of neutrophils.

Example  8. psoriasis In patients  Identification of eosinophils and neutrophils

8-1. Identification of eosinophil-cytotoxic granulocyte expression

The expression of cytotoxic granulocyte genes in eosinophils in skin tissues obtained from patients with psoriasis was confirmed using real - time PCR.

First, skin tissue was obtained in lesions of 42 patients (30 males, 12 females) diagnosed with psoriasis. The clinic was performed with the consent of all patients and was approved by the ethics committee of the Gil Medical University Foundation. Real-time PCR was performed under the same conditions and methods as described in Example 1, except that the obtained tissue and the primers described in Table 1 and Table 4 were used. At this time, as a control group, a normal skin tissue obtained when a benign tumor patient was operated (13 men and 9 women) was used.

Name of the primer The sequence (5 '- > 3') SEQ ID NO: PRG2-F AAA CTC CCC TTA CTT CTG GCT SEQ ID NO: 37 PRG2-R GCA GCG TCT TAG CAC CCAA SEQ ID NO: 38 EDN-F TGT GGT AAC CCA AAT ATG ACC TG SEQ ID NO: 39 EDN-R GGT CTC GTC GTT GAT CTC TGT SEQ ID NO: 40 ECP-F CCT CAT AAC AGA ACT CTC AACE SEQ ID NO: 41 ECP-R GCA ACT ACA TAG AAC CTC CTT SEQ ID NO: 42

As a result, as shown in Fig. 12, the expression of cytotoxic granulocytes PRG2 and EDN in eosinophils was significantly increased in the lesion tissues of psoriasis patients (Fig. 12).

8-2. Neutrophil penetration confirmation

The skin tissues obtained in Example 8-1 were stained with H & E stain using conventional methods to observe eosinophils and neutrophils.

As a result, as shown in Fig. 13, neutrophil infiltration was observed in the lesion tissue of psoriasis patient, and eosinophil was present in the vicinity of infiltrated neutrophil (Fig. 13).

In conclusion, although the eosinophils did not significantly increase in the psoriasis, the activated eosinophils promoted the activation of the immune response and the neutrophil infiltration in the psoriatic region.

<110> Gachon University of Industry-Academic cooperation Foundation <120> PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING OF          PSORIASIS COMPRISING EXPRESSING OR ACTIVITY INHIBITORS OF          INFLAMMATORY MEDIATOR OF EOSINOPHIL <130> 2018P-03-020 <160> 42 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TLR7-F <400> 1 gctctgtggg agttctgtcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TLR7-R <400> 2 accgtttcct tgaacacctg 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCR3-F <400> 3 gtcatcatgg cggtgttttt c 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCR3-R <400> 4 cagtgggagt aggcgatcac 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 5 ctggtatgac aatgaatacg g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 6 gcagcgaact ttattgatgg 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL1B-F <400> 7 atgatggctt attacagtgg caa 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL1B-R <400> 8 cccttgcaca gtttgactct c 21 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IL23-F <400> 9 gtctccttct ccgcttca 18 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IL23-R <400> 10 ttagggactc agggttgc 18 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL6-F <400> 11 gacagccact cacctcttca ga 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL6-R <400> 12 gtgcctcttt gctgctttca c 21 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-F <400> 13 tcggtaactg acttgaatgt cca 23 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-R <400> 14 ggttgagggt gtctgaagga 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IFNG-F <400> 15 tgtcacattg ggcaaagtt 19 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IFNG-R <400> 16 tcgcttccct gttttagctg c 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL13-F <400> 17 tgtttgtcac cgttggggat 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL13-R <400> 18 tgagtctctg aacccttggc 20 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL1B-2F <400> 19 gcaactgttc ctgaactcaa ct 22 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL1B-2R <400> 20 atcttttggg gtccgtcaac t 21 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL6-2F <400> 21 tagtccttcc taccccaatt 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL6-2R <400> 22 ttggtcctta gccactcctt c 21 <210> 23 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TNF-2F <400> 23 cctgtagccc acgtcgtag 19 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TNF-2R <400> 24 gggagtagac aaggtacaac cc 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL22-F <400> 25 accagaacat ccagaagaat 20 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IL22-R <400> 26 ctcagacgca agcatttc 18 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> IL23-2F <400> 27 ctaagagaag aagaggatga agag 24 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IL23-2R <400> 28 ctggctgttg tccttgag 18 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL17-F <400> 29 tcagcgtgtc caaacactga g 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL17-R <400> 30 cgccaaggga gttaaagact t 21 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CXCL2-F <400> 31 ccaaccacca ggctacagg 19 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CXCL2-R <400> 32 gcgtcacact caagctctg 19 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL5-F <400> 33 aagtgtgtgc caacccagag 20 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CCL5-R <400> 34 caagctggct aggactagag c 21 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL11-F <400> 35 agctagtcgg gagagcctac 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL11-R <400> 36 aaggaagtga ccgtgagcag 20 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PRG2-F <400> 37 aaactcccct tacttctggc t 21 <210> 38 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PRG2-R <400> 38 gcagcgtctt agcacccaa 19 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> EDN-F <400> 39 tgtggtaacc caaatatgac ctg 23 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> EDN-R <400> 40 ggtctcgtcg ttgatctctg t 21 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ECP-F <400> 41 cctcataaca gaactctcaa ca 22 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> ECP-R <400> 42 gcaactacat agaacctcct t 21

Claims (10)

A pharmaceutical composition for preventing or treating psoriasis comprising, as an active ingredient, an expression or activity inhibitor of EDN (eosinophil-derived neurotoxin) protein,
Wherein the expression inhibitor is at least one selected from the group consisting of antisense nucleotide, siRNA and shRNA complementarily binding to the mRNA of the gene encoding the EDN protein,
Wherein the activity inhibitor is an antibody that binds complementarily to the EDN protein.
The pharmaceutical composition for preventing or treating psoriasis according to claim 1, wherein the EDN protein increases neutrophil expression or activity.
The method of claim 1, further comprising an inhibitor of the expression or activity of IL-8 (interleukin-8)
Wherein the expression inhibitor is at least one selected from the group consisting of antisense nucleotides, siRNA and shRNA complementarily binding to the mRNA of the gene encoding the IL-8 protein,
Wherein the activity inhibitor is an antibody that binds complementarily to IL-8 protein.
delete delete As a health functional food for preventing or ameliorating psoriasis containing an EDN protein expression or activity inhibitor as an active ingredient,
Wherein the expression inhibitor is any one or more selected from the group consisting of antisense nucleotides, siRNA and shRNA complementarily binding to the mRNA of the gene encoding the EDN protein,
Wherein said activity inhibitor is an antibody that binds complementarily to an EDN protein.
A cosmetic composition for the prevention or improvement of psoriasis comprising an EDN protein expression or activity inhibitor as an active ingredient,
Wherein the expression inhibitor is any one or more selected from the group consisting of antisense nucleotides, siRNA and shRNA complementarily binding to the mRNA of the gene encoding the EDN protein,
Wherein the activity inhibitor is an antibody that binds complementarily to the EDN protein.
1) treating the differentiated eosinophils with a TLR7 ligand and the test substance;
2) measuring the level of expression or activity of the EDN protein in the differentiated eosinophils treated with the test substance; And
3) selecting a test substance having decreased expression or activity level of EDN protein in eosinophil cells compared with a control group not treated with the test substance in step 2).
9. The method of claim 8, wherein the differentiated eosinophil cell is an eosinophilic granule-containing cell.
9. The method according to claim 8, further comprising the step of measuring the expression or activity level of IL-8 protein in step 2).

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