Compositions comprising inhibitors for inhibiting activity or expression of nod1, and screening methods for the inhibitors

Abstract

To provide a novel composition for preventing or treating skin inflammation, particularly acne vulgaris, which contains an NOD1 inhibitor as an active ingredient, while preserving skin health, and also to provide a screening method for a substance capable of preventing or treating skin inflammation.SOLUTION: A screening method of the present invention comprises: (a) a step of treating human sebaceous gland cells with a test substance; (b) a step of assessing whether the function of NOD1 (i.e., activity or expression) is inhibited; and (c) a step of selecting, based on the comparison result of (b), a test substance that inhibits the function of NOD1 as a substance capable of preventing or treating the skin inflammation.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a composition comprising an inhibitor that inhibits the activity or expression of nucleotide binding oligomerization domain protein-1 (NOD1), and a method for screening for said inhibitor. [Background technology]

[0002] Cutibacterium acnes (C. acnes) is a gram-positive anaerobic bacillus that is a component of the normal skin flora. C. acnes is known to be a causative or exacerbating factor for acne vulgaris and other skin diseases.

[0003] Conventionally, in Japan, the treatment of acne vulgaris has been centered on antibiotic therapy. In 2008, adapalen, which has an anti-inflammatory effect and corrects abnormal keratinization by exhibiting a retinoid-like effect, and in 2015, benzoyl peroxide, which has a strong bactericidal effect, were introduced, and the range of options has somewhat expanded to include treatment with these drugs alone or in combination in addition to conventional antibiotic treatment, but the current situation is that there are still few options (Non-Patent Document 1).

[0004] In recent years, there has been concern that long-term use of antibacterial drugs alone may lead to the emergence of drug-resistant C. acnes (Non-Patent Document 1). In addition, there is concern that long-term use of antibacterial drugs with bactericidal properties may alter the human skin flora, which is composed of normal skin bacteria (Non-Patent Document 2). Therefore, there is an urgent need to identify new therapeutic targets and establish new treatment methods to avoid the long-term use of antibacterial drugs.

[0005] Human sebocytes are the main cells that reside in the sebaceous gland and have sensors against bacterial infections called pattern recognition receptors (PRRs). In acne vulgaris, the proliferation of C. acnes in the sebaceous gland and inflammation caused by the PRRs of human sebaceous gland cells have been cited as a factor in the worsening of symptoms.

[0006] There are several types of PRRs that function as sensors against the above-mentioned bacterial infections, one of which is the signal transduction type PRRs. Signal transduction type PRRs are mainly membrane-bound Toll-like receptors (TLRs), C-type lectin receptors (CLRs), and cytoplasmic NOD-like receptors (Nucleotide binding oligomerization receptors). NODs are known to be classified into NLR, RIG-I-like receptor (RLR) and AIM2-like receptor (ALR). Among them, NODs are known to recognize the structure of a fragment of peptidoglycan present on the surface of most bacterial cell walls and play an important role in innate immune responses. NODs can be divided into NOD1, which has one CARD domain at the N-terminus, and NOD2, which has two CARD domains.

[0007] To date, Toll-like receptor 2 (TLR2) (Non-Patent Document 3) and NOD-like receptor P3 (NLRP3) (Non-Patent Document 4) have been identified as PRRs expressed in human sebaceous gland cells, and inhibition of these PRRs has been proposed as a therapeutic target for acne vulgaris. For example, inhibitors of TLR2, a PRR, have been developed (Patent Document 1).

[0008] The NLR family members NOD1 and NOD2 are also thought to play an important role in innate immunity as sensors that detect bacterial invasion by recognizing components of bacteria in the cytoplasm. Among them, NOD1 detects some gram-positive bacteria such as acne bacteria in addition to gram-negative bacteria, but does not detect many skin resident bacteria such as Staphylococcus epidermidis (hereinafter S. epidermidis). It is also known that skin resident bacteria constantly produce antimicrobial peptides via skin PRRs and maintain the health of the skin (Non-Patent Document 5). However, it was unknown whether NOD1 was expressed in human sebaceous gland cells. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication 2010-99015 [Non-patent literature]

[0010] [Non-Patent Document 1] Biological Sample Analysis Vol.42,No3,152-156(2019) [Non-Patent Document 2] Sci Transl Med Volume 13, 625(2021) [Non-Patent Document 3] Life Sciences 139,123-131(2015) [Non-Patent Document 4] Journal of Investigative Dermatology Volume 134, 2747-2756(2014) [Non-Patent Document 5] Journal of Investigative Dermatology Volume 131, 1974-1980(2011) Summary of the Invention [Problem to be solved by the invention]

[0011] The present inventors have newly discovered that NOD1 is expressed as a PRRs in human sebaceous gland cells. It has been revealed that NOD1 in human sebaceous gland cells not only induces inflammation like other PRRs, but also induces inflammation additively with TLR2. NOD1 recognizes some gram-positive bacteria such as Propionibacterium acnes in addition to gram-negative bacteria, but does not recognize many resident skin bacteria. In other words, NOD1 inhibitors suppress excessive inflammation caused by proliferated Propionibacterium acnes, but do not inhibit the homeostatic role of resident skin bacteria. Therefore, NOD1 inhibitors are believed to be useful as new preventive or therapeutic agents for acne vulgaris. Therefore, based on the discovery of NOD1 as a new therapeutic target, the present invention aims to provide compositions containing inhibitors that inhibit NOD1 activity or inhibit the expression of its gene / protein, and methods for screening for these inhibitors. [Means for solving the problem]

[0012] As a result of extensive research, the inventors have discovered a new composition containing inhibitors that inhibit NOD1 activity and / or the expression of its gene / protein, and have completed the following invention. That is, the present invention includes the following. 1 The present invention relates to a composition containing an inhibitor of NOD1 as an active ingredient. 2. The composition according to 1 above, wherein the inhibitor against NOD1 comprises a NOD1 activity inhibitor and / or a NOD1 expression inhibitor. 3. The inhibitor against NOD1 is a NOD1 inhibitor which inhibits NOD1 activity, and The present invention relates to the composition according to 1 above, which contains an antisense nucleic acid or a small interfering RNA capable of specifically inhibiting the expression of NOD1 and / or serves as a NOD1 expression inhibitor. 4. The composition according to 1 above, further comprising an inhibitor of TLR2 as an active ingredient. 5. The composition according to any one of 1 to 4 above, which is a pharmaceutical composition having an effect of preventing or treating skin inflammation. 6. The composition according to 5 above, wherein the skin inflammation is acne vulgaris. 7. The composition according to 5 above, wherein the composition is a pharmaceutical or quasi-drug for external use. 8. The composition according to 7 above, wherein the skin inflammation is acne vulgaris. 9. The composition according to any one of items 1 to 4, which is a cosmetic composition having an effect of preventing or treating skin inflammation. 10. The composition according to claim 9, wherein the skin inflammation is acne vulgaris. 11. A screening method for a substance that prevents or treats skin inflammation, comprising the steps of: (a) contacting a NOD1 activator with human sebaceous gland cells treated with a test substance; (b) quantifying the gene expression level and / or protein expression level of an inflammatory cytokine and comparing these expression levels with the expression levels when the NOD1 activator is contacted with human sebaceous gland cells not treated with the test substance; and (c) selecting a test substance that reduces the gene expression level and / or protein expression level of an inflammatory cytokine as a substance that can prevent or treat the skin inflammation based on the comparison result of (b). 12. A screening method for a substance that prevents or treats skin inflammation, comprising the steps of: (a) treating human sebaceous gland cells with a test substance; (b) quantifying the NOD1 gene expression level and / or NOD1 protein expression level and comparing these expression levels with those of human sebaceous gland cells not treated with the test substance; and (c) selecting a test substance that reduces the NOD1 gene expression level and / or NOD1 protein expression level based on the comparison result of (b) above as a substance that can prevent or treat the skin inflammation. 13. A screening method for a substance that prevents or treats skin inflammation, comprising the steps of: (a) contacting a NOD1 activator with human sebaceous gland cells treated with a test substance; (b) quantifying the gene expression level and / or protein expression level of NOD1 and comparing these expression levels with those in a case where the NOD1 activator is contacted with human sebaceous gland cells not treated with the test substance; (b1) quantifying the gene expression level and / or protein expression level of inflammatory cytokines and comparing these expression levels with those in a case where the NOD1 activator is contacted with human sebaceous gland cells not treated with the test substance; and (c) selecting, based on the comparison result between (b) and (b1), a test substance that reduces the gene expression level and / or protein expression level of NOD1 and a test substance that reduces the gene expression level and / or protein expression level of inflammatory cytokines as a substance that can prevent or treat the skin inflammation. 14. The method according to any one of claims 11 to 13, wherein the skin inflammation is acne vulgaris. Effect of the Invention

[0013] The present invention provides a novel composition for preventing or treating skin inflammation caused by Propionibacterium acnes, particularly acne vulgaris, while maintaining the health of the skin. The present invention also provides a method for screening a substance capable of preventing or treating skin inflammation. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows the expression of NOD1 and NOD2 in human sebaceous gland cells. Figure 1A shows an electrophoretic photograph detecting the expression of NOD1, NOD2, and GAPDH genes. Figure 1B shows a graph showing the absolute expression levels of NOD1, NOD2, and NLRP3 genes. [Diagram 2]Figure 2 shows inflammation induction in human sebaceous gland cells by stimulation with NOD1 or NOD2. Figure 2A is a graph showing the change in gene expression of the inflammatory cytokine IL-8 (gene name, CXCL8) by stimulation with quantitative ligands (Tri-DAP and MDP) for different times. Figure 2B is a graph showing the change in gene expression of the inflammatory cytokine IL-8 by stimulation with different concentrations of ligands (Tri-DAP and MDP) for 4 hours. Figure 2C is a graph showing the change in protein expression of the inflammatory cytokine IL-8 by stimulation with different concentrations of ligands (Tri-DAP and MDP) for 24 hours. Figure 2D is a Western blotting photograph ([1]) showing the change in phosphorylation of p65, p38, JNK and ERK proteins by stimulation with quantitative ligand (Tri-DAP) for different times, and a graph ([2]) showing the phosphorylation ratio of p65, p38, JNK and ERK proteins. [Diagram 3] Figure 3 is a graph showing inflammation induction at gene expression levels and protein expression levels of the inflammatory cytokine IL-8 in human sebaceous gland cells with or without combined treatment with ligands Tri-DAP and LTA. Figure 3A is a graph showing changes in the gene expression level of the inflammatory cytokine IL-8. Figure 3B is a graph showing changes in the protein expression level of the inflammatory cytokine IL-8. [Figure 4] Fig. 4 is a graph showing the suppression of inflammation induction by ligand stimulation at gene expression levels and protein expression levels when human sebaceous gland cells are treated with or without the NOD1 inhibitor ML130. Fig. 4A is a graph showing the change in gene expression level of the inflammatory cytokine IL-8 when stimulated with a ligand when treated with or without the NOD1 inhibitor ML130. Fig. 4B is a graph showing the change in protein expression level of the inflammatory cytokine IL-8 when stimulated with a ligand when treated with or without the NOD1 inhibitor ML130. [Diagram 5]Fig. 5 shows the suppression of inflammation-inducing protein phosphorylation levels by ligand stimulation in human sebaceous gland cells treated with or without the NOD1 inhibitor ML130. Fig. 5A shows a Western blotting photograph showing the changes in phosphorylation of p65 and p38 proteins. Fig. 5B shows a graph showing the phosphorylation ratios of p65 and p38 proteins. [Figure 6] Figure 6 is a graph showing the suppression of inflammation induction by ligand stimulation at gene expression levels and protein expression levels when human sebaceous gland cells are treated with or without NOD1 siRNA. Figure 6A is a graph showing the change in gene expression level of inflammatory cytokine IL-8 when treated with or without NOD1 siRNA. Figure 6B is a graph showing the change in protein expression level of inflammatory cytokine IL-8 when treated with or without NOD1 siRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Below, we will specifically explain a new composition for preventing or treating skin inflammation, which contains a NOD1 inhibitor as an active ingredient, and a method for screening substances that can prevent or treat skin inflammation. However, the present invention is not limited to the specific embodiments below, and can be modified as desired within the scope of the technical concept.

[0016] [NOD1 and NOD2] NOD1 and NOD2, which are members of the NLR family, recognize components of bacteria in the cytoplasm. Specifically, NOD2 recognizes MDP, which is present in the peptidoglycan of most bacteria. In contrast, NOD1 recognizes iE-DAP, which is present in the peptidoglycan of gram-negative bacteria and some gram-positive bacteria such as Propionibacterium acnes, but does not recognize many skin flora such as S. epidermidis. NOD1 and NOD2 induce the activation of NF-κB and MAPK signaling pathways via RIPK2 (receptor-interacting serine-threonine kinase 2), thereby inducing the expression of various cytokines (e.g., IL-8, etc.) and antimicrobial peptides, and thus activating innate immunity. NOD2 also expresses a variety of hematopoietic cells (e.g., T cells, B cells, macrophages, dendritic cells, mast cells) and non-hematopoietic cells (e.g., Paneth cells, stem cells, goblet cells, intestinal cells). In contrast, NOD1 is expressed in cell types such as gastric, colonic, and oral epithelial cells, pancreatic, lung, kidney, and splenic macrophages, and dendritic cells, but expression of NOD1 has not yet been demonstrated in human sebaceous cells.

[0017] The present inventors have newly discovered that NOD1 is expressed as a PRRs in human sebaceous gland cells.

[0018] The inventors extracted RNA from three different lots of human sebaceous gland cells and THP-1 cells as a positive control, and prepared cDNA using the obtained RNA. The obtained cDNA was subjected to polymerase chain reaction (PCR) using DNA polymerase and each synthetic primer, and the expression of NOD1, NOD2 and GAPDH genes was detected (see FIG. 1A). Furthermore, absolute quantification was performed to confirm the absolute expression levels of NOD1, NOD2 and NLRP3 genes (see FIG. 1B). In FIG. 1B, it can be seen that the absolute expression level of NOD1 gene is higher than that of NOD2 and NLRP3 genes.

[0019] Furthermore, the inventors confirmed that inflammation was induced by stimulation with NOD1. As shown in FIG. 2A, human sebaceous gland cells were stimulated with a quantitative amount of NOD1 ligand (Tri-DAP) or NOD2 ligand (MDP) for different times, and then the gene expression of the inflammatory cytokine IL-8 was confirmed according to the RNA extraction method, cDNA synthesis method, and real-time quantitative PCR. As a result, only NOD1 stimulation was observed to increase the expression, peaking at 4 hours. As shown in FIG. 2B and C, human sebaceous gland cells were stimulated with different concentrations of ligand (Tri-DAP or MDP) for 4 hours, and as a result, only NOD1 stimulation was confirmed to increase the gene expression of the inflammatory cytokine IL-8, and 24 hours after stimulation, only NOD1 stimulation was confirmed to increase the protein expression level of the inflammatory cytokine IL-8. Figure 2D is a Western blotting photograph ([1]) showing the changes in phosphorylation of p65, p38, JNK and ERK proteins in human sebaceous gland cells following stimulation with a quantitative ligand (Tri-DAP) for different periods of time, and a graph ([2]) showing the phosphorylation ratios of p65, p38, JNK and ERK proteins.

[0020] From the results shown in Figures 1 and 2, NOD1 is also present in human sebaceous cells. Furthermore, when human sebaceous cells are stimulated by ligands, the phosphorylation ratios of p65 and p38 proteins increase, and IL-8 is produced in response to inflammatory stimuli, demonstrating that NOD1 functions as a PRR in human sebaceous cells.

[0021] [NOD1 inhibitors] The "NOD1 inhibitor" which is an active ingredient in the composition of the present invention includes a substance that inhibits the activity of NOD1 protein (hereinafter also referred to as a NOD1 activity inhibitor) and / or a substance that inhibits the expression of NOD1 (hereinafter also referred to as a NOD1 expression inhibitor). Note that, as used herein, the term "inhibit" means to completely or partially suppress or reduce the target of inhibition (e.g., activity or expression). As mentioned above, since inflammation is caused by stimulation of NOD1, it can be assumed that inhibitors of NOD1 will have an effect of suppressing inflammation.

[0022] (NOD1 activity inhibitor) Examples of NOD1 activity inhibitors that can be used in the composition of the present invention include substances that antagonize bacteria such as Propionibacterium acnes and inhibit NOD1 activity by inhibiting the binding of NOD1 to the bacteria, and specific antibodies against NOD1. Specific examples include 2-aminobenzimidazoyl derivatives such as ML130 and CID-1088439, with ML130 being more preferred. Dual inhibitors of NOD1 and NOD2, such as ethyl-1-(4-toluenesulfonyl)-1H-indole-2-carboxylate and ethyl-1-(4-methylbenzyl)-1H-indole-2-carboxylate, also inhibit NOD1 activity. The NOD1 activity inhibitor can be selected, identified or confirmed using the screening method described below.

[0023] (NOD1 expression inhibitor) Examples of NOD1 expression inhibitors that can be used in the composition of the present invention include antisense nucleic acids or small interfering RNAs (siRNAs) capable of specifically inhibiting the expression of NOD1, microRNAs, ribozymes capable of specifically cleaving NOD1 mRNA, and expression vectors capable of expressing these in mammalian cells. Examples of NOD1 expression inhibitors include immunosuppressants such as cyclosporine A. NOD1 expression inhibitors can be selected, identified, or confirmed using the screening method described below.

[0024] The antisense nucleic acid refers to a nucleic acid that can specifically hybridize with a transcription product (mRNA or initial transcription product) of NOD1 and inhibit the translation of NOD1. The nucleic acid may be DNA, RNA, or a DNA / RNA chimera.

[0025] The length of the antisense nucleic acid is not particularly limited as long as it can specifically hybridize with the NOD1 transcription product, but from the viewpoint of ease of synthesis and antigenicity, the lower limit is usually 10 bases or more, preferably 15 bases or more, and the upper limit is usually 100 bases or less, preferably 30 bases or less, more preferably 24 bases or less. Therefore, as the antisense nucleic acid, for example, an oligonucleotide of 10 bases or more and 100 bases or less, preferably 15 bases or more and 30 bases or less, more preferably 15 bases or more and 24 bases or less can be used.

[0026] Furthermore, the target sequence of the antisense nucleic acid is not particularly limited as long as it is a sequence to which the antisense nucleic acid hybridizes to inhibit translation of NOD1, and may be the entire sequence of the mRNA or a partial sequence (usually 10 bases or more, preferably 15 bases or more; and usually 100 bases or less, preferably 30 bases or less, more preferably 24 bases or less), or may be an intron portion of the initial transcription product, but when an oligonucleotide is used as the antisense nucleic acid, it is desirable for the target sequence to be located from the 5' end of the NOD1 mRNA to the C-terminus of the coding region.

[0027] The antisense nucleic acid may be synthesized by a method known per se, for example, an oligonucleotide having a desired base sequence may be synthesized using a commercially available automatic DNA / RNA synthesizer.

[0028] The siRNA is a double-stranded oligoRNA having a sequence complementary to a partial sequence (usually 18 bases or more, preferably 21 bases or more, and usually 30 bases or less, preferably 27 bases or less, more preferably 23 bases or less; in the case of an initial transcript, including an intron portion) in the coding region of the NOD1 transcript (mRNA or initial transcript), and is not particularly limited as long as it can specifically recognize and cleave the transcript to inhibit the expression of NOD1. The length of the siRNA is usually 21 to 23 bases. Those skilled in the art can determine the sequences of the sense strand and antisense strand of the siRNA that can be used to inhibit the expression of human NOD1 based on the mRNA sequence of human NOD1. siRNA may be synthesized by a method known per se. For example, siRNA can be synthesized by synthesizing a sense strand and an antisense strand using an automatic DNA / RNA synthesizer and then annealing them.

[0029] In addition, any expression vector used in the art can be used, for example, plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC19), etc. 4) Yeast-derived plasmids (e.g., pSH19, pSH15), bacteriophages such as λ phage, retroviruses, vaccinia viruses, animal viruses such as baculovirus, as well as pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, etc.

[0030] [Skin inflammation] As used herein, the term "skin inflammation" includes, but is not limited to, inflammation associated with acne vulgaris (acne), rosacea, atopic dermatitis, contact dermatitis, drug eruption, psoriasis, seborrheic dermatitis, connective tissue disease (e.g. lupus and scleroderma), other autoimmune disorders such as blistering diseases (e.g. bullous pemphigoid and pemphigus), pigmentation diseases (e.g. post-inflammatory hyperpigmentation, melasma and vitiligo), urticaria or urticarial papules, tinea corporis or fungal infections of fingernails or toenails.Inflammation is a key step for most of these diseases. A composition containing an inhibitor of NOD1 as an active ingredient provided by the present invention is particularly effective in preventing and treating acne vulgaris, ie, acne.

[0031] [Composition containing a NOD1 inhibitor as an active ingredient] The composition containing the NOD1 inhibitor of the present invention as an active ingredient can be provided specifically in the form of a pharmaceutical composition or a cosmetic composition.

[0032] Pharmaceutical Composition In the case of a pharmaceutical composition, the composition of the present invention can be administered orally or parenterally, either as is or in combination with a pharmacologically acceptable carrier. As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as formulation materials are used, and are incorporated as excipients, lubricants, binders, disintegrants in solid preparations, and solvents, solubilizers, suspending agents, isotonicity agents, buffers, soothing agents, etc. in liquid preparations. In addition, formulation additives such as preservatives, antioxidants, colorants, and sweeteners can also be used as necessary.

[0033] The pharmaceutical composition of the present invention may be administered orally in the form of, for example, tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules and microcapsules), syrups, emulsions, suspensions, etc., and may be administered parenterally in the form of, for example, liniments, injections, infusions, drops, etc. It is also effective to prepare a sustained-release preparation by combining with an appropriate base (e.g., butyric acid polymer, glycolic acid polymer, butyric acid-glycolic acid copolymer, mixture of butyric acid polymer and glycolic acid polymer, polyglycerol fatty acid ester, etc.).

[0034] The content of the NOD1 inhibitor in the pharmaceutical composition of the present invention varies depending on the form of the preparation, but is usually 2 to 85% by weight, preferably 5 to 70% by weight, based on the total weight of the preparation.

[0035] The method of preparing the NOD1 inhibitor into the above-mentioned dosage form can be applied to the known preparation method generally used in the field.In addition, when preparing into the above-mentioned dosage form, if necessary, various preparation additives such as excipients, binders, disintegrants, lubricants, etc., which are generally used in the pharmaceutical field when preparing into the dosage form, sweeteners, surfactants, suspending agents, emulsifiers, etc., can be appropriately added in appropriate amounts.

[0036] For example, when the NOD1 inhibitor is formulated into a tablet, it can be produced by adding excipients, binders, disintegrants, lubricants, etc., and when it is formulated into a pill or granule, it can be produced by adding excipients, binders, disintegrants, etc. Also, when it is formulated into a powder or capsule, it can be produced by adding excipients, when it is formulated into a syrup, it can be produced by adding sweeteners, and when it is formulated into an emulsion ... When the composition is prepared as a suspension, it may be prepared by adding a suspending agent, a surfactant, an emulsifier, etc.

[0037] Examples of excipients include lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, licorice powder, mannitol, sodium bicarbonate, calcium phosphate, and calcium sulfate. Examples of binders include 5 to 10% by weight starch paste solution, 10 to 20% by weight gum arabic solution or gelatin solution, 1 to 5% by weight tragacanth solution, carboxymethylcellulose solution, sodium alginate solution, glycerin, and the like. Examples of disintegrants include starch, calcium carbonate, and the like. Examples of lubricants include magnesium stearate, stearic acid, calcium stearate, and purified talc. Examples of sweeteners include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, simple syrup, and the like. Examples of the surfactant include sodium lauryl sulfate, polysorbate 80, sorbitan mono fatty acid ester, polyoxyl 40 stearate, and the like. Examples of suspending agents include gum arabic, sodium alginate, sodium carboxymethylcellulose, methylcellulose, bentonite, and the like. Examples of emulsifying agents include gum arabic, tragacanth, gelatin, polysorbate 80, and the like.

[0038] Furthermore, when the NOD1 inhibitor is prepared in the above-mentioned dosage forms, colorants, preservatives, fragrances, flavorings, stabilizers, thickening agents, and the like that are commonly used in the pharmaceutical field can be added in appropriate amounts, if desired.

[0039] The pharmaceutical composition of the present invention containing an inhibitor against NOD1 is stable, has low toxicity, and can be used safely. The daily dose varies depending on the condition and weight of the patient, the type of inhibitor against NOD1, the route of administration, etc., but a person skilled in the art can determine an appropriate amount by taking these factors into consideration. In addition, the administration may be once a day or divided into two or three times a day.

[0040] When an inhibitor against NOD1 is administered parenterally, it is usually administered in the form of a liquid (e.g., an injection). The single dose varies depending on the subject, the target organ, the symptoms, the administration method, etc., but it is convenient to administer, for example, about 0.1 mg to about 100 mg per kg of body weight by intravenous injection in the form of an injection. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc., and sustained-release preparations include iontophoresis transdermal preparations, etc. Such injections are prepared by a method known per se, that is, by dissolving, suspending, or emulsifying an inhibitor against NOD1 in a sterile aqueous or oily liquid. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other auxiliary drugs (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and the like, which may be used in combination with suitable solubilizing agents, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (e.g., polysorbate 80, HCO-50), etc. Examples of oily solutions include sesame oil, soybean oil, etc., which may be used in combination with solubilizing agents (e.g., benzyl benzoate, benzyl alcohol, etc.), etc. In addition, the solution may be mixed with buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol, etc.), etc. The prepared injection solution is usually filled into ampoules.

[0041] The pharmaceutical composition of the present invention is useful for treating skin conditions such as atopic dermatitis and seborrheic dermatitis. In the case of using a NOD1 inhibitor in combination with another drug, the administration form of the NOD1 inhibitor and the concomitant drug is not particularly limited, and it is sufficient that the NOD1 inhibitor and the concomitant drug are combined at the time of administration. Examples of such administration forms include (1) administration of a single preparation obtained by simultaneously formulating an inhibitor against NOD1 and a concomitant drug, (2) simultaneous administration of two preparations obtained by separately formulating an inhibitor against NOD1 and a concomitant drug via the same administration route, (3) administration of two preparations obtained by separately formulating an inhibitor against NOD1 and a concomitant drug via the same administration route with a time lag, (4) simultaneous administration of two preparations obtained by separately formulating an inhibitor against NOD1 and a concomitant drug via different administration routes, and (5) administration of two preparations obtained by separately formulating an inhibitor against NOD1 and a concomitant drug via different administration routes with a time lag (e.g., administration of an agent consisting of the pharmaceutical composition of the present invention followed by a concomitant drug, or administration in the reverse order). The dosage of the concomitant drug can be appropriately selected based on the dose used in clinical practice. The mixing ratio of the inhibitor against NOD1 and the concomitant drug can be appropriately selected depending on the subject of administration, the administration route, the target disease, symptoms, combination, etc. For example, when the subject of administration is a human, 0.01 to 100 parts by weight of the concomitant drug may be used per 1 part by weight of the NOD1 inhibitor.

[0042] Since the pharmaceutical composition is used for the prevention and treatment of acne vulgaris, that is, acne, it is expected to be highly effective as an external composition for external medicines and external quasi-drugs.

[0043] (Cosmetic composition) The composition of the present invention is also a cosmetic composition. The cosmetic composition of the present invention can be easily manufactured by a method known in the art by including the NOD1 inhibitor of the present invention and one or more excipients and additives that are generally used in the field of cosmetic composition manufacturing.

[0044] The cosmetic composition of the present invention contains an inhibitor against NOD1 as an active ingredient, and can be prepared together with a dermatologically acceptable excipient in the form of a basic cosmetic composition (skin lotion, milky lotion, cream, serum, cleansing foam and cleansing water such as face wash, pack, body oil), color cosmetic composition (foundation, lipstick, mascara, makeup base), hair product composition (shampoo, rinse, hair conditioner, hair gel) and soap. The excipient in the cosmetic composition is not limited to the above, but may include, for example, a skin softener, a skin penetration enhancer, a colorant, a fragrance, an emulsifier, a thickener and a solvent. In addition, it may further include a fragrance, a colorant, a bactericide, an antioxidant, a preservative and a moisturizer, and may include a thickener, an inorganic salt, a synthetic polymeric substance, etc. for the purpose of improving physical properties.

[0045] The excipient in the cosmetic composition may include, but is not limited to, a skin softener, a skin penetration enhancer, a colorant, a fragrance, an emulsifier, a thickener, and a solvent. In addition, a fragrance, a colorant, a bactericide, an antioxidant, a preservative, and a moisturizer may be further included, and a thickener, an inorganic salt, a synthetic polymeric substance, and the like may be included for the purpose of improving physical properties. For example, when a cream is produced from the cosmetic composition of the present invention, an inhibitor against NOD1 may be added to a general oil-in-water (O / W) cream base to produce the cream. Here, a fragrance, a chelating agent, a colorant, an antioxidant, a preservative, and the like may be added, and a synthetic or natural material such as a protein, a mineral, or a vitamin for the purpose of improving physical properties may be added.

[0046] The content of the NOD1 inhibitor contained in the cosmetic composition of the present invention is preferably 0.0005 to 10% by weight, more preferably 0.001 to 5% by weight, based on the total weight of the entire composition, but is not limited thereto. If the content is less than 0.001% by weight, the intended preventive or therapeutic effect on skin inflammation cannot be expected, and 10% by weight or more is preferable. Exceeding this amount may pose safety or manufacturing problems for all dosage forms.

[0047] Furthermore, the cosmetic composition of the present invention can be produced by mixing the NOD1 inhibitor with other ingredients known to be effective in alleviating skin inflammation and protecting the skin.

[0048] Furthermore, as described above, these TLR2 inhibitors are known to be effective as therapeutic targets for acne vulgaris (acne), and therefore the composition of the present invention containing a NOD1 inhibitor as an active ingredient may further contain a TLR2 inhibitor.

[0049] [Method for screening substances that prevent or treat skin inflammation] The present invention provides a method for screening for a substance that prevents or treats skin inflammation, which mainly comprises the following steps: (a) treating human sebaceous gland cells with a test substance; (b) assaying for inhibition of NOD1 function (i.e., activity or expression); and (c) selecting the test substance that inhibits the function of NOD1 based on the comparison result of (b) above as a substance capable of preventing or treating the skin inflammation. In this screening method, the term "skin inflammation" has the same meaning as that described in the description of the composition containing the NOD1 inhibitor as an active ingredient. The screening method of the present invention will now be described in detail.

[0050] The test substance subjected to the screening method may be any known compound or novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, organic low molecular weight compound, compound library prepared using combinatorial chemistry technology, random peptide library prepared by solid phase synthesis or phage display method, or natural components derived from microorganisms, animals, plants, marine organisms, etc. The test substance may be labeled or unlabeled, and a mixture containing a specified ratio of labeled and unlabeled substances can also be used as the test substance. Examples of labeling substances include fluorescent substances such as FITC and FAM, luminescent substances such as luminol, luciferin, and lucigenin, 3 H,14 C. 32 P, 35 S, 123 Examples of such affinity substances include radioisotopes such as I, and affinity substances such as biotin and streptavidin.

[0051] (Screening method for NOD1 activity inhibitors - Screening method A) A method for screening for a substance that inhibits the activity of NOD1 (a NOD1 activity inhibitor) will be described.

[0052] Screening method A for a NOD1 activity inhibitor comprises the following steps (a), (b) and (c): (a) contacting a NOD1 activator with human sebaceous gland cells treated with a test substance; (b) quantifying the gene expression level and / or protein expression level of the inflammatory cytokine, and comparing these expression levels with the expression levels when the NOD1 activator is contacted with human sebaceous gland cells that have not been treated with the test substance; (c) selecting a test substance that reduces the mRNA expression level of the gene for an inflammatory cytokine and / or the protein expression level of the inflammatory cytokine based on the comparison result of (b) above as a substance capable of preventing or treating the skin inflammation.

[0053] In step (a) of the screening method A, first, human sebaceous gland cells are treated with a test substance. Then, a NOD1 activator is contacted with the human sebaceous gland cells treated with the test substance. Examples of the NOD1 activator include NOD1 ligands. Tri-DAP is a representative NOD1 ligand.

[0054] When the test substance is an inhibitor of NOD1 activity, its addition inhibits NF-κB and M Activation of the APK pathway is inhibited, which in turn inhibits the expression of various inflammatory cytokines induced by activation of the NF-κB and MAPK pathways. In step (b) of screening method A, first, the gene expression levels and / or protein expression levels of these inflammatory cytokines are measured. The gene expression levels and protein expression levels are measured by known methods taking into consideration the type of cells used, etc. For example, the expression level of the inflammatory cytokine IL-8 gene can be measured by a method known per se, using the product of the IL-8 gene, such as a transcription product (mRNA) or a translation product (protein). For example, the expression level of the transcription product can be measured by preparing total RNA from cells and subjecting it to RT-PCR, Northern blotting, or the like. In addition, the expression level of the translation product can be measured by preparing an extract from cells and subjecting it to an immunological method. Examples of immunological methods that can be used include radioisotope immunoassay (RIA), ELISA (Methods in Enzymol. 70:419-439 (1980)), and fluorescent antibody techniques. Next, the expression levels of the IL-8 gene and / or protein in the cells contacted with the test substance are compared with the expression levels of the IL-8 gene and / or protein in control cells not contacted with the test substance. The comparison of the expression levels is preferably performed based on the presence or absence of a significant difference. The expression levels of the IL-8 gene and / or protein in the control cells not contacted with the test substance may be expression levels measured before or simultaneously with the measurement of the expression levels of the IL-8 gene and / or protein in the cells contacted with the test substance, but from the viewpoint of experimental accuracy and reproducibility, it is preferable that the expression levels are measured simultaneously.

[0055] In step (c) of screening method A, a test substance that reduces the gene expression level and / or the protein expression level of an inflammatory cytokine is selected as a substance capable of preventing or treating the above-mentioned skin inflammation.

[0056] (Screening method for NOD1 expression inhibitors - Screening methods B and C) A method for screening for a substance that inhibits the expression of NOD1 (a substance that inhibits NOD1 expression) will be described.

[0057] Screening method B for a NOD1 expression inhibitor comprises the following steps (a), (b), and (c): (a) treating human sebaceous gland cells with a test substance; (b) quantifying the NOD1 gene expression level and / or NOD1 protein expression level and comparing these expression levels with those of human sebaceous gland cells not treated with the test substance; and (c) selecting a test substance that reduces the NOD1 gene expression level and / or the NOD1 protein expression level based on the comparison results of (b) above as a substance capable of preventing or treating the skin inflammation.

[0058] In step (a) of screening method B, first, human sebaceous gland cells are treated with a test substance.

[0059] When the test substance is an inhibitor of NOD1 expression, its addition affects the expression level of the NOD1 gene and / or the expression level of NOD1 protein. In step (b) of screening method B, first, the expression level of NOD1 gene and / or the expression level of NOD1 protein in human sebaceous gland cells contacted with a test substance is measured. The expression level can be measured by a method known per se, taking into consideration the type of cells used, etc. The specific measurement method is the same as step (b) of screening method A. Next, the expression level of the NOD1 gene and / or protein in the cells contacted with the test substance was compared with the expression level of the NOD1 gene and / or protein in the control cells not contacted with the test substance. The expression levels of the NOD1 gene and / or protein in control cells not contacted with the test substance may be expression levels measured in advance or simultaneously with the measurement of the expression levels of the NOD1 gene and / or protein in cells contacted with the test substance, but from the viewpoint of experimental accuracy and reproducibility, it is preferable that the expression levels are measured simultaneously.

[0060] In step (c) of screening method B, a test substance that reduces the expression level of the NOD1 gene and / or the expression level of NOD1 protein is selected as a substance capable of preventing or treating the above-mentioned skin inflammation.

[0061] Screening method C for a NOD1 expression inhibitor comprises the following steps (a), (b), (b1) and (c): (a) contacting a NOD1 activator with human sebaceous gland cells treated with a test substance; (b) quantifying the amount of NOD1 gene expression and / or the amount of NOD1 protein expression, and comparing these expression levels with the expression levels when the NOD1 activator is contacted with human sebaceous gland cells that have not been treated with the test substance; (b1) quantifying the gene expression level and / or protein expression level of an inflammatory cytokine, and comparing these expression levels with the expression levels when the NOD1 activator is contacted with human sebaceous gland cells that have not been treated with a test substance; and (c) selecting a test substance that reduces the gene expression level of NOD1 and / or the protein expression level of NOD1, and a test substance that reduces the gene expression level of an inflammatory cytokine and / or the protein expression level of an inflammatory cytokine, as a substance capable of preventing or treating the skin inflammation, based on the comparison result between (b) and (b1) above.

[0062] In step (a) of screening method C, first, human sebaceous gland cells are treated with a test substance. Then, a NOD1 activator is contacted with the human sebaceous gland cells treated with the test substance. The NOD1 activator may be the same as that in screening method A.

[0063] When the test substance is an inhibitor of NOD1 expression, its addition affects the expression level of the NOD1 gene and / or the expression level of NOD1 protein. In step (b) of screening method C, first, the NOD1 gene expression level and / or NOD1 protein expression level in human sebaceous gland cells that have been subjected to step (a) is measured. The expression level can be measured by a method known per se, taking into consideration the type of cells used, etc. The specific measurement method is the same as step (b) of screening method A. Next, the expression level of the NOD1 gene and / or protein in the cells contacted with the test substance is compared with the expression level of the NOD1 gene and / or protein in control cells not contacted with the test substance. The comparison of the expression levels is preferably performed based on the presence or absence of a significant difference. The expression level of the NOD1 gene and / or protein in the control cells not contacted with the test substance may be an expression level measured in advance or simultaneously with the measurement of the expression level of the NOD1 gene and / or protein in the cells contacted with the test substance, but it is preferable that it is an expression level measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.

[0064] In addition to inhibiting NOD1 expression, it is believed that signaling downstream of NOD1 in the signal transduction pathway is also normally affected, and activation of the NF-κB and MAPK pathways and the expression of various inflammatory cytokines induced by activation of the NF-κB and MAPK pathways are also inhibited. Step (b1) of screening method C is the same as step (b) of screening method A. That is, step (b1) of screening method C is further supplemented by step (b) of screening method A. (b) and, based on the results of both, it can be determined whether the test substance can be used as a substance for preventing or treating skin inflammation. However, the order of steps (b) and (b1) of screening method C does not matter, and they may be performed simultaneously during screening.

[0065] Specifically, first, the gene expression level and / or the protein expression level of an inflammatory cytokine are measured in step (b1) of the screening method C. The gene expression level and the protein expression level are measured by a known method taking into consideration the type of cells used, etc. Next, the expression levels of the IL-8 gene and / or protein in the cells contacted with the test substance are compared with the expression levels of the IL-8 gene and / or protein in control cells not contacted with the test substance. The comparison of the expression levels is preferably performed based on the presence or absence of a significant difference. The expression levels of the IL-8 gene and / or protein in the control cells not contacted with the test substance may be expression levels measured before or simultaneously with the measurement of the expression levels of the IL-8 gene and / or protein in the cells contacted with the test substance, but from the viewpoint of experimental accuracy and reproducibility, it is preferable that the expression levels are measured simultaneously.

[0066] In step (c) of screening method C, a test substance that reduces the gene expression level of NOD1 and / or the protein expression level of NOD1, and a test substance that reduces the gene expression level of an inflammatory cytokine and / or the protein expression level of an inflammatory cytokine are selected as substances that can prevent or treat the above-mentioned skin inflammation. EXAMPLES

[0067] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0068] (Human sebaceous gland cell culture) Human sebocytes were cultured in a 6-well plate at 1.0 × 10 5 cells / well, 5.0 × 10 in 12-well plates 4 Cells were seeded at 200 cells / well and incubated at 37℃, 5% CO 2 The medium used was DMEM / F-12 with an additive. The additive was GlutaMAX TM(2 mM), 2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid (10 mM), human epidermal growth factor (50 ng / mL), and 10% heat-inactivated fetal bovine serum (FBS).

[0069] Example 1 Inhibition of NOD1 Protein Activity Human sebaceous gland cells were treated with 5 μM NOD1 inhibitor ML130 for 30 minutes, and then stimulated with 10 μg / mL Tri-DAP. RNA was extracted from the sample 4 hours after Tri-DAP stimulation using the RNeasy Mini Kit (QIAGEN). The obtained RNA and PrimeScript TM II 1st strand cDNA Synthesis Kit (Takara Bio) was used to generate cDNA using GeneAmp (registered trademark) Reverse transcription polymerase chain reaction (RT-PCR) was performed using a PCR System 9700 (Applied Biosystems) to prepare cDNA. Both procedures were performed according to the method recommended by the manufacturer. The mRNA expression level of the IL-8 gene was measured by TaqMan (registered trademark) gene expression assay using the obtained cDNA using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems). The procedures were performed according to the method recommended by the manufacturer. Real-time quantitative PCR was performed using an Applied Biosystems 7500 real-time PCR system (Applied Biosystems), with an UNG incubation step at 50°C for 2 minutes and an enzyme activation step at 95°C for 10 minutes. After the denaturation step, a cycle including a thermal denaturation step at 95°C for 15 seconds and an annealing / extension step at 60°C for 1 minute was repeated 50 times. Figure 4A shows the induction of gene expression of the inflammatory cytokine IL-8.

[0070] The amount of IL-8 in the cell culture supernatant 24 hours after Tri-DAP stimulation was quantified by ELISA using a Human IL-8 ELISA Kit (Proteintech). The procedure was performed according to the method recommended by the manufacturer. Figure 4B shows the induction of protein expression of the inflammatory cytokine IL-8. In conclusion, ML130 significantly suppressed both the mRNA expression and protein secretion of the inflammatory cytokine IL-8 induced by the NOD1 activator (Tri-DAP).

[0071] Human sebaceous cells were treated with 5 μM NOD1 inhibitor ML130 for 30 min, followed by stimulation with 10 μg / mL Tri-DAP for 1 h. Human sebaceous cells were then lysed in lysis buffer (Tris-HCl (20 mM, pH 7.5), NaCl (150 mM), calcium disodium edetate (10 mM, pH 8.0), 1% Triton-X 100) and adjusted with sample buffer (Tris-HCl (250 mM, pH 6.8), 20% 2-mercaptoethanol, 8% sodium dodecyl sulfate, 20% sucrose, bromophenol blue (40 μg / μL)). The adjusted samples were electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel, and proteins were transferred to a polyvinylidene difluoride membrane at 100 mV for 1 h using a semi-dry blotting apparatus (Bio-Rad Laboratories). The membrane was blocked with blocking solution (5% skim milk in Tris-buffered saline (TBS-T) containing 0.1% Tween-20) for 1 h. After washing three times with TBS-T, the membrane was exposed to each antibody overnight at 4°C. After washing three times with TBS-T, the membrane was exposed to HRP-conjugated secondary antibodies at room temperature for 2 h. Phosphorylation of p65 and p38 proteins was confirmed by Western blot analysis using ECL Prime Western blotting detection reagent (Cytiva) or ImmunoStar LD (Wako) according to the manufacturer's recommended method (Figure 5A and B). That is, ML130 inhibited the phosphorylation of p65 and p38, which is an inflammatory cell signaling induced by Tri-DAP.

[0072] These results demonstrate that the use of ML130 to inhibit activity against the NOD1 protein is an effective means of suppressing inflammation caused by P. acnes and other bacteria.

[0073] Example 2 Inhibition of NOD1 Gene Expression and Protein Expression 19.2 nM of siRNA for the NOD1 gene (hereinafter, also referred to as siNOD1) (manufactured by Horizon Discovery) was transfected into human sebaceous gland cells using Lipofectamin 2000 (manufactured by Thermo Fisher Scientific). For samples taken 48 hours after transfection, it was confirmed that the expression level of the NOD1 gene had decreased by approximately 50% according to the above-mentioned RNA extraction method, cDNA synthesis method, and real-time quantitative PCR. Furthermore, for cell lysates taken 48 hours after transfection, it was confirmed that the expression level of NOD1 protein had also decreased by 30% according to the above-mentioned Western blotting method. Furthermore, assuming that NOD1 expression is inhibited and that signal transduction downstream of NOD1 in the signal transduction pathway is also affected, we confirmed the induction of gene expression of the inflammatory cytokine IL-8 4 hours after Tri-DAP stimulation according to the above-mentioned RNA extraction method, cDNA synthesis method, and real-time quantitative PCR (Figure 6A). For the cell culture supernatant 24 hours after Tri-DAP stimulation, we confirmed the induction of protein expression of the inflammatory cytokine IL-8 according to the above-mentioned ELISA method (Figure 6B). As a result, the use of siNOD1 completely abolished the mRNA expression and protein secretion of the inflammatory cytokine IL-8 induced by the NOD1 activator (Tri-DAP). It was enabled. As described above, it has been demonstrated that the method of inhibiting NOD1 gene expression and protein expression by using siNOD1 is an effective means of suppressing inflammation caused by P. acnes and other bacteria.

[0074] <Cosmetic formulation examples> The lotion and cosmetic cream can be prepared according to the formulation examples in Tables 1 and 2 below. [Table 1]

[0075] [Table 2] [Industrial Applicability]

[0076] The screening method for a substance that prevents or treats skin inflammation of the present invention allows selection of a substance that directly or indirectly inhibits the function of NOD1, thereby providing a means for suppressing skin inflammation caused by Propionibacterium acnes, particularly skin inflammation caused by acne vulgaris, and is expected to have an effect of preventing or treating skin inflammation caused by acne vulgaris.

Claims

1. A composition comprising an inhibitor of NOD1 as an active ingredient.

2. The composition of claim 1 , wherein the inhibitor against NOD1 comprises a NOD1 activity inhibitor and / or a NOD1 expression inhibitor.

3. The composition described in claim 1, wherein the inhibitor against NOD1 includes a NOD1 inhibitor which is an inhibitor of NOD1 activity, and / or an antisense nucleic acid or small interfering RNA which is an inhibitor of NOD1 expression and can specifically inhibit the expression of NOD1.

4. The composition according to claim 1, further comprising a TLR2 inhibitor as an active ingredient.

5. The composition according to any one of claims 1 to 4, which is a pharmaceutical composition having an effect of preventing or treating skin inflammation.

6. The composition of claim 5, wherein the skin inflammation is acne vulgaris.

7. The composition according to claim 5 , wherein the composition is a topical drug or a topical quasi-drug.

8. The composition of claim 7, wherein the skin inflammation is acne vulgaris.

9. The composition according to claim 1 , which is a cosmetic composition having an effect of preventing or treating skin inflammation.

10. The composition of claim 9, wherein the skin inflammation is acne vulgaris.

11. A method for screening for a substance that prevents or treats skin inflammation, comprising the steps of: (a) contacting a NOD1 activator with human sebaceous gland cells treated with a test substance; (b) quantifying the gene expression level and / or protein expression level of the inflammatory cytokine, and comparing these expression levels with the expression levels when the NOD1 activator is contacted with human sebaceous gland cells that have not been treated with the test substance; and (c) selecting a test substance that reduces the gene expression level of an inflammatory cytokine and / or the protein expression level of an inflammatory cytokine based on the comparison result of (b) above as a substance capable of preventing or treating the skin inflammation.

12. A method for screening for a substance that prevents or treats skin inflammation, comprising the steps of: (a) treating human sebaceous gland cells with a test substance; (b) quantifying the NOD1 gene expression level and / or NOD1 protein expression level, and comparing these expression levels with those of human sebaceous gland cells not treated with the test substance; and (c) selecting a test substance that reduces the amount of NOD1 gene expression and / or the amount of NOD1 protein expression based on the comparison result of (b) above as a substance capable of preventing or treating the skin inflammation.

13. A method for screening for a substance that prevents or treats skin inflammation, comprising the steps of: (a) contacting a NOD1 activator with human sebaceous gland cells treated with a test substance; (b) quantifying the amount of NOD1 gene expression and / or the amount of NOD1 protein expression, and comparing these expression levels with the expression levels when the NOD1 activator is contacted with human sebaceous gland cells that have not been treated with the test substance; (b1) quantifying the gene expression level and / or protein expression level of an inflammatory cytokine, and comparing these expression levels with the expression levels when a NOD1 activator is contacted with human sebaceous gland cells that have not been treated with a test substance; and (c) selecting, based on the results of the comparison between (b) and (b1), a test substance that reduces the gene expression level of NOD1 and / or the protein expression level of NOD1, and a test substance that reduces the gene expression level of an inflammatory cytokine and / or the protein expression level of an inflammatory cytokine, as a substance capable of preventing or treating the skin inflammation.

14. 14. The method of claim 11, wherein the skin inflammation is acne vulgaris.

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