Inhibitor of expression of cellular senescence-associated secretory phenotype factor, inhibitor of expression of tgfbr1 and / or tgfbr2, and modulator of expression level of senescence-involved gene

Abstract

To provide a novel inhibitor of expression of cellular senescence-associated secretory phenotype factor, a novel inhibitor of expression of TGFBR1 and / or TGFBR2, and a novel modulator of expression level of senescence-involved gene.SOLUTION: The present invention provides an inhibitor of expression of cellular senescence-associated secretory phenotype factor, an inhibitor of expression of TGFBR1 and / or TGFBR2, and a modulator of expression level of senescence-involved gene, each comprising royal jelly as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for suppressing the expression of cellular senescence-associated secretory factor, an agent for suppressing the expression of TGFBR1 and / or TGFBR2, and an agent for regulating the expression level of a gene involved in senescence. [Background technology]

[0002] Cells age when exposed to damage from both inside and outside the body, such as oxidative stress and ultraviolet rays. Senescent cells accumulate with age. Senescent cells release senescence-associated secretory phenotype (SASP) factors. Some SASP factors have the effect of inducing cells surrounding the senescent cells to become senescent cells themselves, promoting the aging of the entire tissue (Non-Patent Document 1).

[0003] Patent Document 1 describes that a component eluted from enzyme-treated royal jelly by a specific method exerts an anti-aging effect by adjusting the expression level of genes involved in the insulin / IGF1 signaling system. In the examples of Patent Document 1, the lifespan extension effect of the component eluted from enzyme-treated royal jelly is shown in a test using the nematode C. elegans. As the mechanism of action, an increase in the gene expression levels of ins-20 and dod-3 involved in the insulin / IGF1 signaling system and a decrease in the gene expression levels of dod-19, dao-4, fkb-4, and ins-9 are shown, but the involvement of SASP factors is not shown. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2012-207004 A [Non-patent literature]

[0005] [Non-Patent Document 1] J Clin Invest.2022 Aug1;132(15):e158450.https: / / doi.org / 10.1172 / JCI158450. [Non-Patent Document 2] Genes Dev.2014,28(22)99-114:http: / / www.genesdev.org / cgi / doi / 10.1101 / gad.235184.113. [Non-Patent Document 3] Int.J.Mol.Sci.2021,22,6814.https: / / doi.org / 10.3390 / ijms22136814 [Non-Patent Document 4] Int.J.Mol.Sci.2019,20,5002;doi:10.3390 / ijms20205002 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a novel agent for suppressing the expression of cellular senescence-associated secretory factors. Further, the present invention aims to provide a novel agent for suppressing the expression of TGFBR1 and / or TGFBR2, and an agent for regulating the expression level of senescence-related genes. [Means for solving the problem]

[0007] The inventors conducted intensive research to achieve the above-mentioned object and discovered that royal jelly has excellent effects of inhibiting the expression of cellular senescence-related secretory trait factors, regulating the expression levels of senescence-related genes, and inhibiting the expression of TGFBR1 and / or TGFBR2, thereby completing the present invention.

[0008] That is, the present invention relates to the following [1] to [7]. [1] An inhibitor of the expression of senescence-associated secretory phenotype (SASP) factors, containing royal jelly as an active ingredient. [2] The expression inhibitor described in [1], wherein the SASP factor is at least one selected from the group consisting of IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14 and PAI1. [3] An agent for suppressing the expression of TGFBR1 and / or TGFBR2, which are TGF-β receptors, containing royal jelly as an active ingredient. [4] An agent for regulating the expression of genes involved in aging, containing royal jelly as an active ingredient. [5] ABCA7, ACTG1, ALDH4A1, AMIGO2, ANKRD50, AP2M1, ASB1, AT P11A、B3GNT2、B4GALT1、BAZ2B、BCAR3、C11orf24、C1QTNF1、C1RL、CALD1、CAM SAP2, CASP8, CCND2, CD274, CDH3, CDK6, CDYL, CEP170, COL1A1, COL4A1, COL 4A2、COL4A5、COL4A6、COL5A1、COLGALT1、CORO1C、CPA4、CRIM1、CRNN、CSF3、C SNK1E, CSRP1, CSTF3, CXCL1, CXCL10, CXCL14, CXCL2, CXCL3, CXCL5, CYP24A 1、DECR1、DHX16、DRAM1、DUSP4、DUSP5、DYNC1H1、EDN1、EMC1、EPAS1、EREG、FA P, FAT1, FBN2, FNBP1L, FSIP1, FUS, G0S2, GNB4, GPR157, GPR68, GPRC5A, HEG 1、HELZ、HIVEP2、HLA-B、HSPA4L、HSPG2、HUWE1、ID3、IL6、IRF6、ITGB6、IVNS1 ABP, JAG1, JPT2, KCNJ15, KCTD11, KIAA0895L, KIFC3, KRT7, KYNU, LGALS3BP 、LIMK2、LOXL2、LTBP1、LTBP2、MAT2A、MB21D2、MCAM、MDN1、MEST、MICAL2、MOB 3B、MOCOS、MPZL2、MRAS、MRGPRX3、MTAP、MT-CO1、MT-CO2、MTHFD2L、MT-ND2、 MYD88、MYH9、NCEH1、NEFL、NIPAL3、NIPSNAP3A、NOMO2、P4HA1、PALM2AKAP2、P DCD11, PDCD5, PFKFB3, PGAM1, PGK1, PHF20, PLAT, PLOD2, PMEPA1, POLR1A, P OMK, PPP1R3B, PPP1R3C, PRKAR1A, PRORP, PRR5L, PSME3, PTGES, PTGFRN, PTPN 14、PTPRK、RAI14、RGMB、RHOBTB2、RNF152、RRAS、SACS、SBNO2、SDC4、SEMA7A、 SERPINE1, SERPINH1, SGK1, SH2D3A, SHFL, SIPA1L1, SKILL, SLAMF9, SLC16A3The expression level regulator according to [4], wherein the regulator is an inhibitor of increased expression level of at least one gene selected from the group consisting of SLC2A1, SLC35F6, SLC38A4, SMURF2, SOWAHC, SPARC, SPTAN1, STC1, SUSD6, TAGLN2, TDRP, TENM2, TFRC, TGFBI, TGFBR1, TGFBR2, TGM2, THBS1, THEM4, TNC, TNFAIP1, TNFRSF10B, TRAF4, TRIM21, TRIM65, TSEN34, TUBA1A, TUBA1C, TVP23B, TXNDC9, TYMP, UBE2Q2, UFM1, UGP2, USB1, VCAN, WNT5A, YWHAG, ZBED2, ZNF486, and ZNFX1. <h2 style=";text-align:left;direction:ltr">[6] F1、ADK、AIF1L、AKAP12、ALCAM、ALDH1A3、ALDH3A2、ALOXE3、ANK3、ANXA5、AP PL2、AQP3、ARAP1、ARHGEF40、ARNTL2、BEX4、BMP4、BTBD11、CASP1、CAT、CCNI 、CCZ1B、CD109、CD9、CDC14B、CDHR1、CEBPB、CELSR2、CFAP46、CLIP4、CLMN、CN TN1、COG5、CPNE1、CRYBG1、CSRP2、CTDSP2、DDHD2、DENND4C、DSC3、ECH1、ECM 1、EFL1、EIF4B、ELK3、ELOVL4、EML1、EPHB3、ERO1B、ERRFI1、ETV1、FAM110C、F AM117A, FCHSD2, FERMT1, FHL1, FOSL1, FOXQ1, GABRE, GCLC, GPR176, H1-0, H1-10, HADHB, HDAC5, HMGA1, HPGD, HPSE, HSDL2, IMPA2, INSYN2B, IRF2BPL, JA RID2、JPH2、KANK2、KCNG1、KCNK1、KLF11、KLF4、KMO、KRT14、KRT15、KRT5、LH FPL2、LMNA、LMO4、LRP8、LRRC8A、LYST、MAL2、MAN1A1、MAP4K4、MAPKAPK3、MB OAT2、ME1、MMRN2、MOSMO、MPP7、MYO1D、NAPRT、NAV3、NIBAN1、NKX1-2、NQO2、 NR1D1、NRROS、NTSR1、OPN3、OSBPL1A、P3H2、PABPC1、PAG1、PAK6、PCDHGC3、PD E7A、PER3、PFKFB2、PIR、PITPNC1、PKIB、PLA2G4A、PLEKHG6、PPFIBP2、PRNP、 PRR15、PRX、PRXL2A、PSMG3、PTGR1、PTGS1、RAPGEFL1、RNF180、RPS6KA3、SC5D SIX4, SLC1A3, SLC41A1, SLC48A1, SLC7A11, SLC7A8, SLC9A3R1, SMN1, SMO, SMOC2, SNX29, SOX2, SOX9, SRGAP3, STEAP1, SUN1, SYT7, TAB3, TACC2, TAOK3The expression level regulator according to [4], which is an inhibitor of a decrease in expression level of at least one gene selected from the group consisting of TEF, TGFBR3, TIMP1, TKT, TNIK, TNNT1, TOP1MT, TRAPPC9, TRPV3, TSC22D1, TSHZ2, TTC39A, UBASH3B, VWA5A, WLS, XDH, and ZNF395. [7] The agent according to any one of [1] to [6], which is a cosmetic, a quasi-drug, a medicine, or a food or beverage. Effect of the Invention

[0009] The present invention provides a novel agent for suppressing the expression of a cellular senescence-associated secretory factor. The present invention also provides a novel agent for suppressing the expression of TGFBR1 and / or TGFBR2, and an agent for regulating the expression level of a gene involved in senescence. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing a putative mechanism of cellular senescence. [Diagram 2] 1 is a graph showing IL6 expression levels relative to changes in royal jelly concentration. [Diagram 3] Graph (a) shows the expression level of CXCL1 relative to changes in royal jelly concentration, and graph (b) shows the expression level of CXCL2 relative to changes in royal jelly concentration. [Figure 4] 1 is a graph showing the expression level of IL6 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiment for carrying out the present invention will be described in detail, however, the present invention is not limited to the following embodiment.

[0012] In this specification, the term "comprise" includes the meanings of "essentially consist of" and "consist only of."

[0013] A first aspect of the present invention is an agent for suppressing the expression of senescence-associated secretory phenotype (SASP) factors, which comprises royal jelly as an active ingredient.

[0014] Senescent cells release SASP factors extracellularly. Some SASP factors have the effect of inducing cells surrounding the senescent cells to become senescent cells, promoting the aging of the entire tissue. The SASP factor expression inhibitor of the present invention contains royal jelly as an active ingredient, and is therefore capable of suppressing the expression of SASP factors in senescent cells.

[0015] By suppressing the expression of SASP factors in senescent cells, it is possible to prevent surrounding cells from becoming senescent cells, thereby preventing an increase in senescent cells and inhibiting the aging of biological tissues. The SASP factor expression inhibitor of the present invention can also be called an inhibitor of the propagation of senescence.

[0016] In addition, by suppressing the expression of SASP factors, damage to biological tissues can be improved or prevented. The biological tissue may be, for example, skin tissue. In addition, by suppressing the expression of SASP factors, delayed wound healing due to chronic inflammation can be alleviated and tumor formation can be suppressed (Non-Patent Documents 2 and 3).

[0017] Examples of SASP factors include inflammatory cytokines, chemokines, etc. In the SASP factor expression inhibitor of the present invention, the SASP factor may be, for example, IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14, PAI1 (SERPINE1), etc.

[0018] The SASP factor expression inhibitor can inhibit the expression of SASP factors by inhibiting the expression of genes encoding each SASP factor protein, and therefore the SASP factor expression inhibitor of the present invention can be used as an inhibitor of the expression of genes encoding SASP factors.

[0019] The SASP factor expression inhibitor can suppress the expression of SASP factors depending on the concentration or amount of royal jelly used as the active ingredient. That is, the more the active ingredient is used, the more the SASP factor expression can be suppressed.

[0020] A second aspect of the present invention is an expression inhibitor of TGFBR1 and / or TGFBR2, which are TGF-β receptors, containing royal jelly as an active ingredient. TGFBR1 and TGFBR2 are present in the cell membrane upstream of SASP factors. TGF (Transforming growth factor)-β has the function of regulating the action of SASP factors. Therefore, by inhibiting the expression of TGFBR1 and / or TGFBR2, it is possible to inhibit cell cycle arrest and expression of SASP factors present downstream via phosphorylation of SMAD2 / 3 or a SMAD-independent pathway (Non-Patent Document 4).

[0021] The expression inhibitor of TGFBR1 and / or TGFBR2 can inhibit the expression of TGFBR1 and / or TGFBR2 by inhibiting the expression of genes encoding these proteins. Therefore, the expression inhibitor of TGFBR1 and / or TGFBR2 of the present invention can be used as an expression inhibitor of genes encoding TGFBR1 and / or TGFBR2.

[0022] A third aspect of the present invention is an agent for regulating the expression level of a gene involved in aging, comprising royal jelly as an active ingredient. As used herein, a gene involved in aging is a gene whose expression level increases or decreases due to cellular aging.

[0023] Specifically, the agent for regulating the expression level of a gene involved in aging may be an agent for suppressing an increase or decrease in the expression level of a gene involved in aging.

[0024] The inhibitor of increased expression of senescence-related genes has the effect of suppressing the expression of genes selected from Table 1. These genes are expressed more in senescent cells than in non-senescent cells. By using the inhibitor of increased expression of senescence-related genes, the increased expression of these genes can be suppressed. Among these, CCND2 and RRAS are involved in cell cycle arrest.

[0025] [Table 1]

[0026] The inhibitor of the decrease in the expression level of a gene involved in senescence has the effect of increasing the expression of a gene selected from Table 2. These genes are expressed less in senescent cells than in non-senescent cells. The use of the inhibitor of the decrease in the expression level of a gene involved in senescence can inhibit the decrease in the expression of these genes.

[0027] CAT is an antioxidant enzyme and is involved in the suppression of SASP factor expression. TIMP1 is a collagenase inhibitor. CD109 has the functions of epidermis formation and suppression of TGF-β signaling. The regulator of expression levels of aging-related genes according to the present invention can comprehensively regulate the expression levels of the genes shown in Tables 1 and 2, including these genes, and therefore has a comprehensive anti-aging effect.

[0028] [Table 2]

[0029] The expression level regulator of the aging-related gene can regulate the expression level of the aging-related gene depending on the concentration or amount of royal jelly used as the active ingredient. That is, in the case of an expression level increase inhibitor, the more it is used, the more it can suppress the increase in the expression level, and in the case of an expression level decrease inhibitor, the more it is used, the more it can suppress the decrease in the expression level.

[0030] FIG. 1 is a diagram showing the estimated mechanism of cell aging behavior. In FIG. 1, the underlined genes are those whose expression increases with aging and whose expression decreases with the agent of the present invention, or whose expression decreases with aging and whose expression increases with the agent of the present invention. When cells aging due to DNA damage or the like, SASP factors are secreted. When TGF-β binds to TGFBR1 and TGFBR2, which are TGF-β receptors that regulate the function of SASP factors in another cell, damage such as DNA damage is caused in the cell, leading to cellular aging. As shown in the examples described later, replicative aging of cells increases the expression of SASP factors secreted by aged cells, TGF-β receptors present in the cell membrane upstream of SASP factors, CCND2 and RRAS, which are involved in cell cycle arrest, and the like. Adding royal jelly to aged cells changes various signals, such as suppressing the activity of TGF-β receptors, suppressing inflammatory signals including chemokines, and enhancing regulatory signals for keratinocyte differentiation. By adjusting the expression levels of the above and other aging-related genes, the increased expression of SASP factors can be suppressed.

[0031] The SASP factor expression inhibitor, TGFBR1 and / or TGFBR2 expression inhibitor, and aging-related gene expression regulator of the present invention (hereinafter collectively referred to as "agents of the present invention") contain royal jelly as an active ingredient. Royal jelly is a milky white jelly-like substance made by mixing secretions secreted from the hypopharyngeal gland and mandibular gland by 3-12-day-old worker bees among honeybees. Major physiologically active ingredients in royal jelly include, for example, organic acids such as saturated fatty acids and unsaturated fatty acids that are specific to royal jelly, proteins, amino acids, peptides, lipids, sugars, vitamin B, vitamins such as folic acid, nicotinic acid, and pantothenic acid, and various minerals.

[0032] The royal jelly in the present invention includes raw royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, non-enzyme-treated royal jelly, royal jelly extract, royal jelly fermentation product, etc. Furthermore, royal jelly may be produced in any of European countries, Oceanian countries, America, Brazil, Japan, China, other Asian countries, etc.

[0033] Dried royal jelly powder is obtained by drying fresh royal jelly to powder. As a drying method, any of the known methods adopted in general food processing, such as natural drying such as ventilation drying or sun drying, forced drying by heating with electricity or the like, and freeze drying, can be used. Freeze drying is preferred.

[0034] The enzyme-treated royal jelly is royal jelly treated with a proteolytic enzyme (protease). Preferably, the enzyme-treated royal jelly is hypoallergenic, in which allergic reactions caused by proteins contained in royal jelly are suppressed by protease treatment. Therefore, the enzyme-treated royal jelly may contain organic acids such as the above-mentioned saturated fatty acids and unsaturated fatty acids, lipids, sugars, vitamins, and various minerals, in addition to the protease decomposition products of proteins contained in royal jelly.

[0035] The royal jelly used to produce the enzyme-treated royal jelly is not particularly limited, and examples thereof include raw royal jelly, royal jelly powder obtained by drying and powdering raw royal jelly, and raw royal jelly extracted with water or aqueous ethanol, etc.

[0036] Enzyme-treated royal jelly can be produced by treating royal jelly raw material with an enzyme having at least endopeptidase activity, an enzyme having at least exopeptidase activity, and / or an enzyme having both endopeptidase activity and exopeptidase activity.

[0037] Examples of proteolytic enzymes having at least endopeptidase activity include endopeptidases derived from animals (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), and microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.).

[0038] Examples of proteolytic enzymes having at least exopeptidase activity include carboxypeptidase, aminopeptidase, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus bacteria, Rhizopus bacteria, etc.), and pancreatin and pepsin which also have endopeptidase activity.

[0039] Among these various enzymes, preferred examples of enzymes having both exopeptidase activity and endopeptidase activity include peptidase produced by Streptomyces griseus (trade name: Actinase AS), peptidase produced by Aspergillus oryzae (trade name: Protease A, Flavorzyme, Proteax), and peptidase produced by Aspergillus melleus (trade name: Protease P). Preferred examples of enzymes having exoprotease activity include peptidase produced by Aspergillus oryzae (trade name: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), peptidase produced by Aspergillus sojae (trade name: Sternzyme B15024), peptidase produced by the genus Aspergillus (trade name: Coclase P), and peptidase produced by Rhizopus oryzae (trade name: Peptidase R). Further preferred examples of enzymes having endoprotease activity include peptidase produced by Bacillus subtilis (trade name: Orientase 22BF, Nucleisin), peptidase produced by Bacillus licheniformis (trade name: Alcalase), peptidase produced by Bacillus stearothermophilus (trade name: Protease S), peptidase produced by Bacillus amyloliquefaciens (trade name: Neutrase), and peptidase produced by the genus Bacillus (trade name: Protamex).

[0040] Enzyme treatment to reduce the allergenicity of royal jelly can be carried out, for example, according to the description in JP-A-2007-295919 and JP-A-2007-295920.

[0041] Royal jelly extract is obtained by extracting royal jelly (including raw, dried and crushed royal jelly) with water or aqueous ethanol or the like.

[0042] The royal jelly fermented product can be produced by a conventional method using microorganisms such as yeast and lactic acid bacteria.

[0043] Royal jelly may be commercially available. Specific examples of commercially available royal jelly include Enzymatically Decomposed Royal Jelly King (manufactured by Yamada Bee Farm), Royal Jelly FD Powder (manufactured by Nakahara Co., Ltd.), Royal Jelly Extract SF (manufactured by Matsuura Pharmaceutical Co., Ltd.), Deproteinized Royal Jelly Powder F (manufactured by Maruzen Pharmaceutical Co., Ltd.), and Deproteinized Royal Jelly Extract (manufactured by API Co., Ltd.).

[0044] The agent of the present invention is highly safe since it uses royal jelly, which has been used as a food ingredient for a long time.

[0045] The content of the above-mentioned active ingredient in the agent of the present invention, in terms of solid content relative to the total amount of the agent, may be, for example, 0.1% by mass or more, 1% by mass or more, 3% by mass or more, 3.5% by mass or more, 5% by mass or more, 7% by mass or more, 10% by mass or more, 15% by mass or more, 20% by mass or more, 25% by mass or more, 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, 50% by mass or more, 55% by mass or more, 60% by mass or more, 65% by mass or more, 70% by mass or more, 75% by mass or more, 80% by mass or more, 85% by mass or more, 90% by mass or more, 93% by mass or more, 95% by mass or more, 98% by mass or more, 99% by mass or more, or 100% by mass. The content of the above active ingredient in the agent is solid content based on the total amount of the agent, for example, 100% by mass or less, 99% by mass or less, 98% by mass or less, 95% by mass or less, 93% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 6 It may be 0 mass% or less, 55 mass% or less, 50 mass% or less, 45 mass% or less, 40 mass% or less, 35 mass% or less, 30 mass% or less, 25 mass% or less, 20 mass% or less, 15 mass% or less, 10 mass% or less, 8 mass% or less, 5 mass% or less, 3 mass% or less, or 1 mass% or less.

[0046] The agent of the present invention can be administered to humans for use in, for example, suppressing the expression of SASP factors, suppressing the expression of TGFBR1 and / or TGFBR2, and adjusting the expression level of aging-related genes in the body. The agent of the present invention may be administered orally or parenterally. Oral administration includes enteral administration. Parenteral administration includes topical administration, particularly transdermal administration.

[0047] The dosage of the agent of the present invention when administered orally may vary depending on the form of the agent and the application method and amount, but for example, for an adult weighing 60 kg, the dosage may be 10 mg to 30,000 mg, preferably 100 mg to 20,000 mg, 150 mg to 15,000 mg, 600 mg to 12,000 mg, 1200 mg to 10,000 mg, or 2,400 mg to 8,000 mg per day in terms of dry solid content of the active ingredient. The dosage may be increased or decreased as appropriate depending on factors such as the health condition of the person taking the agent, the method of administration, and the combination with other agents.

[0048] As long as the effective daily dose is within the above-mentioned range, the agent of the present invention may be administered once a day, or may be administered in divided doses such as twice or three times a day. The agent of the present invention exhibits effects immediately after administration, but continuous administration for 1 to 4 weeks, or for one month or more, six months or more, or one year or more is preferable because the effects can be sustained longer.

[0049] The dosage of the agent of the present invention when administered parenterally may vary depending on the site and range of application. For example, the amount applied to the skin may be 0.01 mg to 50 mg of the active ingredient, preferably 0.02 mg to 40 mg, or 0.02 mg to 35 mg, or 0.025 mg to 30 mg, calculated as the dry solid content.

[0050] The content of the above-mentioned active ingredient in a cosmetic, food or drink, quasi-drug, or pharmaceutical product containing the agent of the present invention is not particularly limited, and may be an effective amount that can achieve the dosage of oral or parenteral administration of the agent.

[0051] The agent of the present invention can be used as cosmetics, food and beverages (particularly food and beverages intended for health, maintaining or improving health, etc. (e.g., health foods, functional foods, nutritional compositions, dietary supplements, supplements, health foods, foods for specified health uses, foods with nutrient functions, or foods with functional claims)), quasi-drugs, pharmaceuticals, etc. The agent of the present invention also encompasses the meaning of an additive that imparts an effect of suppressing the expression of SASP factors, an effect of suppressing the expression of TGFBR1 and / or TGFBR2, and an effect of regulating the expression level of genes involved in aging.

[0052] Cosmetics, foods and beverages, quasi-drugs, or pharmaceuticals containing the agent of the present invention as one component can be produced, for example, by adding the agent of the present invention to intermediate products in the manufacturing process of these products.

[0053] In addition to the royal jelly, the above-mentioned cosmetic product may contain, as necessary, ingredients typically used in cosmetics, such as bactericides, preservatives, surfactants, alcohols, aqueous components, water, colorants, pH adjusters, solubilizers, abrasives, foaming agents, enzymes, flavorings, chelating agents, excipients, thickeners, bases, emulsifiers, solvents, stabilizers, oils, cleaning agents (lactic acid bacteria), whitening agents, moisturizers, antioxidants, UV absorbers, powder components, colorants, and various skin nutrients.

[0054] The cosmetic raw material may be in any form, such as a solid, liquid, or paste. The cosmetic may be a medicated cosmetic (i.e., a quasi-drug). The cosmetic includes any cosmetic that can be applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, and other parts of animals (including humans).

[0055] Cosmetics can take a wide variety of formulations, including aqueous solutions, solubilized solutions, emulsions, powders, oils, gels, ointments, aerosols, water-oil two-layer systems, and water-oil-powder three-layer systems.

[0056] The use of the cosmetics is also arbitrary, and for example, basic cosmetics include facial cleanser, lotion, milky lotion, cream, gel, essence, beauty serum, pack, mask, mist, UV protection cosmetics, etc., makeup cosmetics include foundation, lipstick, blusher, eye shadow, eyeliner, mascara, etc., nail cosmetics include nail polish, base coat, top coat, nail polish remover, etc., and other examples include facial cleanser, (paste or liquid) dentifrice, mouth rinse, oral cosmetics, massage agents, cleansing agents, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling agent, hair tonic agent, hair mist, hair foam, hair liquid, hair gel, hair spray, hand cream, hand soap, hair growth agent, antiperspirant, bath agent, etc.

[0057] The above-mentioned royal jelly can be used as is in the above-mentioned foods and beverages, or, if necessary, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, fragrances, stabilizers, gelling agents, preservatives, sustained-release regulators, surfactants, solubilizers, humectants, etc. can also be blended therein.

[0058] Food and drink includes all foods and drinks that animals (including humans) can consume. The types of food and drink are not particularly limited, and examples thereof include dairy products, fermented foods (yogurt, etc.), beverages (soft drinks such as coffee, juice, and tea drinks, milk drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and alcohol such as fruit wine, etc.), spreads (custard cream, etc.), pastes (fruit paste, etc.), Western confectionery (chocolate, donuts, pies, cream puffs, gum, gummies, jellies, candies, cookies, cakes, puddings, etc.), Japanese confectionery (daifuku, mochi, manju, castella, anmitsu, yokan, etc.), frozen desserts (ice cream, popsicles, sorbets, etc.), foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.), and seasonings (dressing, furikake, umami seasoning, soup base, etc.).

[0059] The method for producing the food or drink is not particularly limited, and any known method can be used as appropriate.

[0060] The dosage unit form when used as a supplement is not particularly limited and can be selected as appropriate. Examples include tablets such as chewable tablets and lozenges, capsules, granules, liquids, powders, syrups, pastes, drinks, gummies, and the like.

[0061] The above-mentioned pharmaceuticals may contain only the above-mentioned royal jelly, or may be mixed with other medicinal ingredients such as vitamins and herbal medicines listed in the Japanese Pharmacopoeia.

[0062] When the agent of the present invention is prepared as a pharmaceutical product, the royal jelly can be prepared, together with pharmaceutical acceptable ingredients, into the form of tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (dispersed medicines), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections (including the case where the agent is mixed with distilled water or an infusion such as an amino acid infusion or an electrolyte infusion at the time of use to prepare a liquid), etc., to form a pharmaceutical preparation.

[0063] The pharmaceutical agent may be administered locally or systemically. There is no particular limitation on the administration method, and the pharmaceutical agent may be administered orally or parenterally. Parenteral administration routes include subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous or intraarterial administration, and transdermal administration.

[0064] In addition to royal jelly, the pharmaceutical of the present invention may contain, as necessary, pharma- ceutically acceptable ingredients such as excipients, binders, disintegrants, lubricants, colorants, suspending agents, thickeners, antioxidants, absorption enhancers, pH adjusters, preservatives, antiseptics, stabilizers, surfactants, sweeteners, flavorings, and fragrances.

[0065] The cosmetics and pharmaceuticals of the present invention also include quasi-drugs.

[0066] The agent of the present invention described above is applicable to mammals including humans (preferably humans). EXAMPLES

[0067] [Preparation of royal jelly] A medium (hereinafter referred to as KGM medium) was prepared by adding keratinocyte growth medium 2 Supplement Mix (PromoCell, C-39016) and 1% penicillin-streptomycin mixed solution (penicillin 10,000u / mL, streptomycin 10,000μg / mL; Nakarai, 09367-34) to keratinocyte basic medium 2 (phenol red-free; PromoCell, C-20216). Unenzymatically decomposed royal jelly was dissolved in KGM medium to a final concentration of 1mg / mL, ultrasonically crushed for 30 minutes, and then filter-sterilized. This was used as royal jelly in the following tests.

[0068] [Cell culture] Normal human epidermal keratinocytes (NHEK, PromoCell, Lot. 470Z031) derived from infants were maintained in KGM medium. NHEK cells that reached subconfluence were subcultured to 3 × 10 4 cells / cm 2 The cells were seeded on a 35 mm dish (Thermo Scientific BioLite130180) so that the cell density was 100%. P5 (cultured for 5 generations) was used as a young model cell, and P20 (cultured for 20 generations) was used as an old model cell. The day after seeding, royal jelly was added to a portion of the P20 at a concentration of 1 mg / ml, and the cells were used for measurement 24 hours after addition. The following measurements were performed on P5, P20, and royal jelly-added P20.

[0069] [Gene expression measurement] Total RNA was extracted using the Total RNA Purification Plus Kit (Norgen Biotek, 48300) according to the manufacturer's protocol. The amount of RNA was quantified using a NanoDropOne microspectrophotometer (ThermoScientific, ND-ONE-W). 2 μl of 5×RTMaster Mix (Toyobo, FSQ201) was added to 8 μL of total RNA prepared to 50 ng, and reverse transcription was performed using a CFX Opus96 real-time PCR system (Bio-rad) under the conditions of 37°C for 15 minutes, 50°C for 5 minutes, and 98°C for 5 minutes to produce cDNA. SsoAdvanced Universal SYBR Green Supermix (Biorad, 172-5271B02) was used to prepare the PCR reaction solution. The primers used and their sequences are shown in Table 3. The RPLP0 gene was used as a reference. Real-time PCR was performed using a CFX Opus96 real-time PCR system (Bio-rad) under the conditions of 95°C for 30 seconds (1 cycle), 95°C for 15 seconds, and 60°C for 30 seconds (40 cycles).

[0070] [Table 3]

[0071] [Examination of royal jelly concentration] For the gene IL6, the concentration of royal jelly added was adjusted to 0 μg / ml, 125 μg / ml, 250 μg / ml, 500 μg / ml, or 1000 μg / ml, and the gene expression level was measured by the same method as above using the primers shown in Table 3. The results are shown in FIG. 2. For the genes CXCL1 and CXCL2, the concentration of royal jelly added was adjusted to 0 μg / ml, 500 μg / ml, or 1000 μg / ml, and the gene expression level was measured by the same method as above using the primers shown in Table 3 (n=3). The results for CXCL1 are shown in FIG. 3(a), and the results for CXCL2 are shown in FIG. 3(b). It was confirmed that the expression levels of IL6, CXCL1, and CXCL2 could be suppressed depending on the concentration of royal jelly added.

[0072] [RNA sequencing] The isolated total RNA was adjusted to 50-100ng / μL, and the quantitative analysis of gene expression and differentially expressed genes (DEG) analysis of the samples were entrusted to Rhelixa Co., Ltd. The RNA samples were confirmed to be not decomposed using a bioanalyzer. RNA seq libraries were created using the NEBNext (registered trademark) Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and the NEBNext (registered trademark) UltraTMII Directional RNA Library Prep Kit (NEB, E7760), respectively, and subjected to the next-generation sequencer NovaSeq6000 (Illumina).

[0073] After that, informatics analysis was performed according to the following procedure. The sequence reads were trimmed using the software Trimmomatic (Version 0.38) based on the following settings: ILLUMINACLIP2:30:10, LEADING20, TRAILING20, SLIDINGWINDOW4:15, MINLEN36, and the software HISAT2 (Version 2.1.0) was used to map to the reference genome. RLE normalization was performed based on the number of reads calculated using the software featureCounts (Version 1.6.3), and differentially expressed genes were extracted using the software DESeq2 (Version 1.24.0).

[0074] Increases or decreases in gene expression were detected with a P value of less than 0.05. The number of mRNAs whose expression increased at P20 compared to P5, i.e., whose expression increased due to replicative senescence, was 2939. The number of mRNAs whose expression decreased at P20 with royal jelly compared to P20, i.e., whose expression decreased by royal jelly treatment in replicative senescent cells, was 740. Of these, the number of mRNAs whose expression increased due to replicative senescence and whose expression decreased by royal jelly treatment in replicative senescent cells was 197. Genes whose expression increased due to replicative senescence and whose expression decreased by royal jelly treatment in replicative senescent cells are shown in Table 4.

[0075] In particular, it was confirmed that the expression levels of the genes encoding the SASP factors IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14, and PAI1, as well as the genes encoding TGFBR1 and TGFBR2, increased with replicative senescence and decreased with the addition of royal jelly. Similarly, it was confirmed that the expression levels of the genes encoding RRAS and CCND2 increased with replicative senescence and decreased with the addition of royal jelly.

[0076] On the other hand, the number of mRNAs whose expression levels were lower at P20 than at P5, i.e., whose expression was decreased due to replicative senescence, was 3097. The number of mRNAs whose expression levels were higher at royal jelly-added P20 than at P20, i.e., whose expression was increased by royal jelly treatment in replicative senescent cells, was 731. Of these, the number of mRNAs whose expression was decreased by replicative senescence and whose expression was increased by royal jelly treatment in replicative senescent cells was 178. The genes whose expression was decreased by replicative senescence and whose expression was increased by royal jelly treatment in replicative senescent cells are shown in Table 5.

[0077] [Table 4]

[0078] [Table 5]

[0079] Table 6 shows the gene expression levels (fold change) of IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14 and PAI1 when the gene expression level in the young model P5 was set to 1.

[0080] [Table 6]

[0081] [Protein expression level] The IL6 levels in the collected culture supernatants were evaluated using a Human IL-6 Quantikine ELISA Kit (R&D Systems, D6050) according to the manufacturer's protocol.

[0082] The results are shown in Figure 4. At P20, it was confirmed that the amount of IL6 protein in the culture supernatant was significantly lower in the royal jelly-added group than in the royal jelly-unadded group. This demonstrated that the addition of royal jelly suppressed the expression of IL6 protein, the expression level of which increases with replicative senescence.

Claims

1. An agent for suppressing the expression of senescence-associated secretory phenotype (SASP) factors, comprising royal jelly as an active ingredient.

2. The expression inhibitor according to claim 1, wherein the SASP factor is at least one selected from the group consisting of IL6, CXCL1, CXCL2, CXCL3, CXCL5, CXCL10, CXCL14 and PAI1.

3. An agent for suppressing the expression of TGF-β receptors TGFBR1 and / or TGFBR2, which contains royal jelly as an active ingredient.

4. An agent for regulating the expression of genes involved in aging, containing royal jelly as an active ingredient.

5. The genes involved in aging are ABCA7, ACTG1, ALDH4A1, AMIGO2, ANKRD50, AP2M1, ASB1, ATP11A, B3GNT2, B4GAL-T1, BAZ2B, BCAR3, C11orf24, C1QTNF1, C1RL, CALD1, CAM-SAP2, CASP8, CCND2, CD274, CDH3, CDK6, CDYL, CEP170, COL1A1, COL4A1, COL4A2, COL4A5, COL4A6, COL5A1, COL-GALT1, CORO1C, CPA4, CRIM1, CRNN, CSF3, CSNK1E, CSRP1, CSTF3, CXCL1, CXCL10, CXCL14, CXCL2, CXCL3, CXCL5, CYP24A1, DECR1, DHX16, DRAM1, DUSP4, DUSP5, DYNC1H1, EDN1, EMC1, EPAS1, EREG, FAP, FAT1, FBN2, FNBPL1, FSIP1, FUS, G0S2, GNB4, GPR157, GPR68, GPRC5A, HEG1, HELZ, HIVEP2, HLA-B, HSP4L, HSPG2, HUWE1, ID3, IL6, IRF6, ITGB6, IVNS1ABP, JAG1, JPT2, KCNJ15, KCTD11, KIAA0895L, KIFC3, KRT7, KYNU, LGALS3BP, LIMK2, LOXL2, LTBP1, LTBP2, MAT2A, MB21D2, MCAM, MDN1, MEST, MICAL2, MOB3B, MOCS3, MPZL2, MRAS, MRGPRX3, MTAP, MT-CO1, MT-CO2, MTHFD2L, MT-ND2, MYD88, MYH9, NCEH1, NEFL, NIPAL3, NIPSNAP3A, NOMO2, P4HA1, PALM2AKAP2, PCDD11, PCDD5, PFKFB3, PGAM1, PGK1, PHF20, PLAT, PLOD2, PMEPA1, POLR1A, POMK, PPP1R3B, PPP1R3C, PRKAR1A, PRORP, PRR5L, PSME3, PTGES, PTGFRN, PTPN14, PTPRK, RAI14, RGMB, RHOTB2, RNF152, RRAS, SACS, SBNO2, SDC4, SEM7A, SERPINE1, SERPINH1, SGK1, SH2D3A, SHFL, SIPAL1L1, SKIL, SLAMF9, SLC16A3,SLC2A1, SLC35F6, SLC38A4, SMURF2, SOWAHC, SPARC, SPTAN1, STC1, SUSD6, TAGLN2, TDRP, TENM2 , TFRC, TGFBI, TGFBR1, TGFBR2, TGM2, THBS1, THEM4, TNC, TNFAIP1, TNFRSF10B, TRAF4, TRIM21, T The expression level regulator according to claim 4, which is an inhibitor of increased expression level of at least one gene selected from the group consisting of RIM65, TSEN34, TUBA1A, TUBA1C, TVP23B, TXNDC9, TYMP, UBE2Q2, UFM1, UGP2, USB1, VCAN, WNT5A, YWHAG, ZBED2, ZNF486, and ZNFX1.

6. The aging-related genes are ABCC2, ABCC3, ABHD5, ABLIM1, ACSL1, ACS2, ADGRA3, ADGRF1, ADK, AIF1L, AKAP12, ALCAM, ALDH1A3, ALDH3A2, ALOXE3, ANK3, ANXA5, APPL2, AQP3, ARAP1, ARHGDEF40, ARNTL2, BEX4, BMP4, BTBD11, CASP1, CAT, CCNI, CCZ1B, CD109, CD9, CDC14B, CDHR1, CEBPB, CELSR2, CFAP46, CLIP4, CLMN, CNTN1, COG5, CPNE1, CRYBG1, CSRP2, CTDSP2, DDHD2, DENNDC4, DSC3, ECH1, ECM1, EFL1, EIF4B, ELK3, ELOVL4, EML1, EPHB3, ERO1B, ERRFI1, ETV1, FAM110C, FAM117A, FCHSD2, FERMT1, FHL1, FOSL1, FOXQ1, GABRE, GCLC, GPR176, H1-0, H1-10, HADHB, HDAC5, HMGA1, HPGD, HPSE, HSDL2, IMPA2, INSYN2B, IRF2BPL, JARID2, JPH2, KANK2, KCNG1, KCNK1, KLF11, KLF4, KMO, KRT14, KRT15, KRT5, LHPL2, LMNA, LMO4, LRP8, LRRC8A, LYST, MAL2, MAN1A1, MAP4K4, MAPKAPK3, MBOAT2, ME1, MMRN2, MOSMO, MPP7, MYO1D, NAPRT, NAV3, NIBAN1, NKX1-2, NQO2, NR1D1, NRROS, NTSR1, OPN3, OSBPL1A, P3H2, PABPC1, PAG1, PAK6, PCDHGC3, PDE7A, PER3, PFKFB2, PIR, PITPNC1, PKIB, PLA2G4A, PLEKHG6, PPFIBP2, PRNP, PRR15, PRX, PRXL2A, PSMG3, PTGR1, PTGS1, RAPGEFL1, RNF180, RPS6KA3, SC5D, SIX4, SLC1A3, SLC41A1, SLC48A1, SLC7A11, SLC7A8, SLC9A3R1, SMN1, SMO, SMOC2, SNX29, SOX2, SOX9, SRGAP3, STEAP1, SUN1, SYT7, TAB3, TACC2, TOK3, TEF,The expression level regulator according to claim 4, which is an inhibitor of a decrease in expression level of at least one gene selected from the group consisting of TGFBR3, TIMP1, TKT, TNIK, TNNT1, TOP1MT, TRAPPC9, TRPV3, TSC22D1, TSHZ2, TTC39A, UBASH3B, VWA5A, WLS, XDH, and ZNF395.

7. The agent according to any one of claims 1 to 6, which is a cosmetic, a quasi-drug, a medicine, or a food or drink.

Images