Novel screening method for Anti-aging substance and epiregulin expression inhibitor

Abstract

To establish a novel aging-related indicator and provide a novel screening method for anti-aging substances using the same.SOLUTION: This screening method is a screening method for anti-aging substances using epiregulin expression as an indicator, including: a culturing step of culturing fibroblasts in a medium having a test substance added thereto; a measurement step of measuring epiregulin expression in the culture after the culturing step; and a selection step of selecting a test substance that exhibits reduced epiregulin expression in comparison to cases without the addition of the test substance.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a screening method for a novel anti-aging substance and an epiregulin expression inhibitor. [Background technology]

[0002] In modern medicine and biology, elucidating the mechanism of aging and preventing premature aging are important themes. Skin is a mirror that reflects the aging process of an individual, and interest in skin is increasing from the perspective of beauty. Changes in the morphology of the skin surface, such as wrinkles, caused by the aging phenomenon are recognized as affecting the quality of life. Currently, the aging process of the skin is known to be divided into "physiological aging" caused by tissue damage associated with aging and "extrinsic aging" caused by environmental factors such as ultraviolet (UV) radiation, smoking, and environmental pollutants. Skin aging contributes to functional defects in skin homeostasis, such as a decrease in elasticity in the dermal connective tissue, which is composed of collagen fibers and elastic fibers.

[0003] The mechanism of photoaging has been widely studied, and several key factors have been reported that lead to extracellular matrix (ECM) degradation affecting the dermal layer while causing significant damage to connective tissue. UV irradiation impairs the transforming growth factor (TGF)-β / Smad pathway in human skin by downregulating TGF-β type II receptors, which results in increased expression of matrix metalloproteinases and inflammatory cytokines, leading to ECM degradation in the dermis.

[0004] On the other hand, in physiological aging, the total number of dermal fibroblasts decreases significantly with age, and aged fibroblasts (senescent fibroblasts) increase in the aged dermal layer. One characteristic of senescent cells is the appearance of senescence-associated secretory phenotypes (SASPs), which are important clues for anti-aging care. Senescent fibroblasts increase the secretion of SASP factors such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, which can cause paracrine tissue dysfunction (Non-Patent Document 1). Furthermore, senescent fibroblasts have a reduced ability to synthesize ECM-related proteins, which are components of collagen fibers and elastic fibers (Non-Patent Document 2). However, the mechanism that controls the secretion of SASP factors and other factors remains unknown. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Journal of Investigative Dermatology(2015)135,1954-1968 [Non-Patent Document 2] American Journal of Pathology, Vol. 168, No. 6, 2006, 1861-1868. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to establish a new indicator associated with aging and to provide a screening method for novel anti-aging substances using the same. [Means for solving the problem]

[0007] The present inventors conducted aging research using skin tissues from young and elderly people and found that the expression of epiregulin (EREG) is increased in the dermis of elderly people and that epiregulin affects the expression of ECM-related genes in the dermis. That is, the present invention has the following configuration.

[0008] The screening method of the present invention is a method for screening for anti-aging substances, and is characterized in that it uses epiregulin expression as an indicator.

[0009] The method is characterized by comprising a culture step of culturing fibroblasts in a medium containing the test substance, a measurement step of measuring the expression of epiregulin in the culture after the culture step, and a selection step of selecting a test substance that suppresses the expression of epiregulin compared to when the test substance is not added.

[0010] The method further comprises a co-culture step of co-culturing passage-aged fibroblasts having a high population doubling level cultured in a medium containing the test substance with normal fibroblasts having a lower population doubling level than the passage-aged fibroblasts, and a second measurement step of measuring the expression of an extracellular matrix-related substance in the normal fibroblasts after the co-culture step. Here, the normal fibroblasts refer to fibroblasts having a low population doubling level (specifically, up to about PDL35) that have not been induced to age. The extracellular matrix-related substance is at least one of COL1A1, COL1A2, FBN1, and FBN2.

[0011] The epiregulin expression inhibitor of the present invention is characterized by containing pearl oyster glycogen. Effect of the Invention

[0012] According to the present invention, screening of anti-aging substances using the expression of epiregulin (EREG), a new indicator, can contribute to the acquisition of new anti-aging substances. The pearl oyster glycogen obtained by this screening method exhibits an effect of suppressing the expression of epiregulin, and can increase the density of collagen fibers in human skin in actual in vivo tests. [Brief description of the drawings]

[0013] [Figure 1]FIG. 1 is a flow chart showing an example of the screening method of the present invention. [Diagram 2] FIG. 1 is a diagram showing the relationship between culture time and PDL. [Diagram 3] FIG. 1 shows the expression levels of EREG, EGF, and HB-EGF. [Figure 4] FIG. 1 shows images of skin tissue, etc. [Diagram 5] FIG. 1 shows the expression levels of ECM-related genes. [Figure 6] FIG. 1 shows photographs of Western blot analysis. [Figure 7] FIG. 1 shows the expression levels of the EREG gene in fibroblasts treated with test substances. [Figure 8] FIG. 1 shows a protocol for one example of co-culture. [Figure 9] FIG. 1 shows collagen images and the like in an in vivo test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The screening method of the present invention uses the expression of epiregulin (EREG) as an index. Epiregulin is a growth factor belonging to the EGF family, and is a soluble protein of about 5 kDa consisting of 46 amino acids. Epiregulin specifically binds to ErbB1 (EGFR) and ErbB4.

[0015] Epiregulin is secreted from fibroblasts, keratinocytes, and macrophages in the skin and plays an important role in inflammation, wound healing, and hair growth processes, and is said to be expressed mainly in the placenta, peripheral blood leukocytes, and cancer tumors of the lung, kidney, colon, etc. However, research related to skin aging is limited, and this invention established epiregulin as a novel factor involved in the deterioration of ECM-related substances with aging.

[0016] An example of the screening method of the present invention will be described with reference to the flow chart of Figure 1. As shown in Figure 1, the screening method comprises a culture step (1) of culturing fibroblasts in a medium containing a test substance, a measurement step (2) of measuring the expression of epiregulin in the culture after the culture step, and a selection step (3) of selecting a test substance that suppresses the expression of epiregulin as compared to when the test substance is not added. Each step will be described below. Note that Figure 1 also shows, as another example, an example of a method further comprising a co-culture step (4) and a second measurement step (5), which will be described later.

[0017] [Culture process (1)] In this step, fibroblasts are cultured in a medium containing a test substance. In the present invention, a "test substance" is a substance that is subjected to screening to determine whether it corresponds to an epiregulin expression inhibitor or an anti-aging agent. The test substance is not particularly limited, and examples thereof include plant-derived components (including plant extracts), animal-derived components (including animal extracts), proteins, sugars (including polysaccharides, oligosaccharides, monosaccharides, etc.), organic compounds, inorganic compounds, etc.

[0018] The medium may be any medium commonly used in the field of cell culture. For example, Dulbecco's modified Eagle medium (DMEM), Eagle's minimum essential medium (EMEM), etc. may be used. The medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. If necessary, the medium may contain a serum substitute such as albumin, and may contain substances such as amino acids and inorganic salts.

[0019] In the culture step (1), the culture temperature is, for example, 30° C. to 40° C., and preferably about 37° C., and the culture is performed in a CO2 incubator. The CO2 concentration is, for example, 2% to 5%, and preferably about 5%. The culture time is, for example, 24 hours to 72 hours.

[0020] The concentration of the test substance in the medium is not particularly limited, but may be, for example, 1 ppm to 1000 ppm, or may be 10 ppm to 100 ppm. The concentration of the test substance in the medium may be set in a stepwise manner from a low concentration to a high concentration.

[0021] The fibroblasts used for the culture are cells that produce collagen fibers, elastic fibers, hyaluronic acid, etc., and for example, commercially available products can be used. The origin of the fibroblasts is not particularly limited, and fibroblasts derived from human neonatal foreskin or human adult can be used.

[0022] Fibroblasts have a relatively high division rate, and senescent fibroblasts can be obtained by repeated subculture. In the screening method of the present invention, epiregulin expression is confirmed in the control group, so it is preferable that epiregulin is expressed to a certain degree in the fibroblasts. From this viewpoint, the fibroblasts used are preferably senescent fibroblasts, and the population doubling level (PDL) is, for example, 30 or more, and preferably 40 or more. In order to obtain fibroblasts that satisfy such a population doubling level, subculture can be performed for a required period (for example, 1 month or more, preferably 3 months or more).

[0023] In the culture step (1), it is sufficient to culture at least fibroblasts in a medium. The fibroblasts may be cultured alone or together with other cells such as epidermal cells, epidermal stem cells, or mesenchymal stem cells.

[0024] [Measurement process (2)] In this step, the expression of epiregulin in the culture (e.g., in fibroblasts) after the culture step (1) is measured. The measurement method is not particularly limited, and the gene expression level or protein level of epiregulin in the culture is measured using a molecular biology method. For example, the gene expression level can be measured by performing real-time PCR (RT-PCR) using the primers described in the Examples. Furthermore, the protein level can be measured by a method such as Western blotting using an antibody specific to epiregulin.

[0025] [Selection process (3)] In this step, a test substance that suppresses epiregulin expression compared to when the test substance is not added is selected. Specifically, the test substance is selected as an epiregulin expression inhibitor. Suppression of epiregulin expression can be determined based on a comparison with a control group in which the culture step (1) and the measurement step (2) were carried out without adding the test substance. Note that the suppression of epiregulin expression may be achieved if the gene expression level or protein level of epiregulin is reduced compared to the control group, and preferably the reduction is significant.

[0026] An example of the procedure for the above-mentioned screening method is shown below. Dermal fibroblasts are cultured in a medium containing the test substance at 37°C, 5% CO2, and in a humidified atmosphere for 48 hours. The gene expression level or protein level of epiregulin in the culture is then measured by a standard method. As a control, the expression level is also measured in dermal fibroblasts cultured in the same manner without the addition of the test substance. If the expression level in the culture containing the test substance is lower than the expression level in the culture without the addition of the test substance, the test substance is selected as an epiregulin expression inhibitor.

[0027] Next, another example of the screening method of the present invention will be described. In addition to the above-mentioned steps (1) to (3), this method further includes a co-culture step (4) and a second measurement step (5) (see FIG. 1). These steps will be described below.

[0028] [Co-culture process (4)] In this step, fibroblasts cultured in a medium containing a test substance (for convenience, referred to as aged fibroblasts) are co-cultured with other fibroblasts having a lower PDL than the fibroblasts (for convenience, referred to as young fibroblasts). The fibroblasts cultured in a medium containing a test substance can be obtained by the procedure of the above-mentioned culture step (1), and for example, some of the fibroblasts after the culture step (1) may be used as the aged fibroblasts. In addition, the young fibroblasts may be fibroblasts having a lower PDL than the aged fibroblasts, for example, a PDL of 20 or less, preferably 10 or less.

[0029] The co-culture may be a non-contact type co-culture in which the aged fibroblasts and the young fibroblasts are not in physical contact with each other, or a contact type co-culture in which the aged fibroblasts and the young fibroblasts are in contact with each other. The non-contact type co-culture may be cultured in a state in which the aged fibroblasts and the young fibroblasts are separated and both cells can share a medium, and may be performed, for example, by the transwell method.

[0030] The transwell method is a method in which a culture vessel is partitioned using a culture insert having a porous membrane at the bottom, and the two types of cells are cultured separately. In this case, from the viewpoint of operability, it is preferable to culture the young fibroblasts on the porous membrane on the inner surface of the culture insert, and the aged fibroblasts on the outer surface of the culture insert. Examples of the material of the porous membrane include polyethylene terephthalate and polycarbonate. The pore size of the porous membrane may be, for example, 0.05 μm to 5 μm, as long as it is impermeable to cells and permeable to the medium.

[0031] The medium, culture conditions, culture time, etc. in the co-culture step (4) are not particularly limited, and those described in the culture step (1) can be appropriately adopted. Note that, as the medium used in the co-culture step (4), a medium containing a test substance is used, and as a control, a medium containing no test substance is used.

[0032] The young fibroblasts used in the co-culture step (4) may be pre-cultured in a medium not containing the test substance. For example, young fibroblasts can be seeded and then cultured in a medium not containing the test substance until they reach subconfluence.

[0033] [Second measurement process (5)] In this step, the expression of extracellular matrix (ECM)-related substances in the juvenile fibroblasts after the co-culture step (4) is measured. Examples of ECM-related substances include, but are not limited to, collagen I (COL1A1, COL1A2), fibrillin (FBN1, FBN2), hyaluronan synthase 2 (HAS2), and versican (VCAN).

[0034] The method for measuring ECM-related substances is not particularly limited, and the expression level of ECM-related genes or the amount of ECM-related proteins in young fibroblasts is measured using molecular biology techniques. For example, gene expression levels can be measured by RT-PCR using the primers described in the Examples. Protein levels can be measured by techniques such as ELISA.

[0035] The results of the second measurement step (5) can be used as a material for selection in the above-mentioned selection step (3). For example, from among the multiple test substances selected as epiregulin expression inhibitors in the selection step (3), a test substance that increases the expression of an ECM-related substance in young fibroblasts compared to a control group in which aged fibroblasts cultured in a medium not containing the test substance are co-cultured with young fibroblasts can be selected as a more suitable candidate substance (epiregulin expression inhibitor) in the second measurement step (5). That is, in this case, the test substance not only directly acts on aged fibroblasts to suppress the expression of epiregulin, but also indirectly acts on the co-cultured young fibroblasts to promote the expression of an ECM-related substance in the cells. The promotion of the expression of an ECM-related substance leads to the promotion of the synthesis of collagen fibers and elastic fibers, and is therefore considered to provide a higher anti-aging effect. The promotion of the expression of an ECM-related substance may be achieved as long as the gene expression level or protein level of the ECM-related substance is increased compared to the control group, and it is preferable that the increase is significant.

[0036] It should be noted that the screening method of the present invention is not limited to the above-mentioned method.

[0037] For example, the screening method of the present invention may have a pre-culture step of culturing fibroblasts in a medium containing no test substance prior to the above-mentioned culture step (1). This step may be carried out, for example, after seeding of fibroblasts, until the medium reaches subconfluence. When the pre-culture step is carried out, the culture step (1) may be carried out by replacing the medium with one containing the test substance, or by adding the test substance to the medium in the pre-culture step.

[0038] Furthermore, the screening method of the present invention may further comprise, after the above-mentioned culturing step (1), a post-culturing step of further culturing the fibroblasts in a medium containing no test substance.

[0039] By screening test substances using the above-mentioned screening method of the present invention, it was found that pearl oyster glycogen and water chestnut fruit extract exhibited an epiregulin expression inhibitory effect. Furthermore, it was found that pearl oyster glycogen exhibited an ECM-related substance expression promoting effect in co-cultured fibroblasts. Based on this finding, the epiregulin expression inhibitor of the present invention will be described below.

[0040] An example of the epiregulin expression inhibitor of the present invention includes pearl oyster glycogen (P. fucata glycogen). Pearl oyster glycogen is glycogen derived from pearl oysters. Glycogen is a polymer formed by polymerization of α-D-glucose, also known as animal starch, through glycosidic bonds to form a polysaccharide structure with many branches.

[0041] For example, methods of extracting pearl oyster glycogen include extracting the pearl oyster flesh with hot water, precipitating it with ethanol, precipitating proteins by adding trichloroacetic acid, dimethylformamide, etc., and then treating it with protease to decompose and remove the proteins, thereby recovering glycogen. In order to recover glycogen efficiently, it is also possible to employ methods such as dialysis, alkaline treatment to decompose proteins, cationic surfactant treatment, and removal of amino acids and amino sugars by ion exchange resins.

[0042] The epiregulin expression inhibitor of the present invention can be used in the form of cosmetics, pharmaceuticals, food and beverages, etc. as a composition containing, for example, 0.001% to 50% by mass (preferably 0.005% to 10% by mass) of pearl oyster glycogen.

[0043] The epiregulin expression inhibitor is preferably a skin external preparation to be applied to the skin, and examples of the formulation include cream, milky lotion, ointment, lotion, etc. These can contain various oils, surfactants, ultraviolet absorbers, powders, pigments, polyhydric alcohols, thickeners, antioxidants, fragrances, preservatives, etc.

[0044] The water content in the epiregulin expression inhibitor is 30% by mass to 95% by mass (preferably 50% by mass to 90% by mass). EXAMPLES

[0045] The present invention will be specifically described below by way of examples, but the present invention is not limited to these examples in any way.

[0046] 1. Consideration of screening indicators 1-1. Examination of gene expression patterns related to aging in skin fibroblasts (i) Cell culture and stimulation Young fibroblasts (PDL7) and aged fibroblasts (PDL43) were prepared from the same lot of normal human dermal fibroblasts (Kurashiki Boseki Co., Ltd.) derived from neonatal foreskin. Figure 2(a) shows the relationship between culture time and PDL, and aged fibroblasts were cultured for more than three months until proliferation slowed (doubling time less than 0.5 / week). To investigate the aging characteristics of the obtained aged fibroblasts, differences in the cellular characteristics of young fibroblasts and aged fibroblasts were confirmed.

[0047] Fibroblasts were plated in 6-well or 12-well plates at 2500 cells / cm per well, respectively. 2 (PDL7) or 7500 cells / cm 2 (PDL43). Then, the cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2 until subconfluent. After confirming 80%-90% confluence, the fibroblasts were cultured in DMEM supplemented with 0.5% FBS for an additional 24 hours. The medium was then changed to DMEM alone containing human recombinant tumor necrosis factor alpha (TNF-α), IL-1β, or EREG (R&D Systems), and the cells were cultured for an additional 48 hours. After culture, gene and protein expression levels were determined as follows:

[0048] (ii) Senescence-associated β-galactosidase staining Fibroblasts were seeded on glass-bottom dishes and cultured overnight. For senescence-associated β-galactosidase (SA-β-Gal) staining, the positive rate of SA-β-Gal was measured using the Cellular Senescence Plate Assay Kit. Specifically, after washing the plate with Hank's balanced salt solution, the cultured cells were stained with SPiDER-β-Gal working solution and Hoechst 33342 for 30 minutes. After washing again with Hank's balanced salt solution, the cells were observed under a fluorescent microscope.

[0049] Figure 2(b) shows images of fibroblasts stained with β-galactosidase and nuclear staining. The scale bar indicates 100 μm. The areas indicated by the white arrows in the figure are the main areas stained with β-galactosidase. As shown in Figure 2(b), PDL43 cells showed higher β-galactosidase activity than PDL7 cells.

[0050] (iii) Quantitative polymerase chain reaction (PCR) analysis Total RNA was extracted from PDL7 and PDL43 cells using the Nucleospin RNA kit (Takara Bio). After RNA isolation, complementary DNA was synthesized using reverse transcriptase (ReverTraAce). Quantitative real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix and the QuantStudio 3 Real-Time PCR System, with 18S rRNA as a reference control. The primer sequences used for PCR amplification are shown in Table 1 below.

[0051] [Table 1]

[0052] Figure 2(c) shows the expression levels of senescence markers and ECM-related genes in PDL7 cells and PDL43 cells. Values ​​are shown as mean ± standard deviation (SD; n = 4). Significance was determined using Student's t-test. As shown in Figure 2(c), in PDL43 cells, the expression levels of the senescence markers p21 and IL-1β mRNA increased, while the expression levels of the ECM-related genes HAS2 gene, VCAN gene, and FBN2 gene decreased.

[0053] These results indicate that in senescent fibroblasts (PDL43), the expression of senescence markers is increased while the expression of ECM-related genes is suppressed, indicating that senescence is induced in PDL43 cells.

[0054] Next, to investigate the relationship between cellular senescence and EREG expression, we examined the expression of EREG and other mRNAs in senescent fibroblasts.

[0055] Figure 3(a) shows the expression levels of EREG, epidermal growth factor (EGF), and heparin-binding epidermal growth factor-like growth factor (HB-EGF) in PDL7 cells and PDL43 cells. Values ​​are shown as mean ± SD (n = 4). Significance was determined using Student's t-test. As shown in Figure 3(a), EREG expression was significantly increased in PDL43 cells compared to PDL7 cells, but no significant changes were observed in the expression of other EGF ligands such as EGF and HB-EGF.

[0056] Next, Figure 3(b) shows the expression level of EREG in PDL7 cells treated with TNF-α and IL-1β. For TNF-α treatment, values ​​are shown as mean ± SD (n = 5), and significance was evaluated using the Bonferroni test after the Kruskal-Wallis test. For IL-1β treatment, values ​​are shown as mean ± SD (n = 4), and significance was evaluated using one-way analysis of variance and the Bonferroni test. As shown in Figure 3(b), the expression level of EREG was also significantly increased by major SASP factors such as TNF-α and IL-1β.

[0057] Thus, EREG expression was increased in senescent fibroblasts and in fibroblasts treated with cytokines. Taken together, these results suggest that EREG may be induced by cellular senescence.

[0058] 1-2. Histological examination using human skin samples (i) Human skin samples OCT-embedded frozen human abdominal skin blocks from six healthy Caucasian women (28, 29, 29, 72, 73, and 84 years old) were purchased from KAC. The skin blocks were divided into frozen and paraffin blocks. Frozen sections (8 μm) were prepared for immunostaining, and paraffin sections (8 μm) were prepared for trichrome staining.

[0059] (ii) Histological examination Trichrome staining of deparaffinized and rehydrated paraffin-embedded skin sections was performed using a Trichrome Stain Kit. Immunohistochemistry was performed by fixing frozen sections in a 1:1 acetone:ethanol solution for 15 min, followed by three washes with PBS and immersion in buffered 3% bovine serum albumin (BSA) in PBS for 1 h at 25°C. After overnight incubation with primary antibodies (in 1% BSA) at 4°C, sections were incubated with secondary antibodies (in 1% BSA) for 1 h at 25°C. EREG polyclonal antibody (♯PA5-119096; Invitrogen) and CDKN2A / p16INK4A monoclonal antibody (♯2D9A12; Abcam) were used as primary antibodies at dilutions of 1:100 and 1:200, respectively. As secondary antibodies, Alexa Fluor 488 goat anti-mouse IgG (♯A11001; Invitrogen) and Alexa Fluor 594 donkey anti-rabbit IgG (♯A-21207; Invitrogen) were used at dilutions of 1:1000 and 1:2000, respectively. After incubation with secondary antibodies, sections were washed three more times with PBS and stained with 5 μg / mL Hoechst 33342 in 1% BSA at 25°C for 15 min. After staining, EREG expression was observed under a fluorescent microscope. Fluorescence data were analyzed using Fiji software (ImageJ version 1.53t).

[0060] Skin tissues were divided into a young group (28 years old, 29 years old, 29 years old) and an elderly group (72 years old, 73 years old, 84 years old) and evaluated. The evaluation results are shown in Figure 4. Figure 4(a) is an image of collagen fibers observed after trichrome staining. Figure 4(b) is an image of skin tissue stained for EREG using immunohistochemistry, and EREG expression was analyzed for tissue co-stained with EREG, aging marker p16INK4A, and cell nuclei. The dotted line in the figure indicates the basement membrane. Figure 4(c) shows the ratio of the average fluorescence intensity of EREG in Figure 4(b). Values ​​are shown as mean ± SD (n = 3), and significance was determined using Student's t test. Figure 4(d) is an image of stained skin, showing the localization of highly expressed EREG. The dotted and solid lines in the figure indicate the boundaries between the basement membrane, papillary dermis, and reticular dermis, respectively.

[0061] As shown in Figure 4, the expression of EREG and the aging marker p16INK4A was elevated in the dermis of elderly subjects (Figure 4(b)). Meanwhile, collagen density was decreased (Figure 4(a)). Analysis of immunofluorescence data showed that EREG protein levels were significantly elevated in the dermis layer of elderly subjects compared with young subjects (Figure 4(c)). In contrast, EREG protein levels in the epidermis were comparable between groups. Furthermore, it was revealed that in two out of three elderly subjects, the expression level of EREG was more significantly increased (localized) in the reticular dermis compared with the papillary dermis (Figure 4(d)). Thus, it was found that epiregulin (EREG) expression was increased in the dermis of elderly skin tissue compared with young skin tissue.

[0062] 1-3. Study of human recombinant epiregulin protein (rhEREG) stimulation (i) Changes in gene expression PDL5 cells and PDL43 cells were stimulated with rhEREG, and changes in the expression levels of each gene due to the stimulation were confirmed.

[0063] Figure 5(a) shows the expression levels of ECM-related genes in PDL5 cells stimulated with rhEREG. Values ​​are shown as mean ± SD (n = 5). For all except FBN2, significance was evaluated using one-way analysis of variance (ANOVA) followed by the Bonferroni test. For FBN2, significance was evaluated using the Kruskal-Wallis test followed by the Bonferroni test. Figure 5(b) shows representative images obtained by Western blot. Figure 5(c) shows quantitative analysis of COL1A1 expression. The intensity of each band was quantified with ImageJ software and normalized to actin. Values ​​are shown as mean ± SD (n = 3). Significance was evaluated by one-way ANOVA and Dunnett's multiple comparison test. Figure 5(d) shows the expression levels of ECM-related genes in PDL43 cells stimulated with rhEREG. Significance was evaluated as in Figure 5(a).

[0064] The results in Figure 5 show that rhEREG stimulation dose-dependently suppressed the mRNA expression of ECM-related substances, collagen I (COL1A1 or COL1A2) and fibrillin (FBN1 or FBN2), in young and aged fibroblasts (Figures 5(a) and (d)). Consistent with the mRNA expression levels, Western blot analysis showed that COL1A1 expression was decreased in young fibroblasts stimulated with rhEREG (Figures 5(b) and (c)).

[0065] It was found that rhEREG stimulation in young fibroblasts (PDL5) and aged fibroblasts (PDL43) significantly suppressed the mRNA and protein expression of ECM-related substances.

[0066] Many growth factors / cytokines, including EGF, are strong collagen inducers and have been reported to promote collagen synthesis. However, the above results showed that the expression of EGF ligands other than EREG (EGF, HB-EGF, etc.) in aged fibroblasts did not change, and rhEREG stimulation in fibroblasts induced downregulation of ECM-related genes. From this, it is speculated that the deterioration of the connective tissue in the dermis layer due to skin aging is due to increased expression of EREG. Previously, it has been reported that EREG expression is increased in the dermis of psoriatic lesions, but has not been examined in the skin of elderly people.

[0067] (ii) Consideration of mechanisms To verify how the mechanism of rhEREG stimulation affects the expression of ECM-related genes in fibroblasts, we evaluated the activity of mitogen-activated protein kinase (MAPK), which plays an important role in regulating collagen expression. The MAPK pathway mainly activates downstream transcription factors and inflammatory cytokines through phosphorylation of extracellular regulated protein kinase (ERK) 1 / 2, Jun N-terminal kinase (JNK), and p38 signaling pathways. Specifically, we analyzed the phosphorylation of epidermal growth factor receptor (EGFR), p38, JNK, and ERK1 / 2 by Western blot when young fibroblasts (PDL4) were treated with rhEREG (50 ng / mL) for 5, 10, 15, 30, and 60 minutes. The specific procedure is as follows.

[0068] Whole cell extracts were prepared in RIPA buffer containing a protease and phosphatase inhibitor cocktail. The resulting protein concentrations were measured using Precision Red Advanced Protein Assay Reagent. Equal amounts of total protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis in 12.5% ​​gels and then transferred to polyvinylidene difluoride membranes. The membranes were then soaked in ImmunoBlock buffer for 1 h at 25°C and incubated overnight at 4°C with primary antibodies at a dilution of 1:1000. The following antibodies were used: phosphor-EGFR (EP774Y; Abcam), EGFR (EP38Y; Abcam), phosphor-p38 (#4511; Cell Signaling Technology), p38 (#9212; Cell Signaling Technology), stress-activated protein kinase (SAPK) / JNK (#9252; Cell Signaling Technology), phosphor-SAPK / JNK (#4668; Cell Signaling Technology), extracellular regulated protein kinase (ERK; #61-7400; Invitrogen), phosphor-ERK (#44-680G; Invitrogen), and glyceraldehyde-3-phosphate dehydrogenase (sc-166574; Santa Cruz Biotechnology).

[0069] Primary antibodies were detected with HRP goat anti-rabbit (#65-6120; Invitrogen) or HRP goat anti-mouse (X1208P; Exalpha Biologicals Inc.) at dilutions of 1:3000 and 1:40000, respectively. For COL1A1 and actin expression analysis, western blotting was performed using an iBind Western Device (Thermo Fisher Scientific). The membrane was treated with COL1A1 (#72026; Cell Signaling Technology) at a dilution of 1:1000, followed by secondary antibody (#65-6120; Invitrogen) at a dilution of 1:3000. Finally, the western blot results were analyzed using a ChemiDOC XRS+ system (Bio-Rad Laboratories).

[0070] Figure 6(a) shows a photograph of the Western blot analysis. Figure 6(b) shows a quantitative analysis of the signal intensity of phosphorylated p38. Values ​​are shown as mean ± SD (n = 3). Significance was evaluated by one-way analysis of variance followed by Dunnett's multiple comparison test.

[0071] As shown in Figure 6, 10-minute treatment significantly increased the phosphorylation of p38, and also significantly increased the phosphorylation of other MAPK downstream molecules (ERK1 / 2) (Figures 6(a) and (b)). These results demonstrated that rhEREG stimulation in fibroblasts rapidly activated the p38 / ERK / JNK signaling pathway.

[0072] It has been reported that collagen synthesis in fibroblasts is negatively regulated via the MAPK pathway. For example, Yang et al. reported that lipo-PGE1 plays an important role as a negative regulator of collagen gene expression and production via ERK / Ets-1 signaling (PLoS One 12:e0179614.). In other words, it is speculated that stimulation of fibroblasts with rhEREG induces phosphorylation of p38, JNK, and ERK1 / 2, thereby acting as a negative regulator of collagen synthesis.

[0073] 2. Screening using epiregulin (EREG) expression as an indicator 2-1. Examination of EREG gene expression (i) Preparation of pearl oyster glycogen as a test substance The flesh of pearl oysters was extracted with two volumes of hot water for one hour, and the residue was removed to obtain the supernatant. Three volumes of ethanol were added to the supernatant. The mixture was centrifuged, and the precipitate was purified to obtain pearl oyster glycogen.

[0074] (ii) Preparation of water chestnut fruit extract (T. japonica fruit extract) as the test substance Water chestnut fruit (manufactured by Tochimoto Tenkaido Co., Ltd.) was extracted with 50% ethanol, and then filtered to remove the residue, to obtain water chestnut fruit extract.

[0075] (iii) Measurement of EREG gene expression level Senescent fibroblasts (PDL43) were plated in 12-well plates at 7500 cells / cm per well. 2 The cells were then cultured in 10% FBS-supplemented DMEM until they reached 80% to 90% confluence, and then further cultured in 0.5% FBS-supplemented DMEM for 24 hours (preculture step).The medium was then changed to DMEM alone (no test substance added), DMEM supplemented with pearl oyster glycogen, or DMEM supplemented with water chestnut fruit extract, and cultured for 48 hours (culture step), after which the expression level of the EREG gene was measured as described above (measurement step).

[0076] Figure 7(a) shows the expression level of the EREG gene in senescent fibroblasts (PDL43) cultured in a medium supplemented with pearl oyster glycogen or water chestnut fruit extract. Values ​​are shown as mean ± SD (n = 3). Significance was evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni test for pearl oyster glycogen, and by Student's t-test for water chestnut fruit extract. As shown in Figure 7(a), both pearl oyster glycogen and water chestnut fruit extract significantly reduced the expression level of the EREG gene compared to the control group.

[0077] 2-2. Examination of effects on other co-cultured fibroblasts The protocol for co-culture is shown in Figure 8. A 6-well plate was used as the bottom dish, and a transparent polyethylene terephthalate membrane with a pore size of 0.4 μm was used as the cell culture insert. Young fibroblasts (PDL4) were cultured in a 6-well plate at 10,000 cells / cm per well. 2 The young fibroblasts were seeded in the inserts at 10°C for 24 hours, and then cultured in DMEM supplemented with 0.5% FBS for another 24 hours. The young fibroblasts were then co-cultured with the aged fibroblasts in each medium (i.e., DMEM without test substance, DMEM supplemented with pearl oyster glycogen, or DMEM supplemented with water chestnut fruit extract) for 24 hours after the 48-hour culture in 2-1(iii) above (i.e., DMEM without test substance, DMEM supplemented with pearl oyster glycogen, or DMEM supplemented with water chestnut fruit extract) (co-culture step). The young fibroblasts in the inserts were then collected and quantitative real-time PCR was performed to measure the expression levels of ECM-related genes (second measurement step).

[0078] Figures 7(b) and (c) show the mRNA expression levels of ECM-related genes in young fibroblasts (PDL4) co-cultured with aged fibroblasts (PDL43) cultured in medium supplemented with or without pearl oyster glycogen or water chestnut fruit extract. Values ​​are shown as mean ± SD (n = 3). Significance was evaluated by one-way analysis of variance and Dunnett's multiple comparison test.

[0079] As shown in Figure 7(b), young fibroblasts co-cultured with aged fibroblasts treated with pearl oyster glycogen significantly increased the expression of ECM-related genes (COL1A1, COL1A2, FBN2). In contrast, young fibroblasts co-cultured with aged fibroblasts treated with water chestnut fruit extract did not show an increase in the expression of ECM-related genes (COL1A1, COL1A2, FBN2) (see Figure 7(c)). In other words, it was shown that pearl oyster glycogen not only acts directly on aged fibroblasts, but also indirectly on other fibroblasts in the co-culture environment.

[0080] As a result of the above screening, pearl oyster glycogen was selected as a more suitable epiregulin expression inhibitor.

[0081] 3. In vivo anti-aging test using pearl oyster glycogen Two types of lotion were prepared: one containing 0.01% by mass of pearl oyster glycogen, and the other not containing pearl oyster glycogen (placebo). Fourteen female subjects (ages 45-61) were asked to apply the lotions twice a day to the forearms for eight weeks (half-body forearm test). The formulations of the two lotions are shown in Table 2 below.

[0082] [Table 2]

[0083] Dermal collagen density and thickness were measured at the sites where the lotion was applied every two weeks using DermaLab (CORTEX TECHNOLOGY). The results are shown in Figure 9. Figure 9(a) shows collagen profiles of the same individual after application of placebo and glycogen-containing lotion. Figure 9(b) plots the percentage change from baseline in the two groups, placebo and glycogen-containing lotion application. Values ​​are shown as means (n=14). Significance was determined using repeated measures ANOVA with pairwise t-tests.

[0084] As shown in Figure 9, when the state of the dermis structure containing collagen fibers in human skin was examined, the lotion containing pearl oyster glycogen rapidly increased collagen fibers in the subject's forearm.

[0085] The results of the above-mentioned Examples revealed that EREG expression was increased in aged fibroblasts, and further revealed that EREG expression was increased in the dermis layer of elderly people compared to young people. Furthermore, it was found that stimulation of fibroblasts with rhEREG suppressed the expression of ECM-related substances.

[0086] These findings indicate that EREG is a novel factor in the deterioration of ECM-related substances associated with aging. In other words, EREG is useful as an indicator for screening anti-aging substances. Furthermore, it is believed that more useful anti-aging substances can be selected by taking into consideration the effects of co-cultured fibroblasts on ECM-related substances in screening.

[0087] Furthermore, a screening method using EREG as an indicator was used to search for components that could suppress EREG expression, and it was found that pearl oyster glycogen is useful as an EREG expression inhibitor. Pearl oyster glycogen reduced EREG expression in aged fibroblasts and indirectly increased the expression of ECM-related substances in co-cultured young fibroblasts. In addition, in an in vivo test in which a lotion containing pearl oyster glycogen was actually applied to the skin, it was found to rapidly increase the amount of dermal collagen.

[0088] Thus, knowledge has been gained that is useful for the development of anti-aging skin care that targets EREG in aged skin, and the present invention provides a new approach. The present invention suggests the possibility of future therapeutic and preventive strategies for premature skin aging that target EREG expression, and is believed to be of great value in the fields of pharmaceuticals, cosmetics, and other fields.

Claims

1. A method for screening for anti-aging substances using epiregulin expression as an indicator.

2. The screening method according to claim 1, characterized in that the method comprises a culture step of culturing fibroblasts in a medium containing a test substance, a measurement step of measuring the expression of epiregulin in the culture after the culture step, and a selection step of selecting a test substance that suppresses the expression of epiregulin compared to when the test substance is not added.

3. The screening method according to claim 2, further comprising a co-culture step of co-culturing the fibroblasts cultured in a medium containing the test substance with other fibroblasts having a lower population doubling level than the fibroblasts, and a second measurement step of measuring the expression of extracellular matrix-related substances in the other fibroblasts after the co-culture step.

4. 4. The method according to claim 3, wherein the extracellular matrix-related substance is at least one of COL1A1, COL1A2, FBN1, and FBN2.

5. An epiregulin expression inhibitor comprising pearl oyster glycogen.

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