Epiregulin expression inhibitor and ECM-related gene expression promoter

Abstract

To provide a component that reduces the expression of epiregulin, which is utilized as a novel aging-related indicator, as well as a component that enhances the expression of ECM-related genes.SOLUTION: It has been discovered that Trapa japonica fruit extract is suitable as an epiregulin (EREG) expression inhibitor. The Trapa japonica fruit extract not only reduces the expression of EREG in senescent fibroblasts but also directly enhances the expression of ECM-related genes.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an epiregulin expression inhibitor and an ECM-related gene expression promoter. [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). [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] The present invention uses epiregulin as a novel aging indicator, and aims to provide a component that suppresses its expression and a component that increases the expression of ECM-related genes. [Means for solving the problem]

[0007] The inventors conducted aging research using skin tissues from young and elderly people and found that 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.

[0008] The present inventors hypothesized that since epiregulin affects the expression of ECM-related genes in the dermis, inhibition of epiregulin expression could be applied to the treatment of various diseases.

[0009] We focused on natural products and searched for ingredients that suppress epiregulin expression, and found that water chestnut fruit extract has this effect.We also found that water chestnut fruit extract directly increases the expression of ECM-related genes.

[0010] That is, the present invention provides an epiregulin expression inhibitor and an ECM-related gene expression promoter, which are characterized by containing water chestnut fruit extract. Effect of the Invention

[0011] INDUSTRIAL APPLICABILITY The present invention can easily provide a component that suppresses the expression of epiregulin (EREG), which is a novel indicator, and also directly increases the expression of ECM-related substances. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing the relationship between culture time and PDL. [Diagram 2] FIG. 1 shows the expression levels of EREG, EGF, and HB-EGF. [Diagram 3] FIG. 1 shows images of skin tissue, etc. [Figure 4] FIG. 1 shows the expression levels of ECM-related genes. [Diagram 5] FIG. 13 shows the expression level of the EREG gene in fibroblasts treated with water chestnut fruit extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention relates to the inhibition of skin aging by water chestnut fruit extract by suppressing the expression of epiregulin and directly increasing the expression of ECM-related genes.

[0014] 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 known 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 the present inventors have established epiregulin as a novel factor involved in the deterioration of ECM-related substances with aging.

[0016] Epiregulin is measured by measuring the expression of epiregulin in a culture (e.g., in fibroblasts). The measurement method is not particularly limited, and the gene expression level or protein level of epiregulin in a 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.

[0017] Examples of extracellular matrix (ECM)-related substances include, but are not limited to, collagen I (COL1A1, COL1A2), fibrillin (FBN1, FBN2), hyaluronan synthase 2 (HAS2), and versican (VCAN). The ECM-related substance in the present invention is characterized as being at least one of COL1A1, COL1A2, FBN1, and FBN2. In the present invention, the term "ECM-related gene" refers to an expression gene of an ECM-related substance.

[0018] 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.

[0019] Water chestnut is the dried fruit of a plant called water chestnut, commonly called water chestnut, whose scientific name is Trapa japonica and which belongs to the family Trapa, family Calaminaceae. Water chestnuts are found in temperate to subtropical regions such as Japan, the Korean peninsula, and China, and are annual plants that grow in ponds and marshes. The fruit is edible. It is used as a tonic and antipyretic, and patent application No. 4-129822 has been filed for use in whitening and preventing rough skin.

[0020] In consideration of extraction efficiency, it is preferable to perform extraction after subjecting water chestnut fruits to processing such as chopping, drying, crushing, etc. Drying may be performed in the sun or using a dryer.

[0021] The solvent used in the extraction is water, a hydrophilic organic solvent, or a mixture thereof. Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 6 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0022] When a mixture of water and a hydrophilic organic solvent is used, if the hydrophilic organic solvent is a lower alcohol, it is preferably added in an amount of 1 to 20 parts by mass per 10 parts by mass of water, if the hydrophilic organic solvent is a lower aliphatic ketone, it is preferably added in an amount of 1 to 15 parts by mass per 10 parts by mass of water, and if the hydrophilic organic solvent is a polyhydric alcohol, it is preferably added in an amount of 1 to 20 parts by mass per 10 parts by mass of water.

[0023] The amount of the solvent used for extraction is preferably about 5 to 100 times, more preferably about 10 to 50 times, the amount of the plant material. To further increase the extraction efficiency, stirring or homogenization may be performed in the extraction solvent. The extraction temperature is preferably about 5°C to a temperature below the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but is preferably about 1 hour to 14 days. The extraction operation is not limited to a single operation. Fresh solvent can be added to the residue after extraction and the extraction operation can be performed again, or the extraction solvent can be brought into contact with the extraction raw material multiple times.

[0024] If necessary, purification treatment such as deodorization and decolorization may be carried out to the extent that the effect is not adversely affected, and the product can be concentrated by removing the solvent using a vacuum concentration device such as an evaporator or by heating. In addition, this extract can be further purified using a synthetic adsorbent (Diaion HP20, Sephabeese SP825, Amberlite XAD4, MCIgel CHP20P, etc.), a dextran resin (Sephadex LH-20, etc.), ultrafiltration, etc.

[0025] The epiregulin expression inhibitor and ECM-related gene expression promoter of the present invention can be used in the form of cosmetics, pharmaceuticals, food and beverages, etc., as a composition containing, for example, 0.00001% by mass to 5% by mass (preferably 0.0001% by mass to 1% by mass) of water chestnut fruit extract.

[0026] The epiregulin expression inhibitor and ECM-related gene expression promoter are preferably used as external preparations for the skin to be applied to the skin, and examples of the forms include creams, milky lotions, ointments, lotions, etc. These can contain various oils, surfactants, ultraviolet absorbers, powders, pigments, polyhydric alcohols, thickeners, antioxidants, fragrances, preservatives, etc.

[0027] The water content in the epiregulin expression inhibitor and the ECM-related gene expression promoter is 30% by mass to 95% by mass (preferably 50% by mass to 90% by mass). EXAMPLES

[0028] 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.

[0029] 1. Epiregulin as a new aging indicator 1-1. Examination of gene expression patterns related to aging in skin fibroblasts (i) Cell culture and stimulation Young fibroblasts (population doubling level (PDL) 7) and senescent fibroblasts (PDL 43) were prepared from the same lot of normal human dermal fibroblasts (Kurashiki Boseki Co., Ltd.) derived from neonatal foreskin. Figure 1(a) shows the relationship between culture time and PDL, and senescent 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 senescent fibroblasts, differences in the cellular properties of young fibroblasts and senescent fibroblasts were confirmed.

[0030] Fibroblasts were seeded in 6-well or 12-well plates at 2500 cells / cm2 (PDL7) or 7500 cells / cm2 (PDL43) per well, respectively. Then, they were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% CO2 until they became subconfluent. After confirming 80%-90% confluence, the fibroblasts were cultured in DMEM supplemented with 0.5% FBS for an additional 24 h. 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 cultured for an additional 48 h. After culture, gene and protein expression levels were determined as follows:

[0031] (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.

[0032] Figure 1(b) shows an image of β-galactosidase stained and nuclear stained fibroblasts. The scale bar indicates 100 μm. The areas indicated by the white arrows in the figure are the main areas stained by β-galactosidase staining. As shown in Figure 1(b), PDL43 cells showed higher β-galactosidase activity than PDL7 cells.

[0033] (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.

[0034] [Table 1]

[0035] Figure 1(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 1(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.

[0036] 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.

[0037] Next, to investigate the relationship between cellular senescence and EREG expression, we examined the expression of EREG and other mRNAs in senescent fibroblasts.

[0038] Figure 2(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 2(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.

[0039] Next, Figure 2(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 Kruskal-Wallis test followed by the Bonferroni 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 2(b), the expression level of EREG was also significantly increased by major SASP factors such as TNF-α and IL-1β.

[0040] 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.

[0041] 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.

[0042] (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).

[0043] 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 3. Figure 3(a) is an image of collagen fibers observed after trichrome staining. Figure 3(b) is an image of skin tissue stained for EREG using immunohistochemistry, and EREG expression was analyzed for tissues co-stained for EREG, aging marker p16INK4A, and cell nuclei. The dotted line in the figure indicates the basement membrane. Figure 3(c) shows the ratio of the average fluorescence intensity of EREG in Figure 3(b). Values ​​are shown as mean ± SD (n = 3), and significance was determined using Student's t test. Figure 3(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.

[0044] As shown in Figure 3, the expression of EREG and the aging marker p16INK4A was elevated in the dermis of elderly subjects (Figure 3(b)). Meanwhile, collagen density was decreased (Figure 3(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 3(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 3(d)). Thus, it was found that epiregulin (EREG) expression was increased in the dermis of elderly skin tissue compared with young skin tissue.

[0045] 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.

[0046] Figure 4(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 4(b) shows representative images obtained by Western blot. Figure 4(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 4(d) shows the expression levels of ECM-related genes in PDL43 cells stimulated with rhEREG. Significance was evaluated as in Figure 4(a).

[0047] The results in Figure 4 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 4(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 4(b) and (c)).

[0048] 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.

[0049] 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.

[0050] 2. Effects of Water Chestnut Fruit Extract (i) Preparation of water chestnut fruit extract (T. japonica fruit extract) 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.

[0051] (ii) Culture of senescent fibroblasts Senescent fibroblasts (PDL43) were seeded at 7500 cells / cm2 on a 30 mm diameter dish. They were then cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) until they reached 80% to 90% confluence, and then cultured in DMEM supplemented with 0.5% FBS for an additional 24 hours (preculture step). After that, the medium was replaced with DMEM alone (no test substance added) or DMEM supplemented with water chestnut fruit extract, and the expression level of the EREG gene was measured by the quantitative polymerase chain reaction (PCR) analysis described above.

[0052] Figure 5(a) shows the expression level of the EREG gene in senescent fibroblasts (PDL43) cultured in a medium supplemented with water chestnut fruit extract. Values ​​are shown as mean values ​​± SD (n = 3). Significance was evaluated using Student's t-test. As shown in Figure 5(a), the expression level of the EREG gene was significantly reduced compared to the control.

[0053] As shown in Figure 5(b), senescent fibroblasts (PDL43) cultured in a medium supplemented with water chestnut fruit extract increased the expression of ECM-related genes (COL1A1, COL1A2). Significance was evaluated by Student's t-test. As shown in Figure 5(b), the expression levels of ECM-related genes were directly and significantly increased compared to the control.

[0054] 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.

[0055] These findings indicate that EREG is a novel factor involved in the deterioration of ECM-related substances associated with aging.

[0056] Furthermore, using EREG as an indicator, the researchers searched for components that could suppress EREG expression and found that water chestnut fruit extract was useful as an epiregulin expression inhibitor. Water chestnut fruit extract not only reduced EREG expression in senescent fibroblasts, but also directly increased the expression of ECM-related genes.

[0057] In this way, we were able to obtain knowledge that can be used to develop anti-aging skin care products that target EREG in aged skin. The present invention suggests the possibility of future strategies for treating and preventing early skin aging that target EREG expression, and the use of water chestnut fruit extract as an EREG expression inhibitor and ECM-related gene expression promoter is believed to be of great value in the fields of pharmaceuticals, cosmetics, and other fields.

Claims

1. An epiregulin expression inhibitor comprising water chestnut fruit extract.

2. An agent for promoting the expression of ECM-related genes, comprising water chestnut fruit extract.

3. The ECM-related gene expression promoter according to claim 2, characterized in that the ECM-related gene is an expression gene for type I collagen (COL1).

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