Dietary Fermented Soybean Suppresses UVB-Induced Skin

Article pubs.acs.org/JAFC

Dietary Fermented Soybean Suppresses UVB-Induced Skin Inflammation in Hairless Mice via Regulation of the MAPK Signaling Pathway Taek Hwan Lee,† Moon Ho Do,‡ Young Lyun Oh,∥ Dong Woon Cho,§ Seung Hyun Kim,† and Sun Yeou Kim*,‡,Δ,# †

College of Pharmacy, Yonsei University, #162-1 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea College of Pharmacy, Gachon University, #191 Hambakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea ∥ Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, Republic of Korea § Sempio Fermentation Research Center, #183 Osongsaengmyeong 4ro, Cheongwongun, ChungCheongbukdo 363-954, Republic of Korea Δ Gachon Medical Research Institute, Gil Medical Center, #21 Namdong-daero 774beon-gil, Namdong-gu, Incheon 405-760, Republic of Korea # Gachon Institute of Pharmaceutical Science, Gachon University, #191 Hambakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea ‡

ABSTRACT: Soybean may be a promising ingredient for regulating UVB-induced inflammatory damage to the skin. We investigated the anti-inflammatory effects of diets supplemented with fermented soybean on UVB-induced skin photodamage and the effectiveness of soybean (S) and fermented soybean (FS) dietary supplementation. To investigate the effects of two major isoflavonesdaidzein and genisteinfrom FS, we used cocultures with keratinocytes and fibroblasts. Genistein treatment strongly inhibited the production of IL-6 and MAPK signaling. Forty hairless male mice divided into four groups were fed with a control diet (group N: normal, group C; +UVB) or diets with 2.5% S+UVB or 2.5% FS+UVB (group S, group FS) for 8 weeks. Macrophage infiltration to the dermis was reduced more in groups S and FS than in group C. The expression levels of iNOS and COX-2 were significantly decreased in group FS (by 7.7% ± 0.4% and 21.2% ± 0.3%, respectively [p < 0.05]). KEYWORDS: soybean, fermented soybean, skin inflammation, ultraviolet B (UVB), dietary supplementation

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important regulator in skin inflammation.13 Activation of AP-1 increases the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α).14 Thus, inhibition of the transcription factor AP-1 and COX-2 protein may be potential therapeutic targets for the treatment of skin inflammation. Therefore, new research in photoinduced skin inflammation has focused on drugs that inhibit these pathways. Consequently, the antiskin inflammatory drugs may use materials to turn off a molecular switch in the skin known as MAPK signaling. Soybean (Glycine max Merr) is an important plant protein source. It has been reported to possess biological properties such as antiobesity,15 antidiabetic,16 anti-inflammatory,17,18 and anticardiovascular disease properties.19 Soybeans also have beneficial effects on aged-skin via an increase of collagen and hyaluronan synthesis,20 reduction of metalloproteinase (MMP) expression,21 and stimulation of fibroblast proliferation.22 Moreover, various soybean products have been reported to have anti-inflammatory effects in cell lines and animal

INTRODUCTION Overexposure to ultraviolet (UV) radiation induces skin problems such as solar lentigo, seborrheic keratosis, erythema, and hyperpigmentation. Cumulative sun exposure may also cause skin cancer.1,2 Especially, UVB irradiation increases DNA damage and oxidative stress.3,4 UVB-induced skin carcinogenesis is associated with cutaneous inflammation.5−7 Therefore, controlling ongoing and chronic inflammation is one approach to reduce the risk of developing skin cancer. People with a weakened immune system are at greater risk of developing skin cancer.8 UV radiation can trigger various pathways related to dermatoheliosis. UV radiation leads to the infiltration of inflammatory cells, mainly neutrophils, into the skin.9 Neutrophils generate proinflammatory cytokines and reactive oxygen species (ROS). The increase in ROS is responsible for modulating the inflammatory response via activation of the mitogen-activated protein kinase (MAPK) pathway.10 MAPK activation is known to mediate skin inflammation and carcinogenesis.1 Activation of the MAPK cascades, including the extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 kinase11 induces the activation of AP-1 (activator protein-1) transcription factor,12 leading to pro-inflammatory gene expression, notably cyclooxygenase-2 (COX-2).1 AP-1 is a particularly © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8962

April 17, 2014 August 6, 2014 August 21, 2014 August 21, 2014 dx.doi.org/10.1021/jf5018252 | J. Agric. Food Chem. 2014, 62, 8962−8972

Journal of Agricultural and Food Chemistry

Article

flow rate was 1.0 mL/min. The gradient was 0.0 min, 8% B; 3.5 min, 15% B; 12.0 min, 17% B; 23.0 min, 24% B; 28.0 min, 50% B; 33.0 min, 100% B; 38.0 min, 100% B; and 40.0 min, 8% B. The re-equilibration time between runs was 20 min. Cell Culture. Normal human dermal fibroblasts (NHDFs) were purchased from ScienCell Research Laboratories (CA, USA). Human keratinocytes (HaCaT) were supplied by professor Tae-Yoon Kim. These cells were grown in DMEM supplemented with 10% FBS, 100 U/mL of penicillin, and 100 U/mL of streptomycin at 37 °C, in a 5% CO2 humidified atmosphere. Co-cultivation of NHDF and HaCaT Cells and Treatment of UVB. NHDF and HaCaT cells were cocultivated according to a previously described method with some modifications.30 Fibroblast NHDF cells were seeded at 6 × 104 cells/mL in 40 mm cell culture dishes. And it was incubated in a DMEM culture medium. After 24 h, keratinocyte HaCaT cells (6 × 104 cells/mL) were added to the NHDF cultures in the DMEM culture medium. After 24 h, the cell mixture was washed with PBS and was treated with 100 mJ/cm2 of UVB irradiation under a BLX-312 UV irradiation system (Vilber Lourmat, Marne-La-Vallée, France). The cell mixture was washed with PBS and changed into fresh serum-free medium. The cell mixture was then treated 10 μM of diadzin, daidzein, genistin, and genistin and incubated at 37 °C in a humidified air atmosphere in the presence of 5% CO2 for 72 h. Finally, conditioned medium and cells were harvested for the ELISA assay and Western blot analysis, respectively. Animals. Seven-week-old albino and hairless male mice (SKH:HR1) (20−27 g; n = 40) were obtained from Japan SLC, Inc. (Hamamatsu, Japan). The experimental protocol for this study was approved by the Institutional Animal Care and Use Committee of Kyunghee University (KHUASP (SU)-12-09). Mice were housed in a temperature- and humidity-controlled room (22 ± 1 °C, 60 ± 5% humidity) with 12 h light/dark cycles. After a week of quarantine, mice were randomly divided into 4 groups with 10 mice/cage and acclimated. During the experimental period, access to food and water was provided ad libitum. UVB Exposure to Hairless Mice. The UV source was supplied by a closely spaced array of five Sankyo Denki sunlamps, which have an irradiance peak at 310 nm (Kanagawa, Japan). Bulbs were positioned 15 cm above the mice. Irradiance (0.1 mW/cm2) was measured with an IL1700 Research Radiometer (International Light, Inc., Newburyport, MA) equipped with a UVB sensor. Mice were exposed to 100 mJ/cm2 UVB radiation (minimal erythemal dose) every day for the first week, and at 200 mJ/cm2 three times per week thereafter for 11 weeks. UVB irradiation was applied to the dorsal skin of mice. Diet. Forty hairless male mice were randomly assigned to four groups of ten mice per cage: (a) normal (N) (control diet only), (b) control (C) (UVB irradiation + control diet), (c) soybean (S) 2.5% (UVB irradiation + diet containing 2.5% soybean), and (d) fermented soybean (FS) 2.5% (UVB irradiation + diet containing 2.5% fermented soybean). All the mice were fed solid formula feed. Animals in each group received the experimental diets for 8 weeks concurrently with the UVB irradiation regimen. Dietary sources were provided (500 g/ cage) every week. To calculate the feed intake, we measured the weight of the remaining feed each week. The animals were allowed free access to food and water. The body weight of mice was measured once a week. Histological Skin Analysis. The animals were euthanized after the final UVB exposure, and biopsies were obtained from the dorsal skin. Biopsies were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Four-micrometer-thick sections were stained with hematoxylin for 10 min, washed, and then stained with eosin for 2 min. After washing with water, the slides were gradually dehydrated in 50%, 70%, 90%, and 100% ethanol. For immunohistochemistry, the sections were incubated in 0.1% protease in PBS for antigen retrieval. Then the sections were incubated in 3% H2O2 in PBS for 10−15 min to block endogenous peroxidase activity. The sections were then incubated with 2% normal horse serum in PBS for 20 min to block nonspecific binding of secondary immunoglobulin. After blocking, the sections were incubated with primary antibody CD11b and iNOS (Cell Signaling Technology, Danvers, MA). After washing in PBS, the

models.3,23,24 However, most studies on soybean and skin inflammation were performed using an atopic dermatitis (AD) model for human AD, the NC/Tnd mouse model.25,26 Research on the effect of soybean on UVB-induced skin inflammation and skin photoaging has been focused on the topical application of isoflavone extract and anthocyanins prior to UVB irradiation of hairless mice.27,28 Despite being considered a good dietary supplement, there is no report on anti-inflammatory effects of soybean long-term supplementation in UVB-treated animal models. Fermentation is widely used in food processing. Fermentation is defined as the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination of both. Through the process of fermentation, daidzin and genistin in soybean are converted into daidzein, and genistein, which have been shown to protect against UVinduced photodamage in human skin fibroblast cells.4 Based on these previous reports, we investigated the anti-inflammatory effects of long-term dietary supplementation with fermented soybean in UVB-induced skin photodamage in hairless mice. Additionally, we compared the effectiveness of soybean (S) and fermented soybean (FS) long-term dietary supplementation in relieving skin inflammation pain caused by UVB irradiation in hairless mice.

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MATERIALS AND METHODS

Chemicals. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and a Masson’s trichrome stain kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). ELISA assay kits to measure levels of IL-16 were purchased from R&D Systems (R&D Systems, Inc., Minneapolis, MN, USA). Standard and Sample Preparation. Daidzin, daidzein, genistin, and genistein were purchased from Sigma-Aldrich (St. Louis, MO). The standard stock solutions were prepared by dissolving 1 mg of each compound in 1 mL of methanol and stored at −20 °C. Soybean was supplied by Aqua Green Technology Co., Ltd. (Jeju, South Korea) in February 2012. A voucher specimen was deposited in the herbarium at the R&D Center of SEMPIO Food Company (Seoul, South Korea). Soybean was stored at −4 °C during the preparation. Soybean was roasted at 100 °C and ground prior to lyophilization. Aureobasidium pullulans (KCCM 12017) was purchased from the Korean Culture Center of Microorganisms (Seoul, South Korea) and Pichia jadinii (KFCC 11487P) was obtained from SEMPIO Food Company. Fermentation Conditions. Fermented soybean was prepared according to a previously described method by Cho et al.29 Briefly, synthetic medium containing soybean (40 g/L), 1.0 g/L ammonium nitrate, 0.5 g/L dipotassium phosphate, 0.1 g/L magnesium sulfate, and 1.0 g/L sodium chloride was autoclaved at 121 °C for 15 min. The two yeast cultures (A. pullulans and P. jadinii) were inoculated into a synthetic medium containing soybean in a 5-L bioreactor. The synthetic medium was incubated at 30 °C with shaking at 20g for 48 h. The samples were then sterilized at 121 °C for 10 min to prevent contamination, after which they were freeze-dried and stored at −18 °C. The yield of the fermented sample was 90%. HPLC Analysis. The HPLC analysis was carried out on a Waters system (Waters Corp., Milford, MA), consisting of a separation module (e2695) with an integrated column heater, an autosampler, and a photodiode array detector (2998). UV absorbance was monitored at 200−400 nm. Quantification was carried out by integration of the peak areas at 254 nm. The injection volume was 10 μL. A column (J’shpere ODS-H80, 250 mm × 4.6 mm; particle size, 4 μm; YMC Co. Ltd., Japan) was installed in a column oven and maintained at 40 °C.28 The mobile phase was composed of water containing 1% acetic acid (solvent A) and acetonitrile (solvent B). The 8963

dx.doi.org/10.1021/jf5018252 | J. Agric. Food Chem. 2014, 62, 8962−8972


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Figure 1. (A) Bioconversion of soy isoflavones by fermentation process. (B) Representative HPLC chromatogram of soybean: standard (a); soybean (b); fermented soybean (c).

slides were incubated in Vectastain ABC reagent (Vector Laboratory, Piscataway, NJ) for 1 h. The color was developed with 3,3′diaminobenzidine (DAB). Images were recorded using a Zeiss

inverted microscope connected to a digital CCD camera (Axiocam, Zeiss, Oberko, Germany). For immunohistofluorescence analysis, the method is the same as above up to the blocking step. Then slides were 8964



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incubated in COX-2 antibodies (Cell Signaling Technology, Danvers, MA) at 4 °C for overnight. After a wash with PBS, the sections were incubated with FTIC-conjugated secondary antibodies (Santa Cruz Biotechnology Inc., CA). Nuclei were counterstained with 4′,6diamidino-2-phenylindole (DAPI). The sections were then observed under a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). Western Blot Analysis. Western blotting was performed using cells and skin tissue lysates. Following treatment, cells and tissues were harvested and washed in PBS. Tissue samples were homogenized with lysis buffer containing 50 mM Tris-Cl, pH 8.0, 0.1% SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 0.5% sodium deoxycholate, 100 Pg/mL PMSF, 1 pg/mL aprotinin, and a phosphatase inhibitor. The lysates were then subjected to centrifugation at 12,000g for 20 min. Cells and tissue lysates were analyzed in triplicate. Protein concentrations were measured using the Bradford reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. Cells and skin tissue lysates containing equal amounts of total protein were separated on 10% or 12% SDS-PAGE gels and then transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, UK). Next, the membranes were blocked with a solution containing 5% nonfat milk in TBST for 1 h at room temperature and then incubated overnight with a primary antibody at 4 °C. The membranes were then washed three times with TBST and incubated with secondary antibodies (Santa Cruz Biotechnology Inc., CA) for 1 h at room temperature. Finally, the immune complexes were detected with a chemidoc XRS+ imaging system (Bio-Rad, CA). Densitometry analysis of the bands was performed using imagequant TL version 8.1 (GE Healthcare, Madison, WI). Statistical Analysis. All experiments were carried out in triplicate. The data are expressed as means ± standard deviation (SD). Statistical comparisons between different treatment groups were performed using one-way analysis of variance (ANOVA) with Tukey’ multiple comparisons test. The level of statistical significance was set at *P < 0.05, **P < 0.01, and ***P < 0.001.

diadzein and genistein are major isoflavones of fermented soybean (Figure 1). To investigate the anti-inflammatory activities of these two compounds, we used coculture systems with keratinocytes and fibroblasts. These different cells were grown in a single dish. When the cell mixtures were treated with 100 mJ/cm2 of UVB irradiation, they were mildly damaged. The cell viability of UVB treated control cells was decreased by 76.6% ± 1.3% compared with UVB nontreated normal cells (Figure 2B). When the cell mixtures were treated with 10 μM of daidzin, daidzein, genistin, and genistein, there was no effect on cell viability in UVB treated cells (Figure 2A, B). UVB irradiation stimulated the cell mixture. As a result, secretion of pro-inflamatory cytokines increased. We measured two major pro-inflamatory cytokines: interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The concentration of IL-6 was higher in the conditioned medium of the UVB control group compared with the normal group. Although the difference was not statistically significant, we confirmed the inhibitory effect of daidzein and genistein on IL-6 production (Figure 2C). TNF-α was not detected in conditioned medium (data not shown). We investigated the MAPK signaling cascade, which is known to regulate inflammation. All compounds (daidzin, daidzein, genistin, and genistein) inhibited UVB-induced phosphorylation of ERK (Figure 2E). In particular, genistein inhibited phosphorylation of p38, ERK, and JNK (Figure 2D− F). Moreover, the inhibitory activity of genistein was significantly different for genistin and genistein (Figure 2D, E) Measurement of Food Efficiency Ratio. There was no significant difference in the average body weight among the four groups of mice after 12 weeks. The groups did not differ significantly in their food efficiency ratios (Figure 3C). FS Dietary Supplementation Improved UVB-Induced Skin Erythema and Inflammation. Mouse dorsal skin physiological changes were recorded after dietary supplementation with S or FS, and after 12 weeks of UVB irradiation. Prolonged skin exposure to UVB leads to skin inflammation erythema and epidermal thickness. In our study, the group FS presented a decrease in UVB-induced erythema (Figure 4A). In addition, the thickness of the epidermis in the UVB treated control group was significantly larger than the normal group. However, this increase in epidermal thickness was lower in the S (79.4 ± 0.8%) and the FS (83.4 ± 0.8%) group (Figure 4B, C). Our histological analysis indicated that UVB radiation induced typical skin inflammation in hairless mice. Prolonged exposure to UVB increased the macrophage population in the upper dermis and epidermis (Figure 4D). Scoring of the four groups was done by Dr. Young Lyun Oh, who is a pathologist at SMC, Korea, based on our H&E staining analysis. The inflammation score was classified as either 0, +, ++, or +++. The score “0” indicates no inflammation, and the number of + symbols indicates the degree of inflammation. Therefore, “+++” represents severe skin inflammation. Group N had a score of “0” and group C was “+++”, group S was “+,” and group FS was “0−+.” Inflammatory symptoms such as intrahorn and subepithelial vacuolization were observed in group C. However, these symptoms were not observed in other groups. Based on immunohistochemical analysis, the population of CD11b+ macrophages was significantly reduced in the group FS compared with the control group (Figure 4D). Particularly,

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RESULTS Soybean and Fermented Soybean Isoflavonoid Content. Daidzin and genistin were converted into daidzein and genistein by a fermentation process in soybean (Figure 1A).31 Daidzin, daizein, genistin, and genistein contents in soybean and fermented soybean were measured by HPLC. The retention time of each compound is presented in Figure 1. The linearity of each compound was calculated using three concentrations of each specific compound. The daidzin, daidzein, genistin, and genistein contents in soybean were 0.1197, 0.0053, 0.2220, and 0.0170 μg/mg, respectively. The Table 1. Isoflavone Content (μg/mg) in Soybean and Fermented Soybean r2 Daidzin Daidzein Genistin Genistein

0.9997 0.9997 0.9997 0.9998

soybean

fermented soybean

± ± ± ±

0.1870 ± 0.0020

0.1197 0.0053 0.2220 0.0170

0.0008 0.0003 0.0002 0.0001

0.2083 ± 0.0021

daidzein and genistein contents in fermented soybean were 0.1870 and 0.2083 μg/mg, respectively (Table 1). Daidzin and genistin peaks were not detected in fermented soybean (Figure 1B). Our HPLC results confirmed the significant bioconversion of the isoflavonoidal glycosides into their corresponding aglycone types in fermented soybean. Daidzein and Genistein Inhibited IL-6 and Phosphorylation of MAPK Signaling in HaCaT Keratinocytes and NHDF Fibroblasts Coculture Systems. We confirmed that 8965



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Figure 2. Effect of Daidzin, Daidzein, Genistin, and Genistein on the production of IL-6 and MAPK signaling in a UV-irradiated HaCaT and NHDF coculture. NHDF cells and HaCaT cells were grown in the same dish (1:1 cellular mixture). After treatment with 10 μM of each of the four compounds for 72 h, cells were observed using an Olympus CKX-41 microscope (×100 in bright field) (A) and cell viability was measured by an MTT assay (B). Conditioned medium was cultivated and the IL-6 concentration was measured (C). For Western blot analysis, the mixtures were treated with 100 mJ/cm2 of UVB irradiation and 10 μM of each of the four compounds, treated at the same time. After 1 h, the mixture cells were harvested and the protein levels were measured (D−G). *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences.

the anti-inflammatory effect was more intense in group FS than in group S. FS Inhibits iNOS and COX-2 Expression. In the immunohistological analysis, group C showed a marked increase in iNOS expression, especially in the epidermis (Figure 5A). We also confirmed the increase in iNOS

expression in group C by Western blot analysis (Figure 5B). Interestingly, dietary supplementation with S and FS inhibited the protein expression of iNOS at 70.3% ± 7.0% and 7.7% ± 0.4%, respectively (p < 0.05) (Figure 5C). The increase of COX-2 expression in group C (Figure 6A) was inhibited by 8966



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of nutraceutical and cosmeceutical treatments for skin photodamage. Evidence suggests that soybeans containing isoflavones may be useful for the regulation of skin problems.33−35 However, no report is available on the anti-inflammatory effect of long-term soybean dietary supplementation on skin inflammation in UVB-irradiated animals. First, we chose the safest and potential soybean as a natural source of functional food for the regulation of photoinduced skin inflammation. Raw soybeans are rich in soyasaponins and isoflavonol glycosides. Namely, soybeans generally contain more isoflavonol glycosides, such as daidzin, genistin, and glycitin, than aglycone type, such as daidzein, genistein, and glycitein.36 Because long-term use of soybean may be associated with allergic reactions involving rash, itching, food protein-induced enterocolitis, constipation, bloating, and nausea,20,21 long-term dietary supplementation is difficult to achieve. Although natural products have many great benefits as dietary supplements, there are some limits in terms of efficacy and side effects. In an attempt to increase the efficacy of soybean supplementation, we established an optimal fermentation process design. The strategy included fermentation optimization using a mixed native yeast culture composed of Pichia jadinii and Aureobasidium pullulans. The optimization standards were based on isoflavonoid glycoside contents and efficacy as well as flavor and taste. Our goal was to increase the efficiency and activity as well as the flavor and taste compared to raw soybeans. In fact, several studies have already reported that biological activities of natural products might be improved by fermentation. Kim et al. reported that fermented red ginseng showed higher antiobesity effect on obesity-mediated metabolic disorders than nonfermented red ginseng.37 Our recent study proved the enhanced antiphotoaging activity of Opuntia f icus-indica by fermentation using two species of yeast, Pichia jadinii and Aureobasidium pullulans.29 For this reason, we designed a powerful strategy, such as soybean fermentation with Pichia jadinii and Aureobasidium pullulans. Based on our HPLC analysis, we found that FS contained only aglycone type. Isoflavone glycosides were not detected in FS. Soy isoflavone aglycones were increased through the fermentation process, which converts glucosides into aglycone by deconjugation of the glycosyl group of a glycoside (Figure 1).31 According to several studies, daidzein and genistein show higher bioavailability and bioactivity than daidzin and genistin. Iovine et al. reported that daidzein and genistein present synergic effects on UVB-mediated DNA damage.4 In fact, aglycones may be absorbed faster and better than glucosides in the human body.38 In conclusion, this fermented soybean may be biochemically and technically superior to its raw material. Dermatitis is a representative problem that is related to skin inflammation. Chronic and overexposure to UVR can induce photosensitivity and photoallergy, which can subsequently lead to phototoxic dermatitis. Particularly, UVB is a potent factor, which induces skin inflammation. Our histological data indicate that the UVB-irradiated group typically showed severe inflammation of the intrahorn. Other groups previously reported that UVB radiation triggered skin inflammation by inflammatory cellular infiltration into the upper dermis and epidermis.39 We also confirmed inflammatory cellular infiltration in the skin dermis of the UVB-induced group (Figure 4). Macrophages, important inflammatory cells, are involved in the initiation of inflammation by the production of various proinflammatory mediators such as nitric oxide (NO) and prostaglandins (PGs). The production of NO and PGs are

Figure 3. (A) Experimental design of the animal study procedures. (B) Composition of diet. (C) Food efficiency ratio during the 8 week intervention. aGroup N: hairless mice fed control diet only; group C: UVB-irradiated hairless mice fed control diet; groups S: UVBirradiated hairless mice fed diets containing 2.5% soybean; groups FS: UVB-irradiated hairless mice fed diets containing 2.5% fermented soybean. bFER (food efficiency rate) = gain of body weight (g)/ amount of food intake (g). Values are mean ± SEM (n = 10). Means with different letters differ by P < 0.05.

long-term dietary supplementation with S and FS by 26.9% ± 0.4% and 21.2% ± 0.3%, respectively (p < 0.05) (Figure 6B, C). FS Regulates MAPK Signaling in the Skin Dermis of UVB-Irradiated Hairless Mice. The UVB-induced inflammatory responses were remarkably reduced by FS long-term treatment. To evaluate the FS anti-inflammatory mechanism on UVB-induced skin inflammation, we investigated the MAPK signaling pathways in the skin dermis by Western blot analysis. UVB radiation increased the phosphorylation of p38, ERK, JNK, and c-Jun in the skin.1,10 As shown in Figure 7, phosphorylation of p38, ERK, JNK, and c-Jun was increased in UVB-irradiated skin dermis. In contrast, long-term FS treatment significantly inhibited the phosphorylation of MAPK (p-p38, 61.3 ± 0.5%; p-ERK, 78.9 ± 3.3%; p-JNK, 14.4 ± 0.5%; and p-c-Jun, 37.6 ± 0.6%) (Figure 7B−E).

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DISCUSSION Dietary supplements such as vitamins, minerals, or essential fatty acids have been reported to restore damaged skin.21,32 Particularly, dietary supplements can help control skin aging as well as inflammation and hyperpigmentation due to overexposure to UVR. However, there is only a paucity of evidence in the literature; therefore, in depth studies are required. Natural and functional food sources will allow the development 8967



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Figure 4. (A) Effects of S and FS on the histology of UVB irradiated hairless mouse skin. (B) Representative features of hairless mice dorsal skin: Group N with no UVB irradiation (a), group C with UVB irradiation (b), group S with UVB irradiation and soybean supplementation (c), and group FS with UVB irradiation and fermented soybean supplementation (d) were recorded using a digital camera (Samsung, Korea). H&E staining (magnification 200×). (C) Measurement of epidermal thickness. (D) Analysis of inflammatory infiltration of macrophages using the marker CD11b in hairless mouse skin after UVB irradiation. White arrows indicate infiltrating inflammatory cells in H&E stained tissue (magnification 200×). Black arrows and arrowhead indicate CD11b+ macrophage cells (magnification 200×). Group N with no UVB irradiation (a, e), group C with UVB irradiation (b, f), group S (c, g), group FS (d, h).

regulated by iNOS and COX-2 enzymes, respectively.40,41 Thus, overexpression of COX-2 in the skin may result in edema and epidermal hyperplasia.28,42 Our immunohistological study indicated that UVB overexposure increased iNOS and COX-2 expression in the dermal cells of the dorsal skin (Figures 5 and 6). Interestingly, a dietary supplement of FS significantly decreased iNOS and COX-2 expression in the skin. Recent studies report a link between iNOS, COX-2, and photo-

inflammation in mouse skin. In particular, the up-regulation of COX-2 is often accompanied by overexpression of iNOS.43 In conclusion, FS inhibited both iNOS and COX-2 expression in dermal skin. FS ameliorated skin inflammation by UVB irradiation, which regulates MAPK signaling. Furthermore, activation of MAPK can cause the induction of iNOS and COX-2 expression and, consequently, may induce skin cancer.44 8968



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Figure 5. (A) Effects of S and FS on iNOS expression in the skin. Representative immunohistochemical images show that iNOS (brown color) is expressed. Nuclei were counterstained with hematoxylin (magnification 200×). Black arrows indicate iNOS expressed cells: group N (a), group C (b), group S (c), and group FS (d). (B) Western blot analysis of iNOS in skin lysates. (C) The protein expression of iNOS was normalized to the band corresponding to α-tubulin. **P < 0.01 and ***P < 0.001 indicate statistically significant differences.

Figure 6. (A) Effects of S and FS on COX-2 expression in the skin. Representative immunohistochemical images show that COX-2 (green staining) is expressed. Nuclei were counterstained using DAPI (blue staining) (magnification 400×). White arrows indicate COX-2 expression; group N (a), group C (b), group S (c), and group FS (d). (B) Western blot analysis of COX-2 in skin lysates. (C) The protein expression of COX-2 was normalized to the band corresponding to α-tubulin. *P < 0.05 and **P < 0.01 indicate statistically significant differences.

inflammation by UVB exposure significantly changed the MAPK signaling pathway. Soybean inhibited the UVB-induced activation of p38, JNK, and c-JUN. Furthermore, FS was more effective in inhibiting p-JNK activation than S. Raw soybean had no effect on the regulation of ERK, but FS treatment strongly affected ERK phosphorylation in the dorsal skin. Thus, the anti-inflammatory effect of FS on UVB-induced skin inflammation could mainly be attributed to the regulation of MAPK signaling. However, additional studies on fermented and raw soybean are warranted to confirm these results. Based on our HPLC data, the chemical patterns of FS were different from that of raw soybean in the polar region, distinguishing the isoflavonoid region. Further studies are required to definitively identify the most biologically active component in FS. Our

UVB-induced skin inflammation is usually conducted under acute UV exposure conditions in in vivo models.10,45,46 After 24 h of UVB exposure, inflammatory signaling was significantly changed. Kim et al. reported that phosphorylation of p38 MAPK was increased by UVB exposure in a time dependent manner at 24 h. Thus, their studies support that p38 MAPK plays an important role in UVB-induced inflammatory responses in the skin of SKH-1 hairless mice.47 Generally, recovery from skin inflammation is easy and spontaneous in acute UVB exposure models. Therefore, identifying the antiinflammatory effects of a product in acute conditions can be challenging. In this study, we focused on chronic skin inflammation, which is induced after long-term UVB exposure (12 weeks). From our results, we showed that chronic skin 8969



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Figure 7. Effects of S and FS on UVB-induced MAPK signaling in skin lysates by Western blot and densitometric analysis. Forty micrograms of total protein from the tissue lysates were used for Western blot analysis. Significance was determined using a one-way ANOVA followed by a Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate statistically significant differences.

species; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun amino-terminal kinase; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; TNF-α, tumor necrosis factor-α; MMP, metalloproteinase; AD, atopic dermatitis; YM, yeast mold; DAPI, 4′,6-diamidino-2-phenylindole; SD, standard deviation; ANOVA, analysis of variance

study only investigated the efficacy of fermented soybean in resolving chronic skin inflammation. However, our findings indicate that future research on the use of natural resources such as soybean in the treatment of chronic skin inflammation may be promising. In conclusion, this study provides meaningful evidence that long-term dietary supplementation with isoflavone-rich, fermented soybean improves skin inflammation induced by chronic exposure to UVB. We suggest that natural fermented soybean will be a safe, potential functional food for skin inflammation due to photodamage.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Tel: +82-32-899-6411, Fax: +82-32-899-8962, E-mail: [email protected]. Funding

This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (iPET, 311035-03-1-HD110), and a grant from the Next-Generation BioGreen 21 Program (No.PJ009511), Rural Development Administration, Republic of Korea. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED UV, ultraviolet; UVR, ultraviolet radiation; UVB, ultraviolet B; S, soybean; FS, fermented soybean; ROS, reactive oxygen 8970



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Dietary Fermented Soybean Suppresses UVB-Induced Skin

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