Protective Effects of Pleurotus Species on UVB-Induced Skin Disorders at Clinically Relevant Plasma Concentrations of the Antioxidant Ergothioneine in Hairless Mice

Abstract

Ergothioneine (ERGO) has antioxidant and anti-inflammatory activities in UV-irradiated skin cells in vitro; however, there is no evidence about the effects of dietary ERGO on UV-induced skin damage or ERGO skin distribution in vivo. This study examined the protective effects of ERGO-rich edible mushrooms Pleurotus species against UVB-induced skin damage and the exposure to ERGO in the plasma and skin. Hos : HR-1 hairless mice were fed with or without freeze-dried cross-bred Pleurotus species (PS) or Pleurotus eringii (PE) and were exposed to UVB. Dietary intake of PS or PE significantly alleviated UVB-induced reductions in skin moisture content, increases in transepidermal water loss and oxidative stress markers, and epidermal thickening at plasma ERGO concentrations of 30–40 μM. Additionally, ingestion of PS significantly suppressed UVB-induced expression of pro-inflammatory cytokines. These results suggest that ingesting PS and PE may protect against UVB-induced skin disorders through antioxidant and anti-inflammatory activities at clinically relevant ERGO concentrations. Ingestion of PS and PE led to an increase in epidermal ERGO concentration to levels that were approx. 100 times higher than the ERGO concentration required for significant suppression of UVB-induced intracellular reactive oxygen species in immortalized human keratinocyte HaCaT cells. This suggests that the beneficial effects of PS and PE may be at least partly due to the antioxidant effects of ERGO in murine skin. Overall, ingestion of ERGO-rich Pleurotus species resulted in efficient distribution of ERGO to the skin and protective effects against UVB-induced skin damage, suggesting that these mushrooms may have beneficial effects in humans.

INTRODUCTION

The skin protects the body from the external environment and helps prevent water loss. As the skin ages, it can become dry and dull, with loss of elasticity and the appearance of fine wrinkles.¹⁾ Skin aging is divided into intrinsic and extrinsic aging; the former is typically under genetic and hormonal influence, while the latter is mainly caused by environmental factors such as UV rays, smoking, diet, chemicals, and trauma.²⁾ Of these, exposure to UV radiation is the primary cause of extrinsic skin aging, a process also known as photoaging. UV radiation from the sun is divided into three classes: UVA (320–400 nm), UVB (290–320 nm), and UVC (200–290 nm). Of the UV radiation reaching ground level, 95% is UVA and <5% is UVB, while UVC is undetectable. However, since the bioactivity of UVB is approx. 1000 times higher than that of UVA, and UV-induced skin erythema, DNA damage, and skin cancer are mainly caused by UVB,³⁾ protecting the skin from UVB may be more important for preventing skin damage.⁴⁾

UVB-induced oxidative damage has been associated with the pathogenesis of skin disorders in humans. UVB induces the uncontrolled release of reactive oxygen species (ROS), which cause oxidative DNA base damage such as the formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG).⁵⁾ Additionally, UVB exposure promotes the release of pro-inflammatory cytokines including interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), and IL-6. These events lead to the activation of immune cells and their subsequent infiltration into the skin.⁶⁾ Therefore, suppressing both ROS and inflammatory cytokines may be critical for mitigating UVB-induced skin damage.⁷⁾

Since some edible mushrooms contain a variety of antioxidants,⁸⁾ eating these mushrooms could potentially help reduce UVB-induced skin damage. Some mushroom species are rich in ergothioneine (ERGO), which is a food-derived amino acid with potent antioxidant activity.⁹⁾ Moreover, ERGO improved sleep quality,¹⁰⁾ and low ERGO levels in the blood and/or plasma are associated with cognitive impairment, low mobility,¹¹⁾ mortality, and risk of cardiovascular disease.¹²⁾ ERGO is synthesized particularly by fungi such as mushrooms and actinobacteria.¹³⁾ Since mammals cannot synthesize ERGO, edible mushrooms are one of the main sources of ERGO in humans. ERGO is well absorbed from the gastrointestinal tract and is distributed to various tissues mainly via its specific transporter OCTN1/SLC22A4, which is expressed throughout various organs.¹⁴⁾ In rats, ERGO concentrations are maintained in the systemic circulation even after fasting for 1 week.¹⁵⁾ This has been attributed in part to minimal metabolism in the body and limited urinary excretion due to OCTN1-mediated renal reabsorption.^14,16)

ERGO has been detected at a concentration of approx. 80 μM in the skin of wild-type mice, but was not detectable in Octn1 gene knockout mice.¹⁴⁾ Thus, OCTN1 may mediate ERGO distribution to the skin tissues. In addition, ERGO protects against UVB-induced cytotoxicity in cultured keratinocytes, possibly by suppressing the generation of ROS and the induction of pro-inflammatory cytokines.¹⁷⁾ ERGO also suppressed the UVB-induced reduction in type I procollagen production in cultured fibroblasts.¹⁸⁾ However, it is unknown whether ingesting ERGO protects skin moisture content and barrier functions against UV-induced damages in vivo.

Pleurotus species are major sources of ERGO among the edible mushrooms,^9,19) but there are no data on the effects of eating these mushrooms on photoaging in vivo. In this study, therefore, we first investigated the protective effect of dietary cross-bred Pleurotus species (PS)²⁰⁾ (Pleurotaceae) and Pleurotus eringii (PE) (Pleurotaceae) on UVB-induced skin injury in hairless mice. We also examined the antioxidant and anti-inflammatory effects of ERGO and ERGO skin distribution following dietary intake of PS and PE mushrooms. Then, we used immortalized human keratinocyte HaCaT cells to examine whether PS extract and pure ERGO could suppress ROS. OCTN1-mediated ERGO uptake in HaCaT cells and OCTN1 localization in human skin epidermal cells were also examined to gain insight into the potential for human application.

MATERIALS AND METHODS

Materials

ERGO and d₉-ERGO were provided by Tetrahedron (Paris, France), and [³H]ERGO (0.3 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA, U.S.A.). PS and PE fruiting bodies were cultivated by Hokuto Corporation (Nagano, Japan). The ERGO concentrations in freeze-dried PS and PE powders were 4.69 and 2.84 mg/g, respectively. PS hot water extract (PSE) was obtained as described previously.²¹⁾ The total of 0.51 g of freeze-dried PSE was obtained from 10 g of PS fruiting bodies. Basal Diet (5755) containing <0.01 μg ERGO/g of chow²²⁾ was obtained from Sankyo Labo Service Corporation (Tokyo, Japan). Anti-TNF-α antibody was purchased from Sigma-Aldrich (SAB4502982, St. Louis, MO, U.S.A.). Anti-β-actin (#4970) and anti-rabbit horseradish peroxidase-conjugated antibodies (#7074) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Anti-OCTN1 antiserum was previously established,²³⁾ and anti-rabbit Alexa 488-conjugated antibody was purchased from Abcam (ab150073, Cambridge, U.K.).

Experimental Animals and UVB Irradiation

Male hairless mice (Hos:HR-1) were obtained from Japan SLC (Shizuoka, Japan) and were kept under standard environmental conditions (12-h light/dark cycle at 21–25°C). Food and tap water were available ad libitum. Animal experiments were conducted based on the Kanazawa University guidelines for animal care and use. Animal studies were approved by the Committee on the Ethics of Animal Experiments of the University of Kanazawa (Permit No.: AP-183968) to minimize animal suffering and loss of life. Mice were purchased at 3 weeks of age and given a Basal Diet with or without 1 or 2.5% (w/w) freeze-dried PS or PE powder as the test diet. The amount of consumed feed in each group was not measured in the present study, but the body weight of each mouse was monitored throughout the experiments. Mice were subjected to one of the following conditions: (1) Control group (Basal Diet); (2) UVB group (UVB irradiation with Basal Diet); (3) UVB +1% PE group (UVB irradiation with Basal Diet containing 1% PE); (4) UVB +2.5% PE group (UVB irradiation with Basal Diet containing 2.5% PE); (5) UVB +1% PS group (UVB irradiation with Basal Diet containing 1% PS); (6) UVB +2.5% PS group (UVB irradiation with Basal Diet containing 2.5% PS). Mice were irradiated with a UVB lamp (VL-215.LM, Vilber-Loumart, Marne-la-Vallée, France) three times a week from the ages of 8–18 weeks, and UVB irradiation was gradually increased every 2 weeks from 50 to 100, 150, 180, and 210 mJ/cm² (Fig. 1A). Skin moisture content and transepidermal water loss (TEWL) from the dorsal skin were evaluated once every 2 weeks from the age of 8 weeks using a skin moisture meter (MC-607, LOZENSTAR, Kanagawa, Japan) and a Tewameter device (TM300, Courage & Khazaka, Cologne, Germany), respectively (Fig. 1A). At the end of the 10-week UVB irradiation, mice were euthanized, and the whole skin, epidermis, and dermis of the dorsal skin and blood were collected. Each blood sample was immediately centrifuged for 5 min at 3000 × g to obtain plasma. Epidermal thickness was measured using hematoxylin and eosin staining (Supplementary Material S1). Oxidative stress was evaluated by measuring 8-OHdG levels in dorsal skin (Supplementary Material S2). Expression of pro-inflammatory cytokine proteins in dorsal skin was quantified by Western blotting and enzyme-linked immunosorbent assay (ELISA) (Supplementary Materials S3, S4), and plasma and skin concentrations of ERGO after ingestion of the test diet were measured using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Supplementary Material S5, Supplementary Table S1).

Fig. 1. Experimental Design for the in Vivo Study

(A) Male hairless mice (Hos:HR-1) were fed the test diet from ages 3 to 18 weeks. The mice were exposed to UVB three times a week from ages 8 to 18 weeks. The dorsal skin moisture content and TEWL were measured every 2 weeks from ages 8 to 18 weeks. After 10 weeks of UVB irradiation, the epidermis, dorsal skin, dermis, and plasma were collected. (B) Changes in body weight during the experimental period. Each value is expressed as mean ± standard deviation (S.D.) (n = 6); n refers to the number of animals. Open circles, control group; closed circles, UVB group; open squares, UVB +1% PE group; closed squares, UVB +2.5% PE group; open diamonds, UVB +1% PS group; closed diamonds, UVB +2.5% PS group.

Cell Culture

To establish human epidermal keratinocyte HaCaT cells (Cosmo Bio Co., Ltd., Tokyo, Japan) in a basal-like-state, they were maintained in a low-calcium medium consisting of MCDB 153 medium (Sigma-Aldrich) supplemented with 10% calcium-depleted fetal bovine serum (FBS) for more than 3 weeks.²⁴⁾ Calcium was removed from FBS using Chelex-100 resin (Bio-Rad, Hercules, CA, U.S.A.) at 4°C for 1 h according to the manufacturer’s instructions. The resin was subsequently removed using a Nalgene Rapid-Flow polyethersulfone membrane filter unit (0.2 μm, Thermo Fisher Scientific, Waltham, MA, U.S.A.).

Measurement of Intracellular ROS

HaCaT cells were preincubated with PSE or ERGO for 24 h before UVB irradiation at 30 mJ/cm² in phosphate-buffered saline (PBS). After exposure to the UVB, the cells were incubated in fresh medium with PSE or ERGO for 6 h. Then, the cells were incubated in fresh medium with 10 μM 2,7-dichlorofluoresceine diacetate (DCFH-DA; Sigma-Aldrich) and 10 μg/mL Hoechst 33342 (Sigma-Aldrich) for 30 min. Intracellular ROS and nuclei, indicated by fluorescence of DCF and Hoechst 33342, respectively, were observed with a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). Areas of fluorescence were quantified using ImageJ software (National Institute of Health, Bethesda, MD, U.S.A.).

Uptake of [³H]ERGO or Unlabeled ERGO into HaCaT Cells

Uptake of ERGO in HaCaT cells was evaluated according to a previously described method with minor modifications.²⁵⁾ HaCaT cells were incubated with [³H]ERGO (0.3 μM) in a transport buffer (125 mM NaCl, 25 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES), 5.6 mM glucose, 4.8 mM KCl, 1.2 mM MgSO₄, 1.2 mM KaH₂PO₄, and 1.2 mM CaCl₂, pH 7.4) at 4 or 37°C. At the end of the uptake, the supernatants were collected, and the cells were washed three times with ice-cold transport buffer. Liquid scintillation spectrometry (LSC-7200, Aloka, Tokyo, Japan) was used to measure [³H]ERGO in the medium and cells. To measure uptake of unlabeled ERGO, HaCaT cells were cultured in a medium supplemented with PSE or ERGO, with the final ERGO concentration adjusted to 10 μM. The supernatant was collected at the end of the incubation, and the cells were washed three times with ice-cold transport buffer, collected with water using a cell scraper, and then sonicated. Unlabeled ERGO in the medium and cells was measured using LC-MS/MS (Supplementary Material S5, Supplementary Table S1). Proteins were quantified with Protein Assay Dye Reagent Concentrate (Bio-Rad). The uptake values were expressed as the cell-to-medium ratio (μL/mg of protein), obtained by dividing the uptake amount by protein amount and medium ERGO concentration.

Knockdown of OCTN1

HaCaT cells were transfected with 5 pmol of small interfering RNA (siRNA) targeted to human OCTN1 (siOCTN1, BONAC Co., Fukuoka, Japan) or non-targeting siRNA (siControl, Nippon Gene, Tokyo, Japan) using lipofectamine RNAiMax in Opti-MEM (Thermo Fisher Scientific). The culture medium was replaced with low-calcium medium 24 h after the transfection, and the cells were cultured for 48 h. Gene expression of endogenous OCTN1 in HaCaT cells transfected with siRNA was assessed using quantitative RT-PCR (Supplementary Material S6, Supplementary Tables S2, S3).

Immunochemical Analysis

HaCaT cells cultured on poly-l-lysine-coated glass coverslips in 24-well plates were washed with PBS containing 0.1% Tween 20 (PBST), and then incubated with 5 μg/mL wheat germ agglutinin (WGA)-rhodamine (Vector Laboratories, Burlingame, CA, U.S.A.) for 30 min. The cells were then washed with PBST and incubated in 4% paraformaldehyde (PFA) in PBS for 20 min. The cells were washed with PBST again, blocked with PBST containing 1% bovine serum albumin (BSA) for 30 min, and incubated with anti-OCTN1 antiserum (1 : 50) in PBST containing 0.1% BSA at room temperature overnight. The cells were washed with PBST three times and then incubated with the anti-rabbit Alexa 488 secondary antibody (1 : 1000) in PBST containing 0.1% BSA for 1 h at room temperature. Then, the cells were washed with PBST three times, mounted with 4′,6-diamidino-2-phenylindole (DAPI), and observed under a confocal laser scanning microscope (LSM980; Carl Zeiss, Jena, Germany).

Human skin samples were obtained from the Human and Animal Bridging Research Organization (Ichikawa, Japan). The human studies were conducted following the Declaration of Helsinki and approved by the Institutional Review Boards of Kanazawa University. All participants provided written informed consent for the use of their samples and medical information for research purposes. The frozen sections were cut at 20 μm using a cryostat (C3050S, Leica, Nussloch, Germany). The sections were incubated with 95% ethanol for 30 min, then incubated with 100% acetone for 1 min. After removing the surrounding liquid, the sections were incubated in PBST containing 1% BSA for 30 min, followed by incubation with anti-OCTN1 antiserum (1 : 50) in PBST overnight at 4°C. The sections were washed with PBST three times, then incubated for 1 h at room temperature with secondary anti-rabbit Alexa 488 antibodies (1 : 1000) in PBST. The sections were then washed with PBST three times, mounted with DAPI, and observed with the LSM980 microscope.

Statistical Analysis

All statistical analyses were conducted using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan).²⁶⁾ All data were assessed using one-way or two-way ANOVA followed by Tukey’s multiple comparison or Dunnett’s test, or a Student’s t test.

RESULTS

Change in Body Weight in Each Group

Change in body weight was measured in each mouse, and that in each group exhibited no statistical difference among each group (Fig. 1B).

Protective Effects of Dietary PS and PE on UVB-Induced Skin Damage

UVB irradiation significantly decreased skin moisture content (Fig. 2A) and increased TEWL (Fig. 2B) in hairless mice. However, ingestion of PS and PE significantly alleviated both the UVB-induced reduction in skin moisture content and the increase in TEWL (Figs. 2A, 2B). Histological analysis revealed that UVB irradiation caused significant epidermal thickening in dorsal skin and that ingesting PS and PE significantly suppressed UVB-induced epidermal thickening (Figs. 2C, 2D).

Fig. 2. Effect of Dietary Intake of PE and PS on UVB-Induced Skin Damage in Hairless Mice

(A, B) The average of five separate measurements of skin moisture content (A) and TEWL (B) was taken from each sample, and the mean and S.D. were calculated (n = 6); n refers to the number of animals. Open circles, control group; closed circles, UVB group; open squares, UVB +1% PE group; closed squares, UVB +2.5% PE group; open diamonds, UVB +1% PS group; closed diamonds, UVB +2.5% PS group. (C) Representative image of H&E-stained skin tissue sections. Scale bar is 300 μm. (D) The average of epidermal thickness at five locations from images of H&E-stained skin was obtained for each mouse, and the mean and S.D. were calculated (n = 6); n refers to the number of animals. (A, B) Significant differences among groups were assessed with two-way ANOVA and Dunnett’s test at each period. Individual data are presented in Supplementary Tables S4 and S5. (D) Significant differences among groups were analyzed by one-way ANOVA and Dunnett’s test. ^*p < 0.05, compared with UVB group, ^#p < 0.05, compared with control group.

Plasma and Skin Concentrations of ERGO after Ingestion of PS and PE

ERGO concentrations in the plasma, epidermis, and dermis were measured 15 weeks after starting the test diet. In the control and UVB groups, the ERGO concentrations in all samples were under the detection limits (plasma: 0.3 μM, epidermis and dermis: 1.2 nmol/g tissue). However, mice that ingested PS and PE had much higher ERGO concentrations in the plasma (30–40 μM) and skin (Fig. 3). In the epidermis, PE intake increased ERGO concentration in a dose-dependent manner, and concentrations were significantly higher in the 1% PS group than the 1% PE group (Fig. 3B). However, no significant differences were observed in concentrations of plasma and dermis between any of the PS and PE groups regardless of dosage (Figs. 3A, 3C). These results demonstrate that ERGO is highly distributed in the epidermis after mushroom ingestion.

Fig. 3. ERGO Concentrations in Plasma and Skin after Ingestion of PE and PS

After 15 weeks on each diet, ERGO concentrations were measured in plasma (A), epidermis (B), and dermis (C). ND, not detectable (limits: plasma, <0.3 μM; epidermis and dermis, <1.2 nmol/g tissue). Each value is expressed as mean ± S.D. (n = 6); n refers to the number of animals. Statistical analysis was conducted using one-way ANOVA and Tukey’s test. ^*p < 0.05, significant difference between mushroom intake groups.

Protective Effect of PS and PE on UVB-Induced Oxidative Stress in Murine Skin

Oxidative stress in murine skin was evaluated by measuring the oxidative DNA damage marker 8-OHdG.²⁷⁾ Levels of 8-OHdG were significantly higher in the UVB group than in the control group. In addition, dietary intake of PS and PE significantly reduced 8-OHdG levels in the dorsal skin of UVB-irradiated mice in a dose-dependent manner (Fig. 4A).

Fig. 4. Effect of Dietary Intake of PE and PS on UVB-Induced Oxidative DNA Damage and Expression of Pro-Inflammatory Cytokines in the Dorsal Skin of Hairless Mice

Levels of 8-OHdG (A), IL-1β (C), and IL-6 (D) were quantified by ELISA, and TNF-α (B) was evaluated with Western blotting. Each value is expressed as mean ± S.D. (n = 6); n refers to the number of animals. Significant differences were assessed by one-way ANOVA and Dunnett’s test. ^*p < 0.05, compared with UVB group, ^#p < 0.05, compared with control group.

Protective Effect of PS and PE on UVB-Induced Inflammation in Murine Skin

Pro-inflammatory cytokine expression was evaluated in the skin of UVB-irradiated hairless mice after ingestion of PS and PE. Expression of IL-1β, TNF-α, and IL-6 was remarkably higher in the UVB group compared with the control group; however, ingestion of PS significantly suppressed the UVB-induced increase in cytokine expression regardless of dosage (Figs. 4B–4D). In addition, 2.5% PE, but not 1%, tended to suppress the increased expression of these UVB-induced cytokines (Figs. 4B–4D).

Effect of PSE and ERGO on UVB-Induced ROS Generation in Human Keratinocytes

The fluorescence intensity of DCF, an indicator of intracellular ROS, was used to investigate the effect of PS and ERGO on UVB-induced ROS generation in HaCaT cells. Intracellular ROS was significantly higher in the UVB-irradiated group than in the control group. In the PSE-treated group, there was significant suppression of UVB-induced ROS production after treatment with 78, 261, and 782 mg/mL of PSE, which included 3, 10, and 30 μM of ERGO, respectively (Figs. 5A, 5C). Treatment with 3 and 30 μM ERGO also significantly reduced UVB-induced ROS generation (Figs. 5B, 5D).

Fig. 5. Effect of PSE and ERGO on UVB-Induced Intracellular ROS Levels in HaCaT Cells

HaCaT cells were pretreated with PSE (A, C) or pure ERGO (B, D) for 24 h before UVB irradiation (30 mJ/cm²). (A, B) Intracellular ROS was detected using DCFH-DA and nuclei were stained with Hoechst 33342. Scale bar: 200 μm. (C, D) The ROS-mediated increase in the DCF-positive area was normalized using the Hoechst 33432-positive area. Values are expressed as means ± S.D. (n = 4); n refers to the number of samples. Significant differences were assessed by one-way ANOVA and Dunnett’s test. ^*p < 0.05, compared with vehicle-treated and UVB-irradiated group, ^#p < 0.05, compared with UVB-untreated group.

Functional Expression of OCTN1 in Human Keratinocytes

ERGO uptake was examined in HaCaT cells to confirm whether OCTN1 is functionally expressed in human keratinocytes. The [³H]ERGO was taken up by HaCaT cells in a time-dependent manner at 37°C, but not 4°C (Fig. 6A), suggesting the involvement of the transporter. ERGO uptake was also observed when HaCaT cells were incubated with PSE (Fig. 6B). This uptake was almost comparable to that seen with pure ERGO when the concentration was matched in the two groups (Fig. 6B). Both gene expression of endogenous OCTN1 and [³H]ERGO uptake were remarkably lower in HaCaT cells transfected with siOctn1 than in cells transfected with siControl (Figs. 6C, 6D), suggesting that OCTN1 mediates ERGO uptake in HaCaT cells. Immunostaining of HaCaT cells and human skin tissues showed that OCTN1 was localized in plasma membranes (Figs. 7A, 7B). It was also detected in the intracellular compartment of HaCaT cells (Fig. 7A). OCTN1 was reported to be expressed in intracellular compartments in other cell lines such as mitochondria in human hepatoma HepG2.²⁸⁾

Fig. 6. Functional Expression of OCTN1 in HaCaT Cells

(A) HaCaT cells were incubated with [³H]ERGO at 4 or 37°C and [³H]ERGO uptake was measured. Individual data are presented in Supplementary Table S6. (B) HaCaT cells were incubated with PSE or ERGO (final ERGO concentration was adjusted to 10 μM), and then ERGO uptake was measured. Individual data are presented in Supplementary Table S7. (C) OCTN1 mRNA was quantified by RT-PCR in HaCaT cells transiently transfected with siControl or siOCTN1. Each value was normalized by the GAPDH mRNA level. (D) On day 3 post-transfection, the cells were incubated with [³H]ERGO for 2 h, and [³H]ERGO uptake was measured. Values are expressed as means ± S.D. (n = 3); n refers to the number of samples. Significant differences were assessed by Student’s t test. ^*p < 0.05, compared with 4°C or siControl group.

Fig. 7. Localization of OCTN1 in HaCaT Cells and Human Epidermis

(A) HaCaT cells were fixed with 4% PFA and immunocytochemistry showed OCTN1 (green), the plasma membrane marker WGA-rhodamine (red), and the nuclear marker DAPI (blue). Scale bar is 50 μm. (B) Frozen human skin sections were fixed with 95% ethanol and 100% acetone, and immunohistochemistry showed OCTN1 (green) and DAPI (blue). Scale bar is 5 μm.

DISCUSSION

This is the first study to demonstrate the effects of ERGO-rich mushrooms on UVB-induced damage and the concomitant, efficient distribution of ERGO to the skin in vivo. Dietary intake of PS and PE significantly alleviated the epidermal thickening, reduction in skin moisture content, and increase in TEWL induced by UVB in mice (Figs. 2A–2D). These results suggest that long-term intake of PS or PE may protect the skin from UVB damage and/or prevent skin disorders. Quantitative evaluation showed that PS and PE reduced epidermal thickening (Fig. 2D), which is consistent with a previous report showing that oral administration of an extract of Coprinus comatus, an edible mushroom containing ERGO, tended to reduce UVB-induced epidermal thickening.⁵⁾ Additionally, oral intake of Flammulina velutipes, another mushroom containing ERGO, increased skin moisture content in humans.²⁹⁾ However, these previous studies did not report ERGO levels in the skin and the involvement of ERGO in the beneficial effect of these mushrooms remained unclear. The present study demonstrated that ingesting PS and PE led to greater ERGO distribution to the skin, suggesting that UVB-induced injury may be ameliorated by the ERGO in these mushrooms. The effect of ERGO on skin moisture content or TEWL was not dose-dependent (Figs. 2A, 2B), which may be due to the small difference in ERGO concentrations in the skin between the 1 and 2.5% groups (Figs. 3B, 3C). The ERGO concentration in plasma (30–40 μM, Fig. 3A) was much higher than the mouse OCTN1 Km value (approx. 5 μM) for ERGO uptake¹⁴⁾; this may have led to saturation of OCTN1-mediated transport, which may at least partially explain the small dose-dependence in plasma and skin concentration of ERGO (Fig. 3).

UVB-induced ROS generation activates NF-κB, which induces pro-inflammatory cytokines including IL-1β, TNF-α, and IL-6 in keratinocytes.⁶⁾ Furthermore, UVB-induced oxidative stress and inflammation promote epidermal thickening,⁷⁾ which, in turn, causes an imbalance in barrier permeability, and the imbalanced skin barrier may lead to reduced skin moisture and/or increased TEWL.³⁰⁾ However, protecting the skin from UVB-induced oxidative stress and inflammation can prevent loss of skin moisture content, reduce TEWL, and mitigate epidermal thickening.⁷⁾ Therefore, UVB-induced skin disorders are closely related to oxidative stress and inflammation. The present study showed that PS ingestion suppressed the UVB-induced formation of the oxidative DNA damage marker 8-OHdG, and expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in mice (Figs. 4A–4D). In addition, ingestion of PS and PE led to increases in ERGO concentrations in the epidermis (324–725 nmol/g tissue) that were approx. 100 times greater than the ERGO concentration (approx. 3 μM) at which UVB-induced intracellular ROS was significantly inhibited in keratinocytes (Fig. 5). These results imply that the antioxidative and anti-inflammatory activities of ERGO may alleviate UVB-induced epidermal thickening, water loss, and skin barrier disruption, although possible involvement of other effects of ERGO than antioxidant or anti-inflammatory activities cannot be denied.

Interestingly, ERGO concentrations were 1.9–4.5 times higher in the epidermis than in the dermis (Figs. 3B, 3C). Hence, epidermal ERGO is likely to protect skin from UVB because the epidermis almost completely absorbs the UVB.^31,32) The efficient distribution of ERGO to the epidermis was compatible with the detection of OCTN1 on the plasma membranes of HaCaT cells and in human epidermal tissue (Figs. 7A, 7B). Knockdown of OCTN1 reduced ERGO incorporation into HaCaT cells (Fig. 6D), implying that ERGO might be taken up by OCTN1 into keratinocytes, which are the main constituents of the epidermis. This ERGO uptake in HaCaT cells (Fig. 6) may be consistent with previous findings of OCTN1 expression and ERGO uptake in normal human epidermal keratinocytes.³³⁾ However, the epidermal ERGO concentration was significantly lower in the 1% PE group than in the other mushroom groups (Fig. 3B). This lower concentration may explain the failure of the 1% PE group to suppress the elevation of pro-inflammatory cytokines (Figs. 3B, 4B–4D).

UV rays induce oxidative stress via light-dependent production of ROS such as singlet oxygen, superoxide radicals, hydrogen peroxide, and hydroxyl radicals.³⁴⁾ UVB activates NADPH oxidase and respiratory chain reactions with the production of superoxide radicals, resulting in highly reactive hydroxyl radical generation.³⁵⁾ However, ERGO inhibits the production of superoxide radicals, hydroxyl radicals, and singlet oxygen.³³⁾ Considering these reports, suppression of the UVB-induced increase in intracellular ROS following treatment of HaCaT cells with PSE and ERGO (Fig. 5) may be due to reduction of the ROS by ERGO in the cells (Fig. 6B). In theoretical studies, the radical scavenging reaction rate of vitamin E for hydroxyl radicals is ≥2.7 × 10⁸ M⁻¹s⁻¹,³⁶⁾ and that of vitamin C, which regenerates α-tocopheroxyl radicals to vitamin E, is 2 × 10⁵ M⁻¹s⁻¹.³⁷⁾ In comparison, ERGO has a reaction rate with hydroxyl radicals that is approx. 40 times higher than that of vitamin E.³⁸⁾ Therefore, ERGO is likely to be a more potent hydroxyl radical scavenger for protecting keratinocytes from UVB irradiation than other food-derived antioxidants.

The plasma ERGO concentration reported in a clinical trial was approx. 3.0 μM without any intervention and approx. 9.5 μM after oral ingestion of ERGO at 20 mg/d for 4 weeks.¹⁰⁾ Blood ERGO concentration was reported to gradually increase until at least 12 weeks after the start of ingestion of ERGO-containing mushrooms.³⁹⁾ Therefore, oral ingestion of PS at 20 mg/d for a longer period than 4 weeks may lead to the effective plasma concentration (30–40 μM) observed in the present study (Fig. 3A). The present study also showed that treatment with 3 μM ERGO significantly reduced UVB-induced ROS production in keratinocytes (Fig. 5B). In a previous study, 10 μM ERGO was reported to reduce the number of TUNEL-positive keratinocytes in solar-simulating UV-irradiated epidermis ex vivo.³³⁾ These in vitro studies may be compatible with the estimated effective ERGO concentration (30–40 μM) after oral ERGO intake which may be enough to exert these biological effects, implying that ERGO intake could protect the skin from UVB, even in humans. Furthermore, no adverse events were observed in a previous clinical trial with oral ERGO administration.^10,40) The European Food Safety Authority’s no observed adverse effect level for synthetic ERGO intake in animal studies is 800 mg/kg/d, which is more than 10 times higher than the dose used in the present study.⁴¹⁾ This suggests that ERGO is a safe food-derived ingredient to protect skin against UV radiation, highlighting the need for an additional clinical study to assess the usefulness of ERGO-containing mushrooms as skin-protecting functional foods. This hypothesis has been at least partially demonstrated by the recent finding of the effects of PS on skin moisturizing function and facial condition in a randomized, double-blind, placebo-controlled trial, in which plasma ERGO concentration increased approx. 30 μM in some subjects after 12 weeks of PS intake.⁴²⁾

CONCLUSION

These data suggest that dietary intake of PS and PE in mice may help protect skin moisture content and barrier functions from UVB radiation. The ERGO found in PS and PE is distributed to the epidermis via OCTN1 functionally expressed on plasma membranes of keratinocytes; it subsequently protects the epidermis against UVB at least partially by reducing oxidative stress and inflammation. Thus, ERGO-rich mushrooms are beneficial foods that show promise for preventing and/or treating photoaging.

Acknowledgments

The present study was partially supported by a Grant-in-Aid for Early-Career Scientists provided to TI (No. 20K15991) and a Grant-in-Aid for Scientific Research (B) provided to YK (No. 22H02781) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Prof. Kazuhiro Ogai for kindly lending us the Tewameter. Finally, we thank Mr. Yudai Araragi for technical assistance and helpful discussions.

Conflict of Interest

The present study was funded by Hokuto Corporation. Three of the authors (MH, JK, and KM) are employees of Hokuto Corporation, which provided mushrooms used in the present study. The other authors (TI, AM, RY, and YK) have no conflicts of interest to disclose.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1)  Cao C, Xiao Z, Wu Y, Ge C. Diet and skin aging—from the perspective of food nutrition. Nutrients, 12, 870 (2020).
• 2)  Tobin DJ. Introduction to skin aging. J. Tissue Viability, 26, 37–46 (2017).
• 3)  Chen X, Yang C, Jiang G. Research progress on skin photoaging and oxidative stress. Postepy Dermatol. Alergol., 38, 931–936 (2021).
• 4)  Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview. Medicines (Basel), 5, 93 (2018).
• 5)  Asahi T, Wu X, Shimoda H, Hisaka S, Harada E, Kanno T, Nakamura Y, Kato Y, Osawa T. A mushroom-derived amino acid, ergothioneine, is a potential inhibitor of inflammation-related DNA halogenation. Biosci. Biotechnol. Biochem., 80, 313–317 (2016).
• 6)  Sárdy M. Role of matrix metalloproteinases in skin ageing. Connect. Tissue Res., 50, 132–138 (2009).
• 7)  Im AR, Ji KY, Nam KW, Chae S. Protective effects of Sosihotang extract against ultraviolet B-induced skin photoageing in hairless mice. J. Pharm. Pharmacol., 72, 1278–1286 (2020).
• 8)  Selvamani S, El-Enshasy HA, Dailin DJ, Malek RA, Hanapi SZ, Ambehabati KK, Sukmawati D, Leng OM, Moloi N. Antioxidant compounds of the edible mushroom Pleurotus ostreatus. Int. J. Biotechnol. Wellness Ind., 7, 1–14 (2018).
• 9)  Ey J, Schömig E, Taubert D. Dietary sources and antioxidant effects of ergothioneine. J. Agric. Food Chem., 55, 6466–6474 (2007).
• 10)  Katsube M, Watanabe H, Suzuki K, Ishimoto T, Tatebayashi Y, Kato Y, Murayama N. Food-derived antioxidant ergothioneine improves sleep difficulties in humans. J. Funct. Foods, 95, 105165 (2022).
• 11)  Kameda M, Teruya T, Yanagida M, Kondoh H. Frailty markers comprise blood metabolites involved in antioxidation, cognition, and mobility. Proc. Natl. Acad. Sci. U.S.A., 117, 9483–9489 (2020).
• 12)  Smith E, Ottosson F, Hellstrand S, Ericson U, Orho-Melander M, Fernandez C, Melander O. Ergothioneine is associated with reduced mortality and decreased risk of cardiovascular disease. Heart, 106, 691–697 (2020).
• 13)  Borodina I, Kenny LC, McCarthy CM, Paramasivan K, Pretorius E, Roberts TJ, van der Hoek SA, Kell DB. The biology of ergothioneine, an antioxidant nutraceutical. Nutr. Res. Rev., 33, 190–217 (2020).
• 14)  Kato Y, Kubo Y, Iwata D, Kato S, Sudo T, Sugiura T, Kagaya T, Wakayama T, Hirayama A, Sugimoto M, Sugihara K, Kaneko S, Soga T, Asano M, Tomita M, Matsui T, Wada M, Tsuji A. Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1. Pharm. Res., 27, 832–840 (2010).
• 15)  Mayumi T, Kawano H, Sakamoto Y, Suehisa E, Kawai Y, Hama T. Studies on ergothioneine. V. Determination by high performance liquid chromatography and application to metabolic research. Chem. Pharm. Bull., 26, 3772–3778 (1978).
• 16)  Cheah IK, Halliwell B. Ergothioneine; antioxidant potential, physiological function and role in disease. Biochim. Biophys. Acta, 1822, 784–793 (2012).
• 17)  Ko HJ, Kim J, Ahn M, Kim JH, Lee GS, Shin T. Ergothioneine alleviates senescence of fibroblasts induced by UVB damage of keratinocytes via activation of the Nrf2/HO-1 pathway and HSP70 in keratinocytes. Exp. Cell Res., 400, 112516 (2021).
• 18)  Tsay GJ, Lin SY, Li CY, Mau JL, Tsai SY. Comparison of single and combined use of ergothioneine, ferulic acid, and glutathione as antioxidants for the prevention of ultraviolet b radiation-induced photoaging damage in human skin fibroblasts. Processes (Basel), 9, 1204 (2021).
• 19)  Dubost NJ, Ou B, Beelman RB. Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity. Food Chem., 105, 727–735 (2007).
• 20)  Ishikawa M, Harada S, Azumi Y, Oku T, Ouchi K, Inatomi S. Breeding of a new strain of Pleurotus sp. Mushroom Science and Biotechnology, 24, 7–15 (2016).
• 21)  Kawai J, Andoh T, Mori K. Suppression of leukotriene B4 production is involved in the anti-pruritic action of Grifola frondosa in pollen allergy-induced ocular itching in mice. Food Agric. Immunol., 32, 310–320 (2021).
• 22)  Nakamichi N, Nakayama K, Ishimoto T, Masuo Y, Wakayama T, Sekiguchi H, Sutoh K, Usumi K, Iseki S, Kato Y. Food-derived hydrophilic antioxidant ergothioneine is distributed to the brain and exerts antidepressant effect in mice. Brain Behav., 6, e00477 (2016).
• 23)  Sugiura T, Kato S, Shimizu T, Wakayama T, Nakamichi N, Kubo Y, Iwata D, Suzuki K, Soga T, Asano M, Iseki S, Tamai I, Tsuji A, Kato Y. Functional expression of carnitine/organic cation transporter OCTN1/SLC22A4 in mouse small intestine and liver. Drug Metab. Dispos., 38, 1665–1672 (2010).
• 24)  Deyrieux AF, Wilson VG. In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line. Cytotechnology, 54, 77–83 (2007).
• 25)  Nakamichi N, Taguchi T, Hosotani H, Wakayama T, Shimizu T, Sugiura T, Iseki S, Kato Y. Functional expression of carnitine/organic cation transporter OCTN1 in mouse brain neurons: possible involvement in neuronal differentiation. Neurochem. Int., 61, 1121–1132 (2012).
• 26)  Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant., 48, 452–458 (2013).
• 27)  Valavanidis A, Vlachogianni T, Fiotakis C. 8-Hydroxy-2′ -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. J. Environ. Sci. Health Part C Environ. Carcinog. Ecotoxicol. Rev., 27, 120–139 (2009).
• 28)  Lamhonwah AM, Tein I. Novel localization of OCTN1, an organic cation/carnitine transporter, to mammalian mitochondria. Biochem. Biophys. Res. Commun., 345, 1315–1325 (2006).
• 29)  Nagae M, Isa A, Ishikawa S, Muta S, Shimizu K. Effect of oral intake of Flammulina velutipes (Enokitake) on skin condition in healthy adult women: a randomized, double-blind, placebo-controlled study on mental and physical health. Cosmetics, 10, 57 (2023).
• 30)  Biniek K, Levi K, Dauskardt RH. Solar UV radiation reduces the barrier function of human skin. Proc. Natl. Acad. Sci. U.S.A., 109, 17111–17116 (2012).
• 31)  D’Orazio J, Jarrett S, Amaro-Ortiz A, Scott T. UV radiation and the skin. Int. J. Mol. Sci., 14, 12222–12248 (2013).
• 32)  Dupont E, Gomez J, Bilodeau D. Beyond UV radiation: a skin under challenge. Int. J. Cosmet. Sci., 35, 224–232 (2013).
• 33)  Markova NG, Karaman-Jurukovska N, Dong KK, Damaghi N, Smiles KA, Yarosh DB. Skin cells and tissue are capable of using l-ergothioneine as an integral component of their antioxidant defense system. Free Radic. Biol. Med., 46, 1168–1176 (2009).
• 34)  Darr D, Fridovich I. Free radicals in cutaneous biology. J. Invest. Dermatol., 102, 671–675 (1994).
• 35)  Masaki H. Role of antioxidants in the skin: anti-aging effects. J. Dermatol. Sci., 58, 85–90 (2010).
• 36)  Navarrete M, Rangel C, Corchado JC, Espinosa-García J. Trapping of the OH radical by α-tocopherol: a theoretical study. J. Phys. Chem. A, 109, 4777–4784 (2005).
• 37)  Scarpa M, Rigo A, Maiorino M, Ursini F, Gregolin C. Formation of α-tocopherol radical and recycling of α-tocopherol by ascorbate during peroxidation of phosphatidylcholine liposomes. An electron paramagnetic resonance study. Biochim. Biophys. Acta, 801, 215–219 (1984).
• 38)  Akanmu D, Cecchini R, Aruoma OI, Halliwell B. The antioxidant action of ergothioneine. Arch. Biochem. Biophys., 288, 10–16 (1991).
• 39)  Watanabe N, Matsumoto S, Suzuki M, Fukaya T, Kato Y, Hashiya N. Effect of ergothioneine on the cognitive function improvement in healthy volunteers and mild cognitive impairment subjects—a randomized, double-blind, parallel-group comparison study. Jpn. Pharmaco. Ther., 48, 685–697 (2020).
• 40)  Cheah IK, Tang RMY, Yew TSZ, Lim KHC, Halliwell B. Administration of pure ergothioneine to healthy human subjects: uptake, metabolism, and effects on biomarkers of oxidative damage and inflammation. Antioxid. Redox Signal., 26, 193–206 (2017).
• 41)  Turck D, Bresson J-L, Burlingame B, et al. Statement on the safety of synthetic L-ergothioneine as a novel food – supplementary dietary exposure and safety assessment for infants and young children, pregnant and breastfeeding women. EFSA J., 15, e05060 (2017).
• 42)  Hanayama M, Mori K, Ishimoto T, Kato Y, Kawai J. Effects of an ergothioneine-rich Pleurotus sp. on skin moisturizing functions and facial conditions: a randomized, double-blind, placebo-controlled trial. Front. Med. (Lausanne), 11, 1–9 (2024).