Journal of the Society of Cosmetic Scientists of Korea (대한화장품학회지)

Society of Cosmetic Scientists of Korea (대한화장품학회)
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Glycation Inhibitory and Antioxidative Activities of Ergothioneine

에르고티오네인의 당화 억제 및 항산화 활성에 관한 연구

  • 배준태 ((주)제이투케이바이오 기술연구소) ;
  • 이청희 ((주)잇츠한불 기술연구원) ;
  • 이근수 ((주)잇츠한불 기술연구원) ;
  • 김진화 ((주)잇츠한불 기술연구원) ;
  • 홍진태 (충북대학교 약학대학)
  • Received : 2019.05.03
  • Accepted : 2019.06.17
  • Published : 2019.06.30
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Abstract

Ergothioneine has been known as an excellent antioxidant and a cellular protector against oxidative damage in vivo. In the present study, ergothioneine was demonstrated to possess antioxidant and anti-glycation activities. The radical scavenging activity of ergothioneine enhanced the viability of human dermal fibroblasts (HDFs) exposed to ultraviolet (UV) light. The UVA irradiation increased the proportion of senescence-associated ${\beta}$-galactosidase (SA-${\beta}$-gal) positive cells in comparison with the normal control group. The treatment of UVA-irradiated HDFs with ergothioneine decreased the level of SA-b-gal (by approximately 45% at an ergothioneine concentration of $400{\mu}M$) compared with the UVA-irradiated HDFs. We also found that ergothioneine inhibited production of glyceraldehyde-derived advanced glycation endproducts (AGEs) in a concentration-dependent manner. The ergothioneine educed carboxymethyl-lysine (CML) expression in comparison to the glyoxal treatment. In addition, in the Western blot analysis, treatment of glyoxal-stimulated HDFs with ergothioneine resulted in a dose-dependent decrease in the expression level of the receptor for AGE (RAGE). These results suggest that ergothioneine may have potent anti-aging effects and could be used as a cosmetic material against cellular accumulation of AGEs.

에르고티오네인은 생체 내에서 우수한 항산화제로서 산화스트레스로부터 세포 보호제로 알려져 있다. 본 연구에서는 에르고티오네인의 라디칼 소거 활성은 자외선에 조사된 사람 섬유아세포 생존율을 향상 시키는 것을 확인하였으며, 세포노화 지표물질인 senescence-associated ${\beta}$-galactosidase (SA-${\beta}$-gal) 활성을 사람 섬유아세포를 이용하여 확인한 결과, 에르고티오네인 $400{\mu}M$ 처리 농도에서 염색된 세포의 수가 약 45%의 SA-${\beta}$-gal의 레벨이 감소하여 세포 노화(senescence)를 억제하는 것을 확인할 수 있었다. 또한, 에르고티오네인은 글리세르알데하이드에 의해 유도된 최종당화산물(AGEs) 생성을 저해하였으며, 카르복시메칠라이신(CML) 발현을 농도의존적으로 저해하는 것으로 나타내었다. 글리옥살에 의해 최종당화산물의 수용체(RAGE)가 발현된 사람 섬유아세포에 에르고티오네인을 처리하였을 때 RAGE의 발현이 농도의존적으로 감소하는 것을 확인하였다. 따라서, 본 연구결과를 바탕으로 에르고티오네인의 항노화 효과와 최종당화산물의 세포 내 축적을 저해할 수 있는 화장품 소재로서 활용가치가 있음을 확인하였다.

Keywords

1.Introduction

Glycation is the non-enzymatic reaction between reducing sugars, such as glucose, and proteins, lipids or nucleic acids[1]. Glycation is a major source of reactive oxygen species (ROS) and reactive carbonyl species (RCS) such as glyoxal and methylglyoxal that are generated by both oxidative (glycoxidative) and non-oxidative pathways[2]. Oxygen, reactive oxygen species and redox active transition metals accelerate advanced glycation end products (AGEs) formation. When an oxidative step is involved, the products are called advanced glycoxidation end products[1,3].

The oxidation process is believed to play an important role in AGEs formation. AGEs are generated by the non‐enzymatic glycation of amino groups of proteins and reducing sugars or other ROS[4]. The accumulation of AGEs in organs is induced by hyperglycemia and is one of the causes of diabetic complications. Moreover, AGEs accumulate in the skin of non‐diabetics and are correlated with aging[5]. Nε‐(carboxymethyl)lysine (CML), one of the principal AGEs in the skin, modifies elastin and collagen[6]. When AGEs accumulate, they induce crosslinking of collagen and reduce skin degradability and dermal regeneration. In addition, AGEs induce fibroblast apoptosis by adducting to receptors for AGE (RAGE) on the cells[7]. These phenomena are thought to be related to skin aging.

A number of antioxidant polyphenols, such as flavonoids and other related compounds, show remarkable promise for a wide range of pharmacological uses, including ROS scavenging activity[8]. Several studies have revealed that the medicinal plants and phenolic compounds have been demonstrated, in a wide collection of in vitro and in vivostudies, to possess significant inhibitory activity against glycation by AGEs mediated pathogenesis[9]. Especially, Mushrooms contain a variety of secondary metabolites, including various phenolic compounds, which have been shown to act as excellent antioxidants[10]. Mushrooms were discovered to be the primary source of ergothioneine (Figure 1), a naturally occurring thiol-containing amino acid, known for its antioxidant properties[11]. Ergothioneine is water-soluble and exerts antioxidant properties through multiple mechanisms, one of which is its powerful ability to scavenge free radicals[12,13].

HJPHBN_2019_v45n2_151_f0001.png 이미지

Figure 1. Chemical structure of ergothioneine.

Therefore, this study examined the effects of ergothioneine on the formation of AGEs, and antioxidant activities in UV-irradiated HDFs.

2. Materials and Methods

2.1. Materials

Ergothioneine, glyceraldehyde and aminoguanidine were obtained from Sigma (USA). Dulbecco’s Modified Eagle Medium ( DMEM), F- 12 Ham Medium, and f et al bovine serum (FBS) were obtained from Gibco (USA). OxiSelect™ Nε-(carboxymethyl) lysine (CML) ELISA kit was purchased from Cell Biolabs (USA). The fluorogenic probes, 5-(6-)chloromethyl-2’, 7’-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and pluronic F-127 were purchased from Molecular Probes (USA). The anti-RAGE antibody and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used for Western blots were purchased from Cell Signaling Technology (USA). The other chemicals and reagents used were of analytical grade.

2.2. In Vitro Glycation assay

The method of Sekiguchi et al. was followed with slight modification[14]. To each sample, an equal volume of double-concentrated PBS was added under low temperature conditions to prepare a neutralized solution. This solution was aliquoted in 50 μL volumes into a 96-well black plate and heated at 37 °C overnight to allow the collagen solution to gelate. To each well containing collagen gel, different concentrations (0.05 – 0.3 mM) of ergothioneine and aminoguanidine, a glycation inhibitor, and 500 mM glyceraldehyde were added and incubated at 37 °C for 24 h to induce glycation. The degree of glycation was assessed by measuring the intensity of AGEs specific fluorescence (at 370 nm excitation and 440 nm emission) on a fluorescence plate reader (Infinite M200 PRO, Tecan Group Ltd., Switzerland).

2.3. Cell Culture

Human dermal fibroblasts (HDFs), isolated from human neonatal foreskin, were purchased from Modern Tissue Technologies Inc. (Seoul, Korea). HDFs were cultured in Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient mixture (DMEM/F-12, 3 : 1 v/v, Sigma) containing 10% fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 μg/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Fibroblast subcultures were isolated by trypsinization and all cells used in this study were between the sixth and tenth passages.

2.4. UV Irradiation

HDFs (1.5 × 105 cells/mL) were seeded into 35ø plates (Corning Inc., USA) and cultured overnight. Cells at 80% confluence were washed twice with PBS prior to irradiation. The UVA source was a Sankyo Denki F5T5BLB (Sankyo Co, Japan), a fluorescent bulb emitting 315 – 400 nm wave with a peak at 360 nm. The UVB source was a Sankyo Denki G6T5E (Sankyo Co, Japan), a fluorescent bulb emitting 280 – 360 nm wave with a peak at 306 nm. The dose of UV radiation, determined with a UV radiometer (International Light Inc., USA) was set at 6.3 J/cm2 (UVA) and 20 mJ/cm2 (UVB), respectively. Control cells were treated identically, except for the exposure to UV light.

2.5. Measurement of Intracellular ROS Formation

Microtiter plate assay HDFs (1.5 × 105 cells/mL) were seeded into 96-well plates and cultured overnight. The CM-H2DCFDA reagent is a non-fluorescent compound that is able to react with free radical compounds, especially hydrogen peroxide, to generate fluorescent DCF. In order to detect ROS, HDFs were loaded with 4.0 μM CM- H2DCFDA plus 2% Pluronic F-127 in HEPES-buffered control salt solution (HCSS, containing 120 mM NaCl, 5 mM KCl, 1.6 mM MgCl2, 2.3 mM CaCl2, 15 mM glucose, 20 mM HEPES and 10 mM NaOH). The cells were incubated for 20 min at 37 °C to which 20 μL of test sample of varying doses were added. After 30 min at 37 °C the cells (HDFs) were irradiated by a UVB source. Fluorescence was determined using a luminescence spectrophotometer LS-45 (Perkin Elmer, UK) with an excitation wavelength of 488 nm and emission wavelength of 525 nm[15].

2.6. Histochemical Method for β-galactosidase Detection

The semiquantitative analysis of senescence-associated β-galactosidase (SA-β-gal) positive cells was performed when the confluence of plated fibroblasts reached 50%. Cells were washed in PBS, fixed in 2% formaldehyde / 0.2% glutaraldehyde for 5 min (room temperature), washed again and the incubated at 37 °C with fresh X-Gal (5-bromo-4chloro-3-indolyl β-D-galactopyranoside) stain solution (Sigma, USA) at pH 7.3. The cells colored blue were considered as indicative of β-galactosidase positive cells; 500 randomly selected cells within a field under the microscope were counted. The percentage of positive cells, which represented the aging rate of the fibroblasts cultures, was calculated as

Senescence rate = the number of blue colored cells / the total cell number × 100%

2.7. Determination of Nε-(Carboxymethyl) Lysine (CML)

Nε-(carboxymethyl) lysine (CML), a major antigenic AGE structure, was measured using an enzyme linked immunosorbant assay (ELISA) kit according to the manufacturer’s protocol. The absorbance of samples was compared with CML-BSA standard provided in the assay kit.

2.8. Western Blot Analysis

Cells were lysed in radio immunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) at 4 °C with sonication for 30 min. The lysates were centrifuged at 15,000 g for 30 min. Loading buffer was added to each volume and boiled for 10 min. Samples were resolved on 12% SDS-PAGE and electro-transferred onto a nitro-cellulose membrane. The membrane was blocked with 5% nonfat milk. Blots were incubated with goat anti-human RAGE IgG, anti-GAPDH antibodies each at a dilution of 1 : 2000 for 12 h at 4 °C. The blots were washed in Tris-buffered saline containing 0.2% Tween 20 and exposed to HRP-conjugated anti-goat IgG or antirabbit secondary antibody (1 : 1500) for 60 min, respectively, and the membranes were detected with the SuperSignal West Pico chemiluminescence system (Pierce Biotechnology, USA). Relative intensities of protein bands were analyzed by TotalLab software.

2.9. Statistical analysis

Statistical analysis was performed using Student’s t-test after ANOVA. *p < 0.05 based on at least three or more independent experiments was considered to be statistically significant.

3. Results and Discussion

Ergothioneine (a water-soluble thiol) is a histidine derivative with sulfur on the imidazole ring and a trimethylated amine[16]. Numerous in vitro assays demonstrated the antioxidant and cytoprotective capabilities of ergothioneine against a wide range of cellular stressors[17]. Ergothioneine has been shown to be radioprotective and scavenge singlet oxygen, hydroxyl radicals, hypochlorous acid, and peroxyl radicals as well as inhibit peroxynitrite-dependent nitration of proteins and DNA[11]. Oxidative stress has been widely accepted to mediate the deleterious effects of solar radiation in the skin during photoaging.

The formation of AGEs occurs through multiple processes related in part through ROS[18]. Interestingly, in vitroexposure of AGEs to UVA irradiation leads to formation of ROS, such as superoxide anion, hydrogen peroxide and hydroxyl radicals[19]. Extracellular matrix (ECM) proteins have been regarded as one of the major target structures for glycation. The most abundant collagen type in the skin is type I, whereas collagen IV is being found in the basal membrane. Intermolecular crosslinks of adjacent collagen fibers change its biomechanical properties leading to stiffness and decreased flexibility, thus increasing its susceptibility to mechanical stimuli[20]. Collagens are important proteins for the skin. Thinner and wrinkled skins, the typical signs of normal aging, are the consequence of reduced collagen. Protein glycation contributes to skin aging as it deteriorates the existing collagen by crosslinking.

To evaluate the inhibitory effect of ergothioneine on protein glycation, we used a glycation-inducing reaction system with collagen and glyceraldehydes for rapid detection of fluorescent AGEs was used. Aminoguanidine was employed as a known AGEs inhibitor.

In collagen glycation assay, ergothioneine exhibited significant AGEs inhibitory abilities in a dose-dependent manner. As shown in Figure 2, ergothioneine or aminoguanidine at various concentrations for AGEs formation was incubated with collagen and glyceraldehydes at 37 °C for 24 hours. The percentage inhibition of AGEs formation by ergothioneine (0.05 – 0.3 mM) was 7.1% to 50.5%, respectively. Both ergothioneine and aminoguanidine inhibited the formation of glycated collagen dose-dependently in a similar manner. However, the inhibitory activity of ergothioneine on glycation reaction observed is less than that of aminoguanidine. Aminoguanidine, a small hydrazine like compound, has been synthesized and become one of the most promising pharmacological interventions for glycation inhibition[21].

HJPHBN_2019_v45n2_151_f0002.png 이미지

Figure 2. Anti-glycation effects of ergothioneine on glyceraldehyde-derived AGE formation. Collagen gel was incubated with glyceraldehydes in the absence (control) and presence of various concentrations from both ergothioneine and aminoguanidine (0.05 – 0.3 mM) for 24 h, respectively. aminoguanidine was used as a positive control. Fluorescence of samples was measured at Ex 330 nm and Em 420 nm. Results were expressed as the average of triple determinations with SD *p < 0.05, significantly different from control.

UV irradiation of human keratinocytes and fibroblasts in the presence of AGEs led to increased ROS formation and decreased proliferation in vitro[22]. In this study, the possible photo-protective potential of ergothioneine against UVB-induced ROS formation in HDFs were examined. Ergothioneine is an antioxidant in vivo and a cellular protector against oxidative damage[23]. To determine of intracellular ROS formation, we investigated H2DCF to evaluate the intracellular ROS scavenging ability of ergothioneine in UVB-irradiated HDFs.

Assays designed to assess the oxidation of H2DCF to DCF have been used extensively in order to measure intracellular H2O2 or oxidative stress. H2DCF is oxidized to DCF by hydroxyl radicals, nitrogen dioxide radicals (NO2−), and bicarbonate radical anions[24]. In this study, the basal and UVB-induced levels of fluorescence for CMH2DCFDA loaded HDFs after the ergothioneine treatment are shown in Figure 3. Our preliminary study demonstrated that ergothioneine alone had no significant effect on either the cell viability or morphology (data not shown). The basal level of ROS in HDFs in the normal culture condition was about 115 – 125 AU, while that of the CM-H2DCFDA solution was about 45 AU under the same conditions (*p < 0.05). UVB exposure (20 mJ/ cm2) induced an increase in fluorescence of about 2.8 fold over basal levels (fluorescence value: 350 AU) in CMH2DCFDA-loaded cells. After treatment with the ergothioneine in the culture medium for 2 h, the value of DCF fluorescence decreased remarkably in a dose dependent manner.

HJPHBN_2019_v45n2_151_f0003.png 이미지

Figure 3. The effects of ergothioneine on the production of intracellular ROS in human dermal fibroblasts. HDFs were incubated with 4 μ M CM-H2DCFDA for 20 min, and irradiated by UVB 20 mJ/cm2. Various concentrations of ergothioneine (EGT) were used for treatment. ROS generation was assessed by luminescence spectrophotometer. The values of DCF fluorescence are significant (*p < 0.05). Values are means ± SD of at least triple.

AGEs have been shown to affect various functions of skin cells in vitro. They decrease proliferation and enhance apoptosis of HDFs, an effect which is at least partly the receptor for AGE (RAGE) dependent and correlates with the activation of NFκB and caspases[25]. AGEs are able to induce premature senescence in HDFs in vitro[26].

We investigated the effect of ergothioneine on UVA-induced expression of β-galactosidase, an enzyme specific to cellular ageing, and examined the expression level of the enzyme in cells in the non-irradiated and the irradiated groups with UVA (10 J/cm2) for 72 hours. Cells entering replicative senescence are known to show changes in cell morphology, a decrease in growth rate and increases in SA-β-gal activity[27]. The change of cell morphology was examined by histochemical staining. As shown in Figure 4, the treat ment of UVA-irradiated HDFs with 400 μM ergothioneine decreased the senescent cells (formed after UV irradiation) by approximately 40%.

 HJPHBN_2019_v45n2_151_f0004.png 이미지

Figure 4. (A) Effects of ergothioneine on UVA-induced photoageing feature of HDFs tested by cytochemical staining of SA-β-gal (light microscopy, × 200). (B) Senescent cells were measured by HDFs pretreated with ergothioneine (EGT) and indicated by percentage of decrease in comparison with that of UVA-irradiated HDFs as a control. The activity is significant (*p < 0.05) and values are means ± SD of at least triple.

Glyoxal is an intermediate glycation product generated from fructosyl‐lysine and N‐terminal amino acid residue‐derived fructosamines. It reacts with proteins to rapidly form AGEs[28]. Nε-CML has been used as a biomarker for the formation of non-fluorescent AGEs. As shown in Figure 5A, treatment with glyoxal clearly increased the amount of Nε-CML in the fibroblasts without any significant cytotoxity (data not shown). Figure 5B shows that the effect of ergothioneine on Nε-CML in glyoxal-glycated BSA was 2.41-fold higher than in non-glycated BSA. It was interesting to note that ergothioneine (50 – 200 μM) significantly reduced the concentration of Nε-CML in glyoxal-glycated BSA (12.6% and 54.0%). These results suggest that ergothioneine can protect against AGEs formation.

 HJPHBN_2019_v45n2_151_f0006.png 이미지

Figure 5. Effects of ergothioneine on N ε-CML formation to HDFs induced by glyoxal. (A) Cells were treated with various concentrations of glyoxal for Nε-CML production, (B) Inhibitory effect of ergothioneine (EGT) on glyoxal‐induced Nε-CML formation. Values are means ± SD of at least triple. *p < 0.05 compared with control.

RAGE is the most studied receptor for advanced glycation end products. RAGE is almost ubiquitary expressed in the organism, typically at low levels, and its expression is upregulated under various pathologic conditions[7]. In the skin, RAGE expression was observed in both epidermis and dermis, and it was increased in sun-exposed compared with UV irradiation-protected areas. Keratinocytes, fibroblasts, dendritic cells and to a lesser extent endothelial cells and lymphocytes express RAGE[7].

In this study, the effect of ergothioneine on RAGE expression of HDFs was investigated by western blot analysis, in a dose-dependent manner. The treatment of HDFs with various concentrations of glyoxal (50 – 400 μM) increased the expression of RAGE (data not shown). Glyoxal-induced RAGE expressions at protein levels were significantly decreased after ergothioneine treatment and indicating that ergothioneine reduces the induction mainly caused by glyoxal (Figure 6). Therefore, it was suggested that ergothioneine might inhibit AGE formation by involving downregulation of RAGE protein content.

HJPHBN_2019_v45n2_151_f0005.png 이미지

Figure 6. Effects of ergothioneine on RAGE expression in HDFs. The HDF cells were treated with different doses of ergothioneine for 24 h. Total RAGE expression was determined by western blotting. The band intensities were quantified by densitometry, normalized to the level of GAPDH. Values are means ± SD of at least triple. *p < 0.05 compared with control.

Regarding the inhibitory mechanism of ergothioneine on AGEs production, the antioxidative effect and the trapping of intermediate glycation products are considerable. In terms of AGEs production, oxidation is involved in the production of dicarbonyl intermediates and AGEs formation from Amadori products[4]. Hence, the antioxidative effect of ergothioneine is thought to inhibit the production of intermediate glycation such as glyoxal.

4. Conclusion

In this research, we evaluated the antioxidant capacity of intracellular ROS in UV-irradiated HDFs and AGEs-inhibitory activity of ergothioneine. Ergothioneine as a strong antioxidant agent suppressed AGEs production and RAGE‐induced fibroblast. To the best of our knowledge, this is the first report to demonstrate the protective effects of ergothioneine against AGEs formation in HDFs. Although the metabolic pathway of ergothioneine is still unclear, but it is indicate that ergothioneine is a potent inhibitor of AGEs formation and AGEs cross-linking with collagen in vitro. These results suggest that ergothioneine may have potent anti-aging effects and can be used as new functional material against cellular accumulation of AGEs.

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