6-Shogaol Protects Human Melanocytes against Oxidative Stress through Activation of the Nrf2-Antioxidant Response Element Signaling Pathway

Skin is a major target of oxidative stress. Increasing evidence suggests that oxidative stress is the cause of melanocyte disappearance in vitiligo, which is an acquired pigmentary skin disorder characterized by patches of skin that have lost pigmentation. New herbal extracts with antioxidant activity are therefore being studied. 6-Shogaol (6-SG), an active compound from ginger, is capable of attenuating oxidative stress-induced ageing and neurotoxicity. Subsequently, to investigate whether 6-SG could protect melanocytes from oxidative stress, cultured human primary epidermal melanocytes (HEMn-MPs) were treated with hydrogen peroxide (H2O2) in the presence or absence of 6-SG. The 6-SG exhibited protective effects against H2O2-induced cell death by reducing oxidative stress. In addition, the 6-SG treatment activated the Nrf2-antioxidant response element signaling pathway by upregulating the mRNA expression of the antioxidant enzyme heme oxygenase 1 (HO-1), and protein expression of Nrf2, NAD(P)H: quinine oxidoreductase 1 (Nqo1), and HO-1. Furthermore, the 6-SG also displayed protective effects on melanocytes against Rhododendrol-induced oxidative stress. We concluded that 6-SG protects melanocytes against oxidative stress in vitro, and its protective effect is associated with the activation of the Nrf2-antioxidant response element signaling pathway. 6-SG, therefore, has potential for use in the prevention of melanocyte loss in the early stages of vitiligo or other pigmentary disorders.


Introduction
Skin is the largest surface barrier organ, providing protection from harmful environmental agents such as pathogens, chemicals, and ultraviolet (UV) light. These external environmental factors directly or indirectly drive the production of various reactive oxidants, which participate in a series of physiological and pathological skin processes. Epidermal melanocytes in the skin are known to be particularly vulnerable to oxidative stress [1]. There is emerging evidence that oxidative stress can perturb the homeostasis in melanocytes and can play a significant role in the pathogenesis of vitiligo.
Vitiligo is an acquired chronic depigmenting disease that affects 0.5-2% of the world population. It is characterized by white depigmented patches in the skin, caused by the loss of functioning melanocytes [2,3]. Vitiligo develops due to the progressive and gradual disappearance of epidermal melanocytes, which is associated with a complex interplay among biochemical, environmental, and immunological events [3,4]. Recently, oxidative stress was shown to play an important role in the development and progression of vitiligo [5,6]. In active vitiligo, high levels of oxidative stress and

6-SG Attenuates H 2 O 2 -Induced Cytotoxicity in Human Primary Epidermal Melanocytes
First, to determine the appropriate hydrogen peroxide (H 2 O 2 ) and 6-SG treatment concentrations, normal human primary epidermal melanocytes (HEMn-Mps) were exposed to different concentrations of H 2 O 2 (Figure S1A) or 6-SG ( Figure S1B) for 24 h, and cell viabilities were determined by a 3-[4-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assay. Based on the cell viability data shown in Figure S1A and Figure S1B, H 2 O 2 concentrations of 0.1 mM and 0.2 mM, and 6-SG concentration of 5 µM were chosen for the subsequent experiments, because 0.2 mM H 2 O 2 significantly reduced cell viability and 5 µM 6-SG did not affect cell viability.
Then, to study the cyto-protective effects, HEMn-MPs were treated for 6 h with 5 µM 6-SG, and then exposed to 0.2 mM H 2 O 2 for 140 h. Cell viability was assessed by observing cell morphology ( Figure 1A) and performing MTT assays ( Figure 1B). Morphological observations with light microscopy indicated that the exposure of melanocytes to 0.2 mM H 2 O 2 for 140 h resulted in obvious membrane blebbing and cell shrinkage. In contrast, 6-SG pre-treatment attenuated these morphological changes in melanocytes ( Figure 1A). In addition to the morphological evaluation, the protective effect of 6-SG against H 2 O 2 was confirmed by the MTT assay. The viabilities of 0.2 mM H 2 O 2 -treated HEMn-MPs reduced to 26.25% for those not treated with H 2 O 2 ( Figure 1B); however, preincubation with 6-SG for 6 h reduced the cytotoxic effects of 0.2 mM H 2 O 2 on melanocytes ( Figure 1A).

6-SG Attenuates Reduced Melanogenesis in H2O2-Treated Human Primary Epidermal Melanocytes
Next, to investigate the effectiveness of 6-SG on the melanogenesis of the melanocytes that have been suffering oxidative stress, the melanin content and mRNA expression level of the key enzyme in melanogenesis were evaluated. After pretreating HEMn-MPs for 6 h with 5 µM 6-SG and exposing them to 0.2 mM H2O2 for another 140 h, the cells with 0.2 mM H2O2 were markedly less pigmented than the control cells, and 6-SG pretreatment greatly improved the medium color ( Figure 2A). Through quantitative analysis, we found that the melanin content in the culture medium and cell lysates was significantly suppressed by the 0.2 mM H2O2 treatment for 140 h, compared to the control cells ( Figure 2B,C). However, the cells pretreated with 6-SG significantly attenuated the H2O2induced suppression of melanogenesis, both in the culture medium and cell lysates ( Figure 2B,C).

6-SG Attenuates Reduced Melanogenesis in H 2 O 2 -Treated Human Primary Epidermal Melanocytes
Next, to investigate the effectiveness of 6-SG on the melanogenesis of the melanocytes that have been suffering oxidative stress, the melanin content and mRNA expression level of the key enzyme in melanogenesis were evaluated. After pretreating HEMn-MPs for 6 h with 5 µM 6-SG and exposing them to 0.2 mM H 2 O 2 for another 140 h, the cells with 0.2 mM H 2 O 2 were markedly less pigmented than the control cells, and 6-SG pretreatment greatly improved the medium color ( Figure 2A). Through quantitative analysis, we found that the melanin content in the culture medium and cell lysates was significantly suppressed by the 0.2 mM H 2 O 2 treatment for 140 h, compared to the control cells ( Figure 2B,C). However, the cells pretreated with 6-SG significantly attenuated the H 2 O 2 -induced suppression of melanogenesis, both in the culture medium and cell lysates ( Figure 2B,C).
Tyrosinase is well known as the key enzyme in melanin biosynthesis, catalyzing the only rate-limiting steps in melanogenesis [26]. After pretreating HEMn-MPs for 6 h with 5 µM 6-SG, and after exposure to 0.2 mM H 2 O 2 for 140 h, the expression level of tyrosinase was evaluated by real-time PCR analysis ( Figure 2D). The mRNA expression level of tyrosinase significantly decreased in the cells treated with 0.2 mM H 2 O 2 compared with control cells, and 6-SG pretreatment significantly blocked the H 2 O 2 -induced decrease in tyrosinase expression ( Figure 2D). Microphthalmia-associated transcription factor (MITF) is known as the main transcription factor regulating tyrosinase expression [27]. Thus, the expression level of MITF was also evaluated by real-time PCR analysis ( Figure 2E). The MITF mRNA Tyrosinase is well known as the key enzyme in melanin biosynthesis, catalyzing the only ratelimiting steps in melanogenesis [26]. After pretreating HEMn-MPs for 6 h with 5 µM 6-SG, and after exposure to 0.2 mM H2O2 for 140 h, the expression level of tyrosinase was evaluated by real-time PCR analysis ( Figure 2D). The mRNA expression level of tyrosinase significantly decreased in the cells treated with 0.2 mM H2O2 compared with control cells, and 6-SG pretreatment significantly blocked the H2O2-induced decrease in tyrosinase expression ( Figure 2D). Microphthalmia-associated

6-SG Reduces H 2 O 2 -Induced Oxidative Stress in Human Epidermal Melanocytes
We used the cell-permeable CellROX ® Green Reagent to detect oxidative stress and observe the real-time visualization of intracellular oxidative stress in living melanocytes by green fluorescence. As shown in Supplementary Video S1, oxidative stress in HEMn-MPs was detected using CellROX ® Green reagent during exposure to 0.2 mM H 2 O 2 for the first 12 h, where an obvious increase in green fluorescence was observed. However, as shown in Supplementary Video S2, in cells with 5 µM 6-SG pretreatment for 6 h, the H 2 O 2 -induced green fluorescence accumulation was markedly attenuated. The presentative images were taken 24 h after the indicated treatments ( Figure 3). In conclusion, 6-SG preincubation markedly attenuates H 2 O 2 -induced cell death and oxidative stress in melanocytes.
transcription factor (MITF) is known as the main transcription factor regulating tyrosinase expression [27]. Thus, the expression level of MITF was also evaluated by real-time PCR analysis ( Figure 2E). The MITF mRNA expression significantly decreased in those cells treated with 0.2 mM H2O2 compared with the control cells, and 6-SG pretreatment significantly reversed the H2O2-induced decrease in MITF expression ( Figure 2E).

6-SG Reduces H2O2-Induced Oxidative Stress in Human Epidermal Melanocytes
We used the cell-permeable CellROX ® Green Reagent to detect oxidative stress and observe the real-time visualization of intracellular oxidative stress in living melanocytes by green fluorescence. As shown in Supplementary Video 1, oxidative stress in HEMn-MPs was detected using CellROX ® Green reagent during exposure to 0.2 mM H2O2 for the first 12 h, where an obvious increase in green fluorescence was observed. However, as shown in Supplementary Video 2, in cells with 5 µM 6-SG pretreatment for 6 h, the H2O2-induced green fluorescence accumulation was markedly attenuated. The presentative images were taken 24 h after the indicated treatments ( Figure 3). In conclusion, 6-SG preincubation markedly attenuates H2O2-induced cell death and oxidative stress in melanocytes.

6-SG Activates Intrinsic Antioxidant Defense Response in Human Epidermal Melanocytes
6-SG is known to have its own antioxidant system. The nuclear factor E2-related factor (Nrf2)antioxidant response element (ARE) is the most important pathway in protecting cells from oxidative stress [28,29]. It is a transcription factor that regulates the expression of antioxidant enzymes in response to oxidative stress [30]. The key downstream ARE antioxidant enzymes of Nrf2 include the heme oxygenase 1 (HO-1) and NAD(P)H: quinine oxidoreductase 1 (Nqo1) [29]. Therefore, after the 140 h treatment with 0.1 mM H2O2 with or without 6-SG pretreatment, the level of the antioxidant gene HO-1 ( Figure 4A,B) and Nrf2 ( Figure 4C) in melanocytes was analyzed by real-time PCR. The results demonstrated that the expression levels of HO-1 and Nrf2 were significantly downregulated in H2O2-treated cells ( Figure 4A,C), and the H2O2-induced downregulation of HO-1 and Nrf2 was reversed by 6-SG pretreatment ( Figure 4B,C). Following that, the expression of Nrf2 and HO-1, as well as Nqo1 protein levels, were evaluated using Western blot analyses in HEMn-MPs, after a 140 h treatment with 0.1 mM H2O2 with or without 6-SG preincubation ( Figure 4D). The H2O2 treatment induced a marked decrease in Nrf2, HO-1, and Nqo1 protein expression, whereas 6-SG pretreatment reversed the H2O2-induced a decrease in these proteins ( Figure 4C).

6-SG Activates Intrinsic Antioxidant Defense Response in Human Epidermal Melanocytes
6-SG is known to have its own antioxidant system. The nuclear factor E2-related factor (Nrf2)-antioxidant response element (ARE) is the most important pathway in protecting cells from oxidative stress [28,29]. It is a transcription factor that regulates the expression of antioxidant enzymes in response to oxidative stress [30]. The key downstream ARE antioxidant enzymes of Nrf2 include the heme oxygenase 1 (HO-1) and NAD(P)H: quinine oxidoreductase 1 (Nqo1) [29]. Therefore, after the 140 h treatment with 0.1 mM H 2 O 2 with or without 6-SG pretreatment, the level of the antioxidant gene HO-1 ( Figure 4A,B) and Nrf2 ( Figure 4C) in melanocytes was analyzed by real-time PCR. The results demonstrated that the expression levels of HO-1 and Nrf2 were significantly downregulated in H 2 O 2 -treated cells ( Figure 4A,C), and the H 2 O 2 -induced downregulation of HO-1 and Nrf2 was reversed by 6-SG pretreatment ( Figure 4B,C). Following that, the expression of Nrf2 and HO-1, as well as Nqo1 protein levels, were evaluated using Western blot analyses in HEMn-MPs, after a 140 h treatment with 0.1 mM H 2 O 2 with or without 6-SG preincubation ( Figure 4D). The H 2 O 2 treatment induced a marked decrease in Nrf2, HO-1, and Nqo1 protein expression, whereas 6-SG pretreatment reversed the H 2 O 2 -induced a decrease in these proteins ( Figure 4C).

6-SG Protects Melanocytes Against Rhododendrol-Induced Oxidative Stress
Rhododendrol [4-(4-hydroxyphenyl)-2-butanol, Rhododenol ® ] was previously used as a skin-whitening cosmetic. These cosmetics were withdrawn from the market in 2013 after Rhododendrol reportedly caused a depigmentation disorder [31]. A previous report suggested that the toxicity of Rhododendrol towards melanocytes is caused by its tyrosinase-catalyzed oxidation and the production of cytotoxic reactive oxygen species (ROS) [32][33][34]. In the present study, we investigated whether 6-SG could protect melanocytes against Rhododendrol-induced cytotoxicity ( Figure 5). After pretreating HEMn-MPs for 6 h with 5 µM 6-SG, and after subsequent exposure to 1 mM-3 mM Rhododendrol for 140 h, cell viabilities were assessed by the MTT assay ( Figure 5A), and the gene expression level of tyrosinase was evaluated by real-time PCR analysis ( Figure 5B). We found that 6-SG pretreatment significantly attenuated Rhododendrol-induced cell death in cultured melanocytes ( Figure 5A). The mRNA expression level of tyrosinase significantly decreased in cells which were treated with 1 mM Rhododendrol, compared with control cells, and 6-SG pretreatment significantly blocked the Rhododendrol-induced decrease in tyrosinase expression ( Figure 5B).

6-SG Protects Melanocytes Against Rhododendrol-Induced Oxidative Stress
Rhododendrol [4-(4-hydroxyphenyl)-2-butanol, Rhododenol ® ] was previously used as a skinwhitening cosmetic. These cosmetics were withdrawn from the market in 2013 after Rhododendrol reportedly caused a depigmentation disorder [31]. A previous report suggested that the toxicity of Rhododendrol towards melanocytes is caused by its tyrosinase-catalyzed oxidation and the production of cytotoxic reactive oxygen species (ROS) [32][33][34]. In the present study, we investigated

Discussion
In this study, we demonstrated that 6-SG attenuated H 2 O 2 -induced cell damage in human epidermal melanocytes by activating the Nrf2-ARE pathway. This study discovered a novel protective effect of 6-SG on human melanocytes, suggesting that 6-SG may have potential for the treatment of pigmentary disorder diseases. In the current study, we used H 2 O 2 (Figures 1-4) and Rhododendrol ( Figure 5) injury HEMn-MPs cellular models to study the effects of 6-SG on oxidative stress. We found that pretreatment with 6-SG provided protection to HEMn-MPs from H 2 O 2 or Rhododendrol-induced damage. Consistent with previous reports, we found that H 2 O 2 is cytotoxic to HEMn-MPs in a dose-dependent pattern ( Figure S1A). The cell viability of HEMn-MPs incubated with H 2 O 2 increased significantly after pretreatment with 6-SG. In addition, 6-SG also attenuated the H 2 O 2 -induced oxidative stress in HEMn-MPs, confirming the protective effect of 6-SG against oxidative stress in HEMn-MPs. The Nrf2-ARE pathway, including HO-1 and Nqo1, protects melanocytes and reduces cytotoxicity caused by oxidative stress [35]. In this study, 6-SG was shown to protect human melanocytes from H 2 O 2 -induced oxidative stress by activating the Nrf2-ARE pathway.
Previous studies have shown that H 2 O 2 is closely related to the onset, as well as the progression of vitiligo [6]. A recent study has shown that H 2 O 2 decreases the antioxidant capability in the epidermis of vitiligo patients [36]. In vitiligo, H 2 O 2 was reported to oxidize melanogenesis-related hormones and factors, such as epidermal ACTH, α-MSH, and β-endorphin, resulting in the loss of their functions via the promotion of pigmentation in melanocytes [37]. Furthermore, H 2 O 2 -mediated oxidation is also reported to affect calcium binding, disrupting calcium homeostasis and l-phenylalanine-uptake in the epidermis in acute vitiligo [38]. In addition, impaired Nrf2 signaling, decreased antioxidative enzyme levels, including HO-1, and increased oxidative stress have been reported in patients with vitiligo [39][40][41][42].
Recently, Nrf2, one of the most critical antioxidant enzymatic systems, and its downstream target genes were found to be up-regulated in non-lesional vitiligo skin biopsies, suggesting that a consistently higher Nrf2-dependent transcriptional activity is required for the maintenance of redox homeostasis in disease-free epidermis. Thus, the activation of antioxidative mechanisms is essential for the protection of melanocytes against oxidative stress in patients with vitiligo.
Ginger is a popular, globally used spice and food preservative, that is also considered to be a life-promoting herb. It has been used in traditional medicine to treat various diseases [12,43]. In the last decade, research on the components of ginger has significantly increased. Among its components, 6-SG has displayed numerous pharmacological properties, including antioxidant, anti-inflammatory, anti-neuroinflammatory, anti-cathartic, anti-neoplastic, and hypotensive [12,44,45]. The present study demonstrates for the first time the antioxidant activity of 6-SG via the induction of Nrf2/ARE-mediated antioxidant enzyme HO-1 and Nqo1 pathway in cultured cells.

Reagents
Hydrogen peroxide (H 2 O 2 ) was purchased commercially from WAKO (Osaka, Japan) and diluted with PBS. 6-SG was purchased commercially from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). Rhododendrol was obtained from Kanebo (Tokyo, Japan) and dissolved in DMSO.

Cell Culture
Normal human neonatal epidermal melanocytes from a moderately pigmented donor (HEMn-MP) were purchased from Invitrogen (Thermo Fisher Scientific, Carlsbad, CA, USA), and cultured in Medium 254 (M-254-500; Thermo Fisher Scientific) supplemented with 1% (v/v) human melanocyte growth supplement (Thermo Fisher Scientific) at 37 • C in an atmosphere containing 5% (v/v) CO 2 . The melanocytes were used at passages 6-8. Cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well 12 h before treatment. Cells were treated with H 2 O 2 at 0.1 mM and 0.2 mM (final concentration), 6-SG at 5 µM (final concentration), or Rhododendrol at 1 mM, 1.5 mM and 3 mM (final concentration) for certain periods prior to extraction of RNA and protein. Because oxidative stress has been well known to trigger an endogenous antioxidant defense system at the early stage (2 h-72 h) by itself [46], we evaluated the antioxidative ability of our agents at a later stage after a longer treatment time (140 h).

Cell Viability Assay
HEMn-MPs (1 × 10 4 cells/well) were cultured in 96-well flat-bottom tissue culture plates. After experimental treatments, cells were washed three times with cold PBS, and cell viability was evaluated using a Cell Count Reagent SF colorimetric assay (Nacalai Tesque, Kyoto, Japan). Briefly, 10 µL of Cell Count Reagent SF was added to each well, and cells were incubated for 2 h at 37 • C. Cell viability was determined colorimetrically by measuring OD 450 with a microplate reader (Model 550: Bio-Rad Laboratories, Hercules, CA, USA). The percentage of viable cells was calculated as follows: percentage viable cells = T/C × 100, where T is the mean OD 450 of the treated group, and C is the absorbance of the control group.

Melanin Content Assay
To determine melanin content, cells were dissolved in 200 µL of 1 N NaOH for 30 min at 100ºC to solubilize the melanin, which was then quantified in cell suspensions by recording the absorbance at 405 nm as described previously [47]. Melanin content was calculated and corrected based on cell number.

Oxidative Stress Assessment
For cultured human primary epidermal melanocytes, oxidative stress was detected by live imaging using CellROX ® Green Reagent (#C10444, Thermo Fisher Scientific Inc., MA, USA). Cells were treated with 5 µM CellROX ® Green Reagent for 30 min and then washed with PBS twice, applied with the indicated treatments, and then subjected to live cell imaging by phase contrast and confocal fluorescence microscopy (Keyence Biozero confocal microscope: Keyence Co., Osaka, Japan).

Statistical Analysis
The experiments were repeated at least three times. Data are presented as mean ± SD. Statistical analysis was conducted using two-way analysis of variance for interactions between variables. Unpaired Student's t-test (Microsoft Excel: Microsoft Corp., Redmond, WA, USA) was used for comparisons between two groups. p-values < 0.05 were considered statistically significant.

Conclusions
In conclusion, our results show that 6-SG pretreatment protected melanocytes against H 2 O 2 -induced oxidative stress via activating the Nrf2 pathway and induction of Nrf2 and HO-1 expression. This study improves our understanding of the molecular mechanism of the beneficial effect of ginger in the treatment of vitiligo patients. These findings demonstrate that 6-SG has potential for use as an antioxidant agent in the prevention and treatment of vitiligo or other oxidative stress-associated pigmentary disorders.