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
]. 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
]. Recently, oxidative stress was shown to play an important role in the development and progression of vitiligo [5
]. In active vitiligo, high levels of oxidative stress and low levels of enzymatic and non-enzymatic antioxidants were revealed in the blood and skin from patients [7
]. Oxidative stress has been discussed as a promising target for vitiligo drug development.
Thus, approaches and treatments using antioxidants to reduce or reverse the oxidative damage in the epidermis and even to achieve re-pigmentation are being studied. With the development of phytoextraction and medicinal chemistry technology, an increasing number of studies on organic antioxidant compounds are being carried out in order to improve the treatment of oxidative stress-associated skin diseases.
Ginger is one of the most widely known spices and a natural antioxidant [11
]. It has been used as a traditional medicinal herb for thousands of years to treat many gastrointestinal, stomachic, and rheumatic disorders [12
]. Recently, ginger root extract was reported to have a neuroprotective ability in β-amyloid-induced Alzheimer’s disease [13
]. Due to its strong antioxidant and neuroprotective effects, ginger is considered to be a promising product for fighting the effects of aging and neurodegenerative diseases [13
]. Melanocytes are melanin-producing cells of the skin that are derived from neural crest cells, and in a previous study, in vitro characterization of epidermal melanocyte cultures from vitiligo revealed common features with neurodegenerative diseases [16
]. Ginger powder in Ayurvedic medicine [17
], and ginger piece erasure in traditional Chinese medicine [18
] have proven effective for the treatment of vitiligo.
Several bioactive compounds have been identified in ginger, including 6-gingerol, 8-gingerol, 10-gingerol, and 6-Shogaol (6-SG) [12
]. Among these ginger compounds, 6-gingerol, the most abundant bioactive compound in ginger, has been extensively studied for its various pharmacological effects including anti-inflammatory, analgesic, antipyretic, chemopreventive, and antioxidant properties [20
]. Interestingly, recent studies have demonstrated that 6-SG exhibited the most potent antioxidant and anti-inflammatory properties [23
]. Given the above, we focused our attention on the properties of 6-SG. The purpose of our study was to investigate whether 6-SG protects human melanocytes from oxidative stress, and to elucidate the underlying molecular mechanism of this protective effect. In the present study, we used H2
-induced and Rhododendrol-induced oxidative stress in the normal human primary epidermal melanocytes as an in vitro model.
In this study, we demonstrated that 6-SG attenuated H2
-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 H2
, Figure 2
, Figure 3
and Figure 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 H2
or Rhododendrol-induced damage. Consistent with previous reports, we found that H2
is cytotoxic to HEMn-MPs in a dose-dependent pattern (Figure S1A
). The cell viability of HEMn-MPs incubated with H2
increased significantly after pretreatment with 6-SG. In addition, 6-SG also attenuated the H2
-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 H2
-induced oxidative stress by activating the Nrf2-ARE pathway.
Previous studies have shown that H2
is closely related to the onset, as well as the progression of vitiligo [6
]. A recent study has shown that H2
decreases the antioxidant capability in the epidermis of vitiligo patients [36
]. In vitiligo, H2
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, H2
-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
]. 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
]. 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
]. 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.
4. Materials and Methods
Hydrogen peroxide (H2O2) 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.
4.2. 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
. The melanocytes were used at passages 6–8. Cells were seeded into 6-well plates at a density of 5 × 105
cells/well 12 h before treatment. Cells were treated with H2
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).
4.3. Cell Viability Assay
HEMn-MPs (1 × 104 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 OD450 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 OD450 of the treated group, and C is the absorbance of the control group.
4.4. 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.
4.5. RNA Isolation and Real-Time RT-PCR Analysis
Total RNA from cell pellets was isolated using the Maxwell® 16 LEV simplyRNA Tissue Kit (Promega, Madison, WI, USA) following manufacturer’s instructions. The integrity of the RNA was verified by gel electrophoresis. Total RNA (100 ng) was reverse-transcribed into first-strand cDNA (ReverTra Ace® qPCR RT Master Mix, TOYOBO, Osaka, Japan). The primers used for real-time PCR were as follows: Tyrosinase, 5′-TGACTCCAATTAGCCAGTTCCT-3′ (sense) and 5′-GACAGCATTCCTTCTCCATCAG-3′ (antisense); HO-1, 5′-CGAGCATAAATGTGACCGGC-3′ (sense) and 5′-CTCTGACAAATCCTGGGGCA-3′ (antisense); and GAPDH, 5′GACAGTCAGCCGCATCTTCT-3′ (sense) and 5′-GCGCCCAATACGACCAAATC-3′ (antisense). Each reaction was performed in triplicate.
4.6. Western Blot Analysis
Proteins from cell pellets were extracted, and 5 μg of extracted proteins was used for Western blotting analysis as described previously [48
]. The following primary antibodies were used at a concentration of 1:1000: anti-Nrf2 (#12721, Cell Signaling Technology, Beverly, MA, USA), anti-Nqo1 (#3187, Cell Signaling Technology), anti-HO-1 (#70081, Cell Signaling Technology), and anti-GAPDH (#2118, Cell Signaling Technology). GAPDH was used as a loading control.
4.7. 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).
4.8. 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.