Molecular Hydrogen Protects Human Melanocytes from Oxidative Stress by Activating Nrf2 Signaling

Journal Pre-proof Molecular Hydrogen Protects Human Melanocytes from Oxidative Stress by Activating Nrf2 Signaling Wei Fang, Luyan Tang, Guizhen Wang, Jinran Lin, Wanqing Liao, Weihua Pan, Jinhua Xu PII:

S0022-202X(20)31206-9

DOI:

https://doi.org/10.1016/j.jid.2019.03.1165

Reference:

JID 2374

To appear in:

The Journal of Investigative Dermatology

Received Date: 11 October 2018 Revised Date:

28 February 2019

Accepted Date: 6 March 2019

Please cite this article as: Fang W, Tang L, Wang G, Lin J, Liao W, Pan W, Xu J, Molecular Hydrogen Protects Human Melanocytes from Oxidative Stress by Activating Nrf2 Signaling, The Journal of Investigative Dermatology (2020), doi: https://doi.org/10.1016/j.jid.2019.03.1165. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

Molecular Hydrogen Protects Human Melanocytes from Oxidative Stress by Activating Nrf2 Signaling Wei Fang1,2,3¶, Luyan Tang1,2¶, Guizhen Wang4 , Jinran Lin1 , Wanqing Liao3 , Weihua Pan3* , Jinhua Xu1,2* 1 Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, China 2

The Shanghai Institute of Dermatology, Shanghai, China

3

Shanghai Key Laboratory of Molecular Medical Mycology, Department of

Dermatology, Changzheng Hospital, Shanghai, China 4

Emergency room, Shanghai Tenth People’s Hospital of Tongji University, Shanghai,

China

¶

These authors contributed equally to this work.

Running title: H2 protects melanocytes against oxidative stress

Corresponding footnote *Corresponding authors 1

Jinhua Xu

Department of Dermatology, Huashan Hospital, Fudan University, NO.12, Middle Urumqi Road, Shanghai, China 200040; The Shanghai Institute of Dermatology, Shanghai, China 200040 Tele: 86-(21)-52887781 Email: [email protected] 2

Weihua Pan

Shanghai Key Laboratory of Molecular Medical Mycology, Department of Dermatology, Changzheng Hospital, Shanghai, China 200003 Tele: 86-(21)-52887781 Email: [email protected]

Molecular Hydrogen Protects Human Melanocytes from Oxidative Stress by Activating Nrf2 Signaling

ABSTRACT Oxidative stress is proven to be critical for the initiation and progression of vitiligo. Molecular hydrogen (H2 ) possesses potent antioxidant activity and has been shown to protect against various oxidative stress-related diseases. In this study, we first investigated the effects and mechanisms of H2 in human melanocytes damaged by hydrogen peroxide (H2 O2 ). We initially found that H2 reduced intracellular reactive oxygen species (ROS) accumulation and malondialdehyde (MDA) levels in both vitiligo specimens and H2 O 2 -treated melanocytes in vitro in a concentration- and time-dependent manner, concomitant with the enhancement of antioxidant enzyme activity. Correspondingly, H2 reversed H2 O2 -induced apoptosis and dysfunction in both normal and vitiligo melanocytes. H2 protected mitochondrial morphology and function in melanocytes under stress and promoted the activation of nuclear erythroid 2-related factor (Nrf2) signaling, while Nrf2 deficiency abolished the protective effect of H2 against H2 O2-induced oxidative damage. Furthermore, H2 positively modulated β-catenin in H2 O 2 -treated melanocytes, and the β-catenin pathway was implicated in H2 -induced Nrf2 activation. Collectively, our results indicate that H2 could be a promising therapeutic agent for vitiligo treatment via attenuating oxidative damage, and its beneficial effect in human melanocytes might involve Wnt/β-catenin- mediated activation of Nrf2 signaling.

KEY WORDS

Hydrogen, vitiligo, melanocyte, oxidative stress, mitochondria, apoptosis, Nrf2 signaling, Wnt/β-catenin signaling.

INTRODUCTION

Vitiligo is an acquired chronic depigmenting disorder caused by the selective destruction of epidermal melanocytes and has a global prevalence of 0.5% to 2.0% (Picardo et al., 2015). Accumulating evidence suggests that oxidative stress is crucial for the onset and progression of vitiligo (Jian et al., 2014.; Li et al., 2016). Vitiligo patients have reduced oxidoreductase levels and activity and high hydrogen peroxide (H2 O 2 ) and peroxynitrite levels in their epidermis (Schallreuter et al., 1991, 1999,

2012). The redox imbalance is closely associated with the disruption of mitochondrial homeostasis, characterized by ultrastructure alterations and functional impairments of mitochondria in vitiligo (Dell'Anna et al., 2003; Prignano et al., 2009; Ding et al., 2015; Sahoo et al., 2017), which results in the collapse of mitochondrial membrane potential (Δψm), reduced ATP generation, and increased reactive oxygen species (ROS) production (Sahoo et al., 2017). Chronic high ROS levels can directly damage lipids and proteins, leading to functional impairment or cell death of melanocytes (Denat et al., 2014). Furthermore, oxidative stress can indirectly compromise the survival and function of melanocytes by regulating keratinocyte-derived chemokines and growth factors (Lee et al., 2005; Li et al., 2016; Richmond et al., 2017). Therefore, antioxidant treatment has become a promising therapeutic option for vitiligo. In the past decade, hydrogen (H2 ) has emerged as a novel therapeutic medical gas. In 2007, Ohsawa et al. first reported that H2 inhalation markedly suppressed brain ischemia/reperfusion injury by reducing cytotoxic oxygen radicals (Ohsawa et al., 2007). Thereafter, similar protective effects of hydrogen were further demonstrated in diverse animal models and human diseases, such as myocardial infarction, diabetes, and skin diseases (Hayashida et al., 2008; Kamimura et al., 2011; Liu et al., 2011; Zhu et al., 2018). The therapeutic effect of H2 might be primarily attributed to its antioxidant activity. H2 selectively neutralizes hydroxyl radicals and peroxynitrite (Ohsawa et al., 2007; Ge et al., 2017). Furthermore, H2 can effectively penetrate target tissue and cells by gaseous diffusion but does not impact physiological parameters, such as pH and oxygen saturation (Abraini et al., 1994; Ono et al., 2012). Due to its potent antioxidant activity and biosafety, H2 might be a potential therapeutic approach for vitiligo. Nuclear erythroid 2-related factor (Nrf2) is a master regulator governing redox homeostasis in mammalian cells (Osburn and Kensler, 2008). Previous studies reported that the activation of Nrf2 was impaired and the activities of antioxidant enzymes were reduced in vitiligo patients, while Nrf2 overexpression afforded protection to melanocytes against oxidative stress (Natarajan et al., 2010; Jian et al., 2011, 2014). Nrf2 signaling has thus become a promising target of potential therapeutic agents for vitiligo (Jian et al., 2016; Chang et al., 2017). In addition, the antioxidant property of H2 is closely associated with the activation of Nrf2 signaling (Ge et al., 2017; Fang et al., 2018). Thus, this study aimed to investigate the hypothesis that H2 might protect melanocytes from oxidative stress-induced damage by activating Nrf2 signaling.

RESULTS

H2 reduced oxidative damage in cutaneous cells isolated from vitiligo patients. We first assessed the effect of H2 on the oxidation levels of cutaneous cells isolated from 3 patients with progressive nonsegmental vitiligo and 3 healthy control subjects. Compared with the healthy group, the vitiligo group displayed a significant increase in the levels of ROS and malondialdehyde (MDA, lipid peroxidation) (Fig. 1a&1b and Fig. S1). Treatment with 35% H2 for 4 hours significantly reduced both the ROS and MDA levels in the vitiligo group but not in the control group (Fig. 1a&1b). These data suggest that H2 can reduce oxidative stress in skin cells of vitiligo patients. H2 evoked antioxidative effects on H2 O2 -treated melanocytes and ke ratinocytes in a concentration-dependent and time-dependent manner. We further evaluated the antioxidative effect of H2 in skin cells using human melanocyte (PIG1 and PIG3V) and keratinocyte (HaCaT) cell lines. Different concentrations (2%, 35%, and 75%) of H2 had no obvious effects on the proliferation of these cell lines (Fig. S2), suggesting that H2 is nontoxic for human skin cells. Pre-incubation with 35% and 75% H2 for 24 h markedly reduced the ROS and MDA levels induced by H2 O2 in melanocytes and keratinocytes (Fig. 1c&1e and Fig. S3). We next examined the effect of the pretreatment time with 75% H2 on the antioxidative effect of H2 and found that at least 4 h of pre-incubation was required to inhibit H2 O2 -induced ROS and MDA levels in skin cells, and stronger protection was observed with extended pretreatment time (Fig. 1d&1f and Fig. S4). Thus, a moderate-high concentration of H2 evoked good protection in melanocytes and keratinocytes against oxidative damage, and its antioxidative effect was positively correlated with its concentration and pretreatment time. We next evaluated the effects of H2 on major antioxidant activities or content in H2 O 2-treated skin cells. After exposure to 1 mM H2 O 2 , superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were significantly inhibited in human melanocytes and keratinocytes (Fig. S5a-c). Meanwhile, H2 O 2-treated cells also displayed a significant reduction in the GSH/GSSG ratio, which is an indicator of oxidative stress (Fig. S5d-f). Pretreatment with 75% H2 for 24 h abolished the H2 O2-induced inhibition of antioxidant enzyme activities and the reduced GSH/GSSG ratio (Fig. S5). Therefore, H2 relieved the damage to the

intracellular antioxidant system in melanocytes and keratinocytes caused by H2 O2 . H2 attenuated H2 O2 -induced destruction and dysfunction of melanocytes and keratinocytes. We next investigated whether H2 could attenuate the effects of H2 O2 on the survival and function of both melanocytes and keratinocytes. H2 O 2 exposure substantially increased the apoptosis rates and inhibited the proliferation of PIG1, PIG3V, and HaCaT cells, while pre- incubation with 75% H2 significantly promoted cell survival and proliferation under oxidative stress (Fig. 2a-2c). Additionally, H2 pretreatment prevented the H2 O2 -induced inhibition of cellular migration and melanin production in both PIG1 and PIG3V cells (Fig. 2d&2e and Fig. S6). The microenvironment of melanocytes and keratinocytes can indirectly regulate the survival and function of melanocytes through secretion of various chemokines and growth factors during the pathogenesis of vitiligo (Lee et al., 2005; Li et al., 2016; Richmond et al., 2017). As expected, H2 O2 treatment led to a drastic upregulation of the chemokines CXCL9, CXCL10, and CXCL16 and decreased the production of stem cell factors (SCF) in keratinocytes (Fig. 2f&2g). However, these detrimental effects of H2 O2 were significantly alleviated by H2 pretreatment (Fig. 2f&2g). Overall, H2 reversed the H2 O2 -derived damage affecting the survival and function of both melanocytes and keratinocytes. H2 ameliorated H2 O2 -induced mitochondrial damage in melanocytes. Mitochondria are the main instigators and the victims of ROS (Raimundo, 2014), and their dysfunction mediates the melanocyte apoptosis induced by oxidative stress. To investigate whether H2 can attenuate the mitochondrial damage caused by H2 O2 , mitochondrial membrane potential (Δψm) and cellular adenosine-triphosphate (ATP) levels were next examined. The control cells showed bright red fluorescence, whereas H2 O 2-treated cells (especially PIG3V cells) showed remarkably enhanced green fluorescence (Fig. 3a&3b), reflecting the collapse of the Δψm. H2 pretreatment significantly ameliorated the H2 O2-induced Δψm collapse (Fig. 3a&3b). In addition, H2 reversed the inhibition of cellular ATP accumulation in H2 O2-treated melanocytes (Fig. 3c). Furthermore, Mito-Tracker Green staining suggested that H2 O 2 exposure significantly reduced mitochondrial content in both PIG1 and PIG3V cells, and H2 pretreatment partially restored the cell mitochondrial content (Fig. 3d and Fig. S7). Cytochrome c and cytochrome b are crucial components of the electron transport chain complexes, whose expression reflects mitochondrial health and function in H2 O 2-treated cells. Quantitative PCR and immunoblotting analyses revealed that H2 O2

exposure caused a notable reduction in the expression of cytochrome c /b in both PIG1 and PIG3V cells, while H2 pretreatment significantly attenuated H2 O2-induced downregulation of these mitochondrial proteins (Fig. 3e&3f and Fig. S8). However, H2 alone did not show any obvious effect on the Δψm, cellular ATP level, mitochondrial content, or mitochondrial protein expression (Fig. 3, Fig. S7, and Fig. S8). Together, the results suggest that H2 might act as a novel mitochondrial protector to enhance the antioxidative capacity of human melanocytes. H2 promoted the activation of the Nrf2/ARE pathway in H2 O2 -treated melanocytes and keratinocytes. We next evaluated the effect of H2 on the Nrf2/ARE pathway in cutaneous cells under

oxidative

stress.

Using

quantitative

PCR,

immunoblotting,

and

immunofluorescence staining, we found that H2 O2 significantly inhibited the expression of Nrf2 in melanocytes and keratinocytes, consistent with previous studies (Fig. 4a and Fig. S9a) (Natarajan et al., 2010; Jian et al., 2014). H2 at 75% dramatically upregulated the expression of cytosolic and nuclear Nrf2 (Fig. 4a) and remarkably promoted its nuclear translocation in H2 O 2 -treated melanocytes (Fig. S9b). Consistently, H2 treatment notably enhanced the expression of the main target genes of Nrf2 (such as heme oxygenase-1 (HO-1) and NADH quinone oxidoreductase 1 (NQO1)) in H2 O 2 -treated cells (Fig. 4a). Thus, H2 might exert its antioxidative effect in melanocytes by promoting the activation of the Nrf2/ARE pathway. Nrf2 knockdown blocked the protection provided by H2 against oxidative damage in melanocytes. To determine whether Nrf2 is required for the protective effects of H2 against oxidative stress in melanocytes, we silenced Nrf2 expression in PIG1 cells with short interfering RNA (siRNA) (Fig. S10) as previously described (Jian et al., 2016). Nrf2 deficiency markedly exacerbated the H2 O2-induced oxidative damage that hindered the survival and function of melanocytes (Fig. 4b-4h and Fig. S11). The H2 -induced reduction in intracellular ROS and MDA levels (Fig. 4b&4c and Fig. S12), inhibition of apoptosis (Fig. 4e&4f), and increase in cell viability (Fig. 4d), migration (Fig. 4g and Fig. S11), and melanin production (Fig. 4h) in H2 O 2 -treated melanocytes were abrogated by Nrf2 silencing. These results suggest that Nrf2 activation is essential for

the antioxidative capacity of H2 in melanocytes. Nrf2 silencing abolis hed H2 -mediated protection of mitochondrial function and content against oxidative stress in human melanocytes. Nrf2 signaling is essential for preserving mitochondrial function against oxidative stress (Kwon et al., 2012; Strom et al., 2016). To determine whether Nrf2 is essential for the protective effects of H2 against H2 O2 -induced mitochondrial damage, mitochondrial content and function were assessed in Nrf2-silenced human melanocytes. Nrf2 deficiency abrogated the maintenance of mitochondrial Δψm (Fig. 5a&5b), cellular ATP accumulation (Fig. 5c), and mitochondrial content (Fig. 5d and Fig. S13) in melanocytes mediated by H2 upon H2 O2 exposure. Meanwhile, Nrf2 silencing also abolished the upregulation of cytochrome c/b induced by H2 pretreatment (Fig. 5e&5f and Fig. S14). Furthermore, Nrf2 deficiency remarkably inhibited the expression of cytochrome c/b in melanocytes even without H2 O2 exposure (Fig. 5e&5f and Fig. S14). These results suggest that Nrf2 activation is essential for mitochondrial homeostasis and the antioxidative capacity of H2 in melanocytes. β-catenin activation was required for Nrf2 regulation in H2 O2 -treated melanocytes and keratinocytes induced by H2 . Canonical Wnt/β-catenin signaling is essential for melanocyte differentiation and melanogenesis in vitiligo and has complex interactions with Nrf2 signaling in several cell types and disease models (Manigandan et al., 2015; Rada et al., 2015; Regazzetti et al., 2015). Thus, we also examined the alterations in the main components of the Wnt/β-catenin pathway in H2 -treated melanocytes and keratinocytes. H2 abolished the H2 O 2-induced downregulation of β-catenin, LEF1, and CDH3 at the transcriptional level (Fig. 6a). The elevation in the β-catenin protein level and the nuclear/cytosolic ratio indicated that β-catenin was activated in H2 -treated melanocytes under oxidative stress (Fig. 6b). Intriguingly, these alterations were not observed in H2 -treated cells without H2 O2 exposure (Fig. 6a&6b). Pretreatment with the β-catenin inhibitor ICG001 (5 μmol/L) markedly impaired Nrf2 expression and nuclear translocation, while Nrf2 silencing had no effect on β-catenin expression or H2 -induced β-catenin

upregulation in melanocytes under oxidative stress (Fig. 6c-6e). Therefore, β-catenin signaling might act as an important upstream regulator of Nrf2 activation in H2 -treated melanocytes.

DISCUSSION

Here, we described molecular hydrogen functioning as a quencher of oxidative stress to maintain mitochondrial homeostasis in human melanocytes through Nrf2 activation. H2 was able to ameliorate oxidative damage in both vitiligo specimens and H2 O 2-treated melanocytes in vitro. The antioxidant action of H2 might be closely associated with its protective roles in maintaining mitochondrial content and function in melanocytes under oxidative stress. Mechanistically, we showed that H2 protected melanocytes against oxidative stress by activating Nrf2 signaling, and the Wnt/β-catenin pathway might be required for H2 -induced Nrf2 activation. These results suggest that H2 might be a promising antioxidative agent for vitiligo treatment. Oxidative stress is critical for the onset and progression of vitiligo, and several antioxidants (such as Ginkgo biloba and simvastatin) have great potential to be developed into novel therapeutic strategies against vitiligo (Parsad et al., 2003; Chang et al., 2017). Previous studies have shown that molecular hydrogen can protect a variety of organs (including skin) against ischemia/reperfusion injury by selectively neutralizing cytotoxic oxygen radicals (Ohsawa et al., 2007; Liu et al., 2010; Fang et al., 2018). In this study, we showed that H2 significantly reduced the intracellular levels of ROS and MDA in both vitiligo specimens and H2 O2 -treated melanocytes and keratinocytes in vitro in a concentration- and time-dependent manner. H2 abolished the H2 O 2-mediated induction of apoptosis, inhibition of migration and melanogenesis, and reduction in antioxidant enzyme activity in melanocytes. Furthermore, H2 also corrected the abnormal expression of keratinocyte-derived chemokines and SCF induced by H2 O 2 , preventing subsequent T cell positioning and promoting melanocyte homeostasis during vitiligo. These data indicate that H2 possesses therapeutic potential for vitiligo. The antioxidant property of H2 was closely associated with the recovery of mitochondrial injury in H2 O2 -treated melanocytes. Mitochondria are the cellular power plants and a major source of intracellular ROS. Exorbitant ROS accumulation, in turn, induces mitochondrial stress and malfunction, which results in Δψm collapse, decreased ATP output, aggravated ROS production, and even cellular apoptosis (Echtay, 2007; Denat et al., 2014; Raimundo, 2014). Melanocytes from perilesional vitiligo skin contain fewer mitochondria with reduced cristae density and vacuolization, indicative of severe damage to mitochondrial morphology and function (Prignano et al., 2009; Ding et al., 2015). In 2017, Sahoo et al. revealed aberrant

assembly of electron transport complexes and inefficient respiration in vitiligo melanocytes, further underscoring the significance of mitochondrial malfunction in vitiligo pathogenesis (Sahoo et al., 2017). Consistently, oxidative stress caused Δψm depolarization, decreased ATP output, and aberrant mitochondrial content in human melanocytes, and these effects were closely associated with H2 O2-induced downregulation of cytochrome b/c. H2 treatment remarkably upregulated cytochrome b/c expression and thus promoted recovery of mitochondrial function and content in melanocytes. Therefore, H2 might act as a novel protector of mitochondria, enhancing the tolerance of human melanocytes to oxidative stress. Nrf2 signaling is essential for the adaptive responses of cells to exogenous and endogenous oxidative stresses (Jaramillo and Zhang, 2013). Nuclear translocation and transcriptional activity of Nrf2 have been shown to be reduced in vitiligo melanocytes (Jian et al., 2011, 2014), while Nrf2 activation is required for the protective effects of several agents (such as aspirin and simvastatin) against vitiligo (Jian et al., 2016; Chang et al., 2017). Our results showed that H2 pretreatment significantly prevented the downregulation and inactivation of Nrf2 in H2 O 2 -exposed melanocytes and keratinocytes. Meanwhile, H2 inhibited H2 O2-induced ROS accumulation and MDA elevation in melanocytes and keratinocytes and ameliorated the effects of oxidative stress on the proliferation, apoptosis, migration, and melanogenesis of melanocytes. The protective effects of H2 were abolished by Nrf2 silencing, indicating that Nrf2 signaling was essential for the survival and function of melanocytes under oxidative stress. Our findings are highly consistent with previous reports showing that Nrf2 signaling is required for the protective effect of H2 in various cells and tissues in response to different stressors (Kawamura et al., 2013; Yu et al., 2015; Tamaki et al., 2016; Murakami et al., 2017). We previously showed that H2 could suppress the formation of pressure ulcers due to cutaneous ischemia/reperfusion by activating Nrf2 expression (Fang et al., 2018). Therefore, the Nrf2/ARE pathway might be a critical component mediating the therapeutic effects of H2 in various oxidative stress-related diseases. Multiple lines of evidence also support the role of the Nrf2 pathway in mitochondrial integrity and homeostasis of various mammalian cells, such as cardiomyocytes and pulmonary epithelia (Piantadosi et al., 2008; Athale et al., 2012). Nrf2 overexpression preserved

mitochondrial morp hology and

function of

cardiomyocytes following H2 O2 exposure (Strom et al., 2016). Our results showed that Nrf2 deficiency in human melanocytes significantly enhanced mitochondrial susceptibility to oxidative stress and notably decreased the expression of cytochrome

b/c. Furthermore, Nrf2 silencing completely abolished the ability of H2 to protect mitochondrial function and content in melanocytes following H2 O 2 exposure. Previous studies reported that Nrf2 was able to maintain mitochondrial function and promote the removal of damaged mitochondrial units in multiple internal organs by induction of the downstream HO-1 and inhibition of NAD(P)H oxidase expression (Slebos et al., 2007; Bolisetty et al., 2013). H2 might maintain mitochondrial homeostasis against oxidative stress by activating the Nrf2/ARE pathway. The canonical Wnt/β-catenin pathway is crucial in regulating cell proliferation, differentiation and apoptosis. UVB was shown to induce differentiation of melanocyte stem cell via upregulating Wnt7a and promoting nuclear translocation of -catenin, and β-catenin activation was required for vitamin D to protect melanocytes against oxidative damage (Yamada et al., 2013; Tang et al., 2018). Major components of the Wnt/-catenin pathway, including LEF1, CDH2, and CDH3, were downregulated in vitiligo lesions compared with control skin (Regazzetti et al., 2015). In this study, we found that H2 antagonized the reduction in the β-catenin level in melanocytes induced by H2 O 2 . It has been reported that H2 suppresses abnormal β-catenin activation against cartilage degradation (Lin et al., 2016). Thus, H2 might regulate the WNT/β-catenin pathway differently in different tissues and/or cell types to protect against various diseases. Moreover, we found that a pharmacological inhibitor of β-catenin abolished the activation of Nrf2 induced by H2 in H2 O2-treated melanocytes, while Nrf2 silencing had no effect on the activity and expression of β-catenin. Previous studies reported that WNT-3A modulates hepatocyte metabolism by activating Nrf2 in a β-catenin- independent manner (Rada et al., 2015), while Nrf2 pathway disruption drove neurogenic impairment in parkinsonian mice via Wnt/β-catenin dysregulation (L'Episcopo et al., 2013). Therefore, Wnt/β-catenin signaling might have complex interactions with Nrf2 signaling depending on the cell type and disease model. In melanocytes, the reduction in the β-catenin protein level upon oxidative stress was relieved by H2 . Meanwhile, the activation of Nrf2 induced by H2 in oxidatively stressed melanocytes was abolished by the Wnt/β-catenin inhibitor ICG001, indicating that Wnt/β-catenin might act upstream of Nrf2 to partially mediate the antioxidant activity of H2 in melanocytes. In summary, our findings showed that H2 might act as an ROS quencher to attenuate oxidative stress and maintain mitochondrial homeostasis in human melanocytes, and this activity might involve activation of the β-catenin/Nrf2 axis.

Thus, molecular hydrogen might represent a promising new therapeutic agent for vitiligo. The main weakness of this study is that a single melanocyte line from a vitiligo patient was utilized to evaluate the effects of hydrogen. More experimental (including clinical melanocyte lines and animal models) and clinical studies are required to elucidate the exact roles and mechanisms of hydrogen in the treatment of vitiligo patients.

MATERIALS & METHODS

Specimens from patients and controls After obtaining informed written consent, full- thickness skin biopsies were taken from the perilesional skin of three patients with active nonsegmental vitiligo. The study was approved by the local ethics committee of Huashan Hospital and was performed in strict compliance with the principles of the Declaration of Helsinki. Cell culture and treatment Primary epidermal cells extracted from a clinical specimen and immortalized human epidermal melanocyte cell lines (PIG1, normal melanocytes; PIG3V, vitiligo melanocytes) were cultured in Medium 254 (Cascade Biologics/Invitrogen, Portland, OR) as previously described (Chang et al., 2017). A human keratinocyte cell line (HaCaT from Cells Center of Shanghai Institutes for Biological Sciences ) was cultured in defined keratinocyte serum- free medium (Gibco, Grand Island, NY). The above cell lines were incubated in a 2%, 35%, or 75% H2 incubator for 2-24 hours and then treated with 1 mM H2 O 2 (Sigma) for 12 hours. Measurement of intracellular ROS Intracellular ROS levels were determined using the fluorescent probe H2DCFDA (Thermo Fisher, Waltham, MA) according to the manufacturer’s protocol. Biochemical assays MDA, GSH, GSSG, and the activities of SOD, catalase, and GPx were detected

using commercially available assay kits (BioVision, Milpitas, CA). Cellular ATP levels (JianCheng Bioengineering Institute, NanJing, Jiangsu, China) were assayed according to the manufacturer’s instructions. Human CXCL9, CXCL10, and CXCL16 Quantikine ELISA kits (R&D Systems, Minneapolis, MN) and an SCF ELISA kit (Elabscience Biotechnology, Wuhan, China) were used to analyze cell supernatant samples according to the manufacturer’s instructions. Determination of cell viability by CCK8 assay Cell viability was measured using the CCK8 assay (Cell Counting Kit-8; Beyotime Biotechnology, Shanghai, China) following the manufacturer ’s instructions. Apoptosis detection by Annexin V-FITC Cell apoptosis was assessed using the Annexin V-FITC Apoptosis Detection Kit (MaiBio, Shanghai, China). Transwell migration assay and melanin content measurement Cell migration was detected in transwell cell culture chambers (Costar 3422; Cambridge, MA, USA) as previously described (Tang et al., 2018). Melanin content was measured as previously described (Shi et al., 2016). Quantitative real-time PCR and immunoblotting Quantitative real-time PCR and immunoblotting (Supplementary Materials and Supplementary Table 1&2) were carried out as previously described (Tang et al., 2018). Laser scanning confocal microscopy Alterations in mitochondrial membrane potential were determined using the JC-1 probe (Abcam, Cambridge, UK). For fluorescence staining of mitochondria, melanocytes were stained with Mito- Tracker Green (Beyotime Biotechnology, Shanghai, China). Subcellular localization analysis of Nrf2 was performed as previously described (Jian et al., 2014).

RNA interference Nrf2 expression was silenced in PIG1 cells with short interfering RNA (siRNA) as previously described (Jian et al., 2016). Statistical analyses All statistical analyses were performed using GraphPad Prism version 5.0 software (GraphPad Software, San Diego, CA). Dual comparisons were performed with a two-tailed Student’s unpaired t test. P values < 0.05 were considered statistically significant.

Data Availability Statement

Datasets

related

to

this

article

can

be

found

at http://dx.doi.org/10.17632/pgtpvhf3wh.2, an open-source online data repository hosted at Mendeley Data (Wei Fang, 2019).

Conflict of Interest Statement

The authors state no conflict of interest.

Funding

This study was supported by a grant from Shanghai Key Medical Discipline for Dermatology (2017ZZ02002), a Shanghai leading talent project grant, and the National Natural Science Foundation of China (31770161).

Acknowledgments

We sincerely thank Dr. Caroline Le Poole (Loyola University Chicago, Maywood, IL, US) for the providing human melanocyte cell lines PIG1 and PIG3V, and we thank Dr. Chunying Li (Xijing Hospital, Fourth Military Medical University,

Xi’an, Shaanxi, China) for helping with cell transference. We extend our sincerest gratitude to the staff of Shanghai Asclepius Meditec Co., Ltd. for their vital technical support in using the hydrogen-oxygen nebulizer device. We also thank Prof. Zhimin Kang of Shanghai Huikang hydrogen medical research center for technical assistance in study design.

CRediT Statement

Conceptualization: WF, WL, WP, JX; Formal Analysis: GW; Resources: JL; Funding Acquisition: WP, JX; Investigation: WF, LT; Supervision: JX, WP; Writing: WF, WP, JX.

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Figure Legends Figure 1. Effects of H2 on intracellular ROS and MDA levels in human cutaneous cells. Intracellular ROS assay (a) and MDA assay (b) in cutaneous cells isolated from the perilesional skin of three vitiligo patients. These cells were then cultured in a 35% H2 incubator for 4 h. Intracellular ROS assay (c&d) and MDA assay (e&f) in melanocytes and keratinocytes following H2 O2 challenge. PIG1, PIG3V, and HaCaT cells were incubated in the presence or absence of H2 (2%-75%) for the indicated times and then incubated in the presence or absence of 1 mM H2 O2 for 12 h. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the control group; # P < 0.05, ## P < 0.01, and ### P < 0.001 compared with the H2 O2 group (without H2 pretreatment). Figure 2. H2 attenuate d H2 O2 -induced degeneration and dysfunction in human melanocytes and keratinocytes. The cells were cultured in 75% H2 for 24 hours and then treated with 1 mM H2 O 2 for 12 hours. (a & b) Cell apoptosis was detected with annexin V-FITC and PI counter staining and analyzed via flow cytometry. (c) CCK8 assay. (d) Cell migration assay and (e) melanin content assay were performed in melanocytes. (f) Quantitative PCR and (g) ELISA assays were carried out in keratinocytes. ** P < 0.01 compared with the control group; # P < 0.05 and ## P < 0.01 compared with the H2 O2 group. Figure 3. Effects of H2 on mitochondrial function and content in PIG1 and PIG3V cells following H2 O2 challenge. Melanocytes were incubated in the presence or absence of 75% H2 for 24 h and then incubated in the presence or absence of 1 mM H2 O 2 for 12 h. (a) Representative images of JC-1 aggregates (red), JC-1 monomers (green), and merged images of both. More JC-1 aggregates suggested intact mitochondria, and the formation of JC-1 monomers in H2 O2-treated cells showed dissipation of the Δψm. Scale bar=20 μm. (b) Ratio of JC-1 aggregates to JC-1

monomers. (c) Cellular ATP assay. (d) Analyses of mitochondrial morphology. Cells seeded on glass coverslips were stained with Mito-Tracker Green as described in Materials and Methods. Scale bar=20 μm. Immunoblotting (e & f) was performed to evaluate the proteins levels of cytochrome c (e) and cytochrome b (f). ** P < 0.01 compared with the control group; ## P < 0.01 compared with the H2 O2 group. Figure 4. Nrf2 was essential for the protective effect of H2 in H2 O2 -treated melanocytes. Cells were incubated in the presence or absence of 75% H2 for 24 h and then incubated in the presence or absence of 1 mM H2 O2 for 12 h. (a) Western blotting of the of Nrf2, HO-1, and NQO-1 in cytosolic fractions and Nrf2 in nuclear fractions of melanocytes and keratinocytes. The intensity of each band was quantified by densitometry analysis. Cytoplasmic protein expression was normalized to that of β-actin, and nuclear protein expression was normalized to that of lamin B1. The nuclear/cytosolic Nrf2 ratio is shown to the right. For (b) to (h), PIG1 cells were transfected with shRNA against Nrf2 or control shRNA for 24 hours and then treated with 75% H2 for 24 hours followed by 1 mM H2 O 2 for 12 hours. Measurement of intracellular ROS (b) and MDA (c) levels. (d) Cell viability determination by the CCK-8 assay. (e&f) Cell apoptosis assay. (g) Cell migration assay. (h) Melanin content assay. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the control group; # P < 0.05, ## P < 0.01, and ### P < 0.001 compared with the H2 O 2 group. Figure 5. Nrf2 silencing abolished the protective effect of H2 on mitochondrial function and content in melanocytes following H2 O2 challenge. PIG1 cells transfected with shRNA against Nrf2 or control shRNA were treated with 75% H2 for 24 hours followed by 1 mM H2 O2 for 12 hours. (a) Alterations in mitochondrial membrane potential (Δψm) were determined using a JC-1 probe. Scale bar=20 μm. (b) Ratio of JC-1 aggregates to JC-1 monomers. (c) Cellular ATP assay. (d) Analyses of mitochondrial morphology. Scale bar=20 μm. Immunoblotting (e and f) was performed to evaluate the proteins levels of cytochrome c (e) and cytochrome b (f). Error bars represent the means±SD of three independent experiments. ** P < 0.01 compared with the control group; ## P < 0.01 compared with the H2 O2 group. Figure 6. β-Catenin activation was required for H2 -induced activation of Nrf2 signaling in melanocytes. Cells were incubated in the presence or absence of 75% H2 for 24 h and then incubated in the presence or absence of 1 mM H2 O2 for 12 h. (a) mRNA levels of β-catenin, LEF1, and CDH3 analyzed by quantitative real-time PCR. (b) Immunoblotting of β-catenin in cytosolic and nuclear fractions. The nuclear/cytosolic β-catenin ratio is shown to the right. (c) Immunoblotting of Nrf2 in cytosolic and nuclear fractions of melanocytes pretreated with ICG001 (β-catenin inhibitor). (d) Immunoblotting of total β-catenin in PIG1 cells with Nrf2 deficiency. (e) Immunoblotting of β-catenin in cytosolic and nuclear fractions of H2 -treated PIG1 cells with Nrf2 deficiency following H2 O 2 challenge. * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the control group; # P < 0.05, ## P < 0.01, and ### P < 0.001

compared with the H2 O2 group.

SUPPLEMENTARY FIGURES

Fig. S1. Effect of hydrogen gas treatment on intracellular ROS level in cutaneous cells from vitiligo patients. Cutaneous cells isolated from the perilesional skin of healthy subjects or vitiligo patients were treated with 35% H2 for 4 hours. Intracellular ROS levels were then measured using the fluorescent probe H2DCFDA. Ctrl group, without H2 treatment. Scale bar=20 μm. Fig. S2. Evaluation of H2 cytotoxicity in human melanocytes and keratinocytes. PIG1 (a, normal melanocytes), PIG3V (b, vitiligo melanocytes), and HaCaT (c, keratinocytes) cells were treated with 2%, 35%, or 75% H2 for 6-24 h. Cell viability was determined with CCK8 assays. Fig. S3. Intracellular ROS measurement in H2 O2 -exposed melanocytes and keratinocytes treated with different H2 concentrations. Scale bar=20 μm. Fig. S4. Intracellular ROS measurement in H2 O2 -exposed melanocytes and keratinocytes treated with 75% H2 for different lengths of time. Scale bar=20 μm. Fig. S5. Effects of H2 on antioxidant enzyme activities or content in H2 O2 -treated melanocytes and keratinocytes. PIG1, PIG3V, and HaCaT cells were incubated in the presence or absence of 75% H2 for 24 h and then incubated in the presence or absence of 1 mM H2 O2 for 12 h. Measurement of SOD (a), CAT (b), GPx (c), GSH (d), GSSG (e), and the GSH/GSSG ratio (f) in melanocytes and keratinocytes. Error bars represent the means±SD of three independent cultures. * P < 0.05 and ** P < 0.01 compared with the control group; # P < 0.05 and ## P < 0.01 compared with the H2 O2 group; ns, not significant. Fig. S6. Effects of 75% H2 on cell migration of H2 O2 -treated melanocytes. Scale bar=20 μm. Fig. S7. Quantitative analyses of mitochondrial content in H2 -treated PIG1 and PIG3V cells following H2 O2 challenge. Cells seeded on glass coverslips were stained with Mito- Tracker Green as described in Materials and Methods. ** P < 0.01 compared with the control group; ## P < 0.01 compared with the H2 O2 group. Fig. S8. Effects of H2 on mRNA levels of cytochrome c and cytochrome b in PIG1 and PIG3V cells following H2 O2 challenge. Fig. S9. H2 promoted Nrf2/ARE signaling activation in H2 O2 -treated melanocytes and keratinocytes. PIG1, PIG3V, and HaCaT cells were incubated in the presence or absence of 75% H2 for 24 h and then incubated in the presence or absence of 1 mM H2 O2 for 12 h. (a) Quantitative real-time PCR analysis of NRF2 expression and that of its downstream target genes. (b) Nrf2 localization in PIG1 and PIG3V cells was determined by immunofluorescence. Scale bar=20 μm. Fig. S10. Nrf2 silencing in PIG1 cells. PIG1 cells were transfected with shRNA

against Nrf2 or control shRNA for 24 hours and then treated with 75% H2 for 24 hours followed by 1 mM H2 O2 for 12 hours. Quantitative real-time PCR (a) and immunoblotting (b&c) were carried out to evaluate Nrf2 expression in melanocytes transfected with Nrf siRNA or the siRNA control. Fig. S11. Nrf2 deficiency abolished the protective effect of H2 on cell migration of melanocytes following H2 O2 challenge. Scale bar=20 μm. Fig. S12. Nrf2 deficiency abolished the anti-ROS effect of H2 on melanocytes following H2 O2 challenge. Scale bar=20 μm. Fig. S13. Nrf2 silencing abolished the protective effect of H2 on mitochondrial morphology and content in melanocytes following H2 O2 challenge. Cells seeded on glass coverslips were stained with Mito- Tracker Green as described in Materials and Methods. ** P < 0.01 compared with the control group; ns, not significant compared with the H2 O2 group. Fig. S14. Effects of Nrf2 silencing on mRNA levels of cytochrome c and cytochrome b in H2 O2 -challenged PIG1 cells pretreated with H2 .