Folic Acid Protects Melanocytes from Oxidative Stress via Activation of Nrf2 and Inhibition of HMGB1

Vitiligo is a cutaneous depigmentation disease due to loss of epidermal melanocytes. Accumulating evidence has indicated that oxidative stress plays a vital role in vitiligo via directly destructing melanocytes and triggering inflammatory response that ultimately undermines melanocytes. Folic acid (FA), an oxidized form of folate with high bioavailability, exhibits potent antioxidant properties and shows therapeutic potential in multiple oxidative stress-related diseases. However, whether FA safeguards melanocytes from oxidative damages remains unknown. In this study, we first found that FA relieved melanocytes from H2O2-induced abnormal growth and apoptosis. Furthermore, FA enhanced the activity of antioxidative enzymes and remarkably reduced intracellular ROS levels in melanocytes. Subsequently, FA effectively activated nuclear factor E2-related factor 2 (Nrf2) pathway, and Nrf2 knockdown blocked the protective effects of FA on H2O2-treated melanocytes. Additionally, FA inhibited the production of proinflammatory HMGB1 in melanocytes under oxidative stress. Taken together, our findings support the protective effects of FA on human melanocytes against oxidative injury via the activation of Nrf2 and the inhibition of HMGB1, thus indicating FA as a potential therapeutic agent for the treatment of vitiligo.


Introduction
Vitiligo is an acquired depigmentation dermatosis with an estimated prevalence of 1% worldwide [1]. To date, the reason for the loss of epidermal melanocytes as the key event in vitiligo is still unclear, which hinders the development of effective therapeutic strategies [2]. Nevertheless, recent studies have indicated the involvement of oxidative stress in the pathogenesis of vitiligo [3]. Along with the deficiency of antioxidant system of melanocytes, especially nuclear factor E2related factor 2 (Nrf2) pathway that is a master regulator of antioxidative response [4,5], reactive oxygen species (ROS) such as hydrogen peroxide (H 2 O 2 ) is excessively accumulated in vitiligo [6]. Accordingly, melanocytes are susceptible to ROS-induced apoptosis in vitiligo [3,5]. Moreover, ROS facilitates the release of high-mobility group protein B1 (HMGB1) that belongs to damage-associated molecular patterns (DAMPs) with strong proinflammatory effects from melanocytes [7][8][9][10]. HMGB1 subsequently causes the production of chemokines in adjacent keratinocytes and the maturation of dendritic cells (DCs) in a paracrine way, which ultimately promotes the formation of cytotoxic T cells (CTLs) that undermine melanocytes in vitiligo [7]. Therefore, oxidative stress plays a vital role in vitiligo via not only direct oxidative damages on melanocytes but also triggering cutaneous T cell response that targets melanocytes.
Folic acid (FA), a synthetic oxidized form of folate with high bioavailability, is well known for its protective effect against neural tube defects [11]. FA digested at intestine is ultimately converted to 5-methyltetrahydrofolate with the help of methylenetetrahydrofolate reductase (MTHFR) and then involved in the remethylation of homocysteine (Hcy) to methionine [12]. In 2014, our group reported an association between single-nucleotide polymorphisms (SNPs) in MTHFR gene and vitiligo susceptibility [13]. Furthermore, Hcy was observed to accumulate in vitiligo and induce the apoptosis of melanocytes via endoplasmic reticulum stress [14]. These findings indicate that the disruption of folate metabolism plays a role in the pathogenesis of vitiligo. Notably, some recent studies have found that FA possesses excellent antioxidant property and is effective in maintaining cellular redox status [15,16]. However, the effect of FA on melanocytes under oxidative stress in vitiligo has not been investigated before.
Herein, we initially observed that FA did protect melanocytes from oxidative damages. Furthermore, the influence of FA on the antioxidant response of melanocytes was evaluated. In addition, the effect of FA on the status of HMGB1 in melanocytes under oxidative stress was also investigated in the present study. 2.2. CCK8 Assay. Cell viability was evaluated by using a CCK8 kit (C008, 7Sea biotech, China) according to manufacturer's manual. Generally, PIG1 cells were seeded into 96-well plates at the density of 2 × 10 4 cells per well before further treatments as indicated. Next, 10 μl of CCK8 solution was added to each well, and the cells were further incubated at 37°C for 2 h. The optical density (OD) was measured at 450 nm by Model 680 Microplate Reader (BioRad, USA). All experiments were performed in triplicate.

Apoptosis Assay.
Cell apoptosis was measured using Annexin V-FITC/PI kit (A005, 7 Seabiotech, China). Briefly, the treated PIG1 cells were stained according to manufacturer's instructions and then detected by flow cytometry (Beckman Coulter, USA) and analyzed with Expo32 software (BeckmanCoulter, USA).
2.5. SOD Activity Assay. After cells were lysed, the total protein was extracted to detect the activity of SOD by using Total Superoxide Dismutase Assay Kit with WST-8 (S0101M, Beyotime, China) following manufacturer's instructions.
2.6. Measurement of Intracellular ROS. The intracellular ROS was measured using a fluorogenic probe for ROS (CM-H2DCFDA) (C6827, Invitrogen, USA) following the protocol reported previously [17]. Briefly, PIG1 cells were seeded into 6-well plates with a density of 5 × 10 5 cells. After indicated treatments, 10 μM of DCFH-DA was added for 30 min, and then, cells were collected for detection of fluorescence intensity of DCF via flow cytometry (Beckman Coulter, USA).
2.7. Immunofluorescence Assay. PIG1 cells were grown in single-layer glass slides (801002, NEST Biotechnology, China) at a density of 5000 cells per dish. After indicated treatments, cells were washed by PBS and fixed with 4% paraformaldehyde. Cells were permeabilized for 15 min in PBS supplemented with 0.1% Triton X-100 at room temperature and labeled with the primary antibody anti-Nrf2 (1 : 200, ab62352, Abcam, USA) at 4°C overnight and corresponding secondary antibody Cy3-tagged goat anti-rabbit IgG (1 : 1000, ab6939, Abcam, USA) for 1 h at room temperature. At last, cells were incubated with the nuclear dye 40, 6-diamidino-2-phenylindole (DAPI) (1 : 1000, 62247, Thermo Fisher Scientific, USA) for 10 min at room temperature in the dark. The fluorescence was detected by using FV-1000/ES laser confocal microscopy (Olympus, Japan).
2.8. Enzyme-Linked Immunosorbent Assay (ELISA). ELISA was performed using the Human HMGB1 ELISA Kit (Shino-Test, Japan) according to manufacturer's 2 Oxidative Medicine and Cellular Longevity instructions. The absorbance (A450) was measured with a plate reader (Bio-Rad).
2.10. RNA Interference. PIG1 cells were seeded at 2 × 10 5 cells per well for 24 h before transfection. Cells were transfected with Nrf2 shRNA or irrelevant shRNA control (Gen-ePharma, China) with Lipofectamine 3000 (Invitrogen) following manufacturer's protocol. The sequences of the shRNAs used in the present study were as follows: shRNA- 2.11. Statistical Analysis. Data analysis was performed using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA). The two-tailed Student's t-test or one-way analysis of variance (ANOVA) was used in our analyses. P values less than 0.05 were considered significant. Data represent as mean ± SD for at least three independent experiments.

Results
3.1. FA Protects Melanocytes from Oxidative Stress. CCK8 assay was initially performed to evaluate suitable doses of FA for use in subsequent experiments. As a result, FA at the concentrations of 10 to 500 μM had no significant influence on the growth of PIG1 cells over an incubation course of three days, but high doses (1000 and 3000 μM) of FA showed toxicity on the third day (Figure 1(a)). To investigate the protective effect of FA on melanocytes under oxidative stress, PIG1 cells were treated with FA at a concentration gradient of 25, 50, and 100 μM, respectively, and costimulated with 800 μM H 2 O 2 for 24 h. It turned out that H 2 O 2induced morphologic changes of shortened or disappeared dendrites in PIG1 cells were significantly rescued by the pretreatment with FA at relatively higher doses of 50 or 100 μM (Figure 1(b)). Consistently, FA reversed the inhibited viability of PIG1 cells caused by H 2 O 2 in a dose-dependent manner (Figure 1(c)). We went on to investigate whether FA protects melanocytes from apoptosis under oxidative stress. Flow cytometry analysis showed that the proportion of apoptotic PIG1 cells was markedly increased after the treatment with H 2 O 2 , whereas the cotreatment with FA at the concentrations of 50 or 100 μM significantly rescued PIG1 cells from H 2 O 2 -induced apoptosis (Figures 2(a) and 2(b)). Moreover, the upregulated protein levels of proapoptotic Bax and cleaved Caspase-3 and the downregulated protein level of Bcl-2 caused by H 2 O 2 were all reversed by cotreated FA (100 μM) in PIG1 cells (Figure 2(c)). Altogether, these results support that FA protects melanocytes from oxidative damages.

FA Potentiates Antioxidant Response in Melanocytes
under Oxidative Stress. Next, the influence of FA on the antioxidant system of melanocytes was evaluated. The accumulation of ROS in PIG1 cells treated with H 2 O 2 was attenuated by the cotreatment with FA at the concentrations of 50 μM or 100 μM (Figures 3(a) and 3(b)). Meanwhile, the repressed activity of superoxide dismutase (SOD), a key antioxidant enzyme that scavenges ROS [17,18], was significantly reversed by FA at 100 μM in PIG1 cells treated with H 2 O 2 (Figure 3(c)).
Since Nrf2 plays a central role in the antioxidant system of melanocytes [3,5], we then evaluated the effect of FA on the activation of Nrf2 pathway. As shown by our Western blot analysis, the cotreatment of FA promotes the expressions of Nrf2, phosphorylated Nrf2 (p-Nrf2) that acts as a transcriptional factor as well as SOD2 and heme oxygenase-1 (HO-1) that are antioxidant proteins transcriptionally regulated by p-Nrf2 in PIG1 cells treated with H 2 O 2 (Figure 3(d)). Subsequent detection of nuclear and cytosolic Nrf2 separately disclosed that FA induced the translocation of Nrf2 from cytoplasm to nucleus in H 2 O 2 -treated PIG1 cells (Figure 3(e)), which was further supported by immunofluorescence assay (Figure 3(f)). Collectively, our findings indicate that FA enhances antioxidant response in melanocytes under oxidative stress.

FA Prevents Melanocytes from Oxidative Damages via
Activating Nrf2 Pathway. To decide whether the protective effect of FA on melanocytes under oxidative stress is dependent on the activation of Nrf2, Nrf2 was silenced by transfecting PIG1 cells with short hairpin RNA (shRNA) of Nrf2 (interference efficiency shown in Figure 4(a)). It was found that FA failed to eliminate intracellular ROS in H 2 O 2 -treated PIG1 cells as long as Nrf2 was deficient (Figures 4(b) and 4(c)). Moreover, Nrf2 knockdown abolished the protection of FA against H 2 O 2 -induced apoptosis on PIG1 cells (Figures 4(d) and 4(e)). Further Western blot assay observed that Nrf2 knockdown abrogated FAinduced upregulation of HO-1 and SOD2 in H 2 O 2 -treated PIG1 cells (Figure 4(f)). Altogether, these findings demonstrate that the activation of Nrf2 pathway mediates the protection of FA on melanocytes against oxidative damages.

FA Reduces the Release of HMGB1 from Melanocytes under Oxidative Stress.
Given the proinflammatory role of HMGB1 secreted by melanocytes under oxidative stress in the immune pathogenesis of vitiligo as described before, the effect of FA on the status of HMGB1 in ROS-triggered melanocytes was examined at last. Consistent with our previous report [7], H 2 O 2 prominently increased the mRNA and protein levels of HMGB1 in PIG1 cells; both of which, however, were repressed by the addition of FA into the culture (Figures 5(a) and 5(b)). Additionally, the cotreatment with FA successfully prevented the release of HMGB1 from H 2 O 2 -treated PIG1 cells ( Figure 5(c)). Therefore, FA is of promising potential in inhibiting oxidative stress-induced inflammatory response mediated by HMGB1 in vitiligo.

Discussion
Oxidative stress is convincingly a key pathogenetic factor of vitiligo and contributes to its onset and progression [19]. Excessive ROS production and insufficient antioxidant response jointly lead to the destruction of melanocytes in    (e, f) The nuclear/cytoplasmic distribution of Nrf2 was detected via Western blot (e) and immunofluorescence (f), respectively. Lamin A/C and tubulin were detected as loading controls in western blot assay. Nrf2 was stained with Cy3 (red) and nuclei were counterstained with DAPI (blue) in fluorescence assay. Scale bar = 10 μm. All data are presented as the mean ± SD across three independent experiments. * P < 0:05, * * P < 0:01, * * * P < 0:001; ns: not significant. 6 Oxidative Medicine and Cellular Longevity   Oxidative Medicine and Cellular Longevity vitiligo [18], which prompts us to seek for measures that rebalance the redox homeostasis of melanocytes. Previous studies have shown that FA keeps the survival of astrocytes and purkinje neurons by decreasing ROS level [20,21]. Meanwhile, rats treated with FA could obtain high levels of antioxidative enzymes like SOD and low ROS levels [16], indicating that FA potentiates antioxidant system both in vitro and in vivo. The current study further showed that FA was able to protect melanocytes from oxidative injury by lowering intracellular ROS levels and upregulating HO-  The expressions of Nrf2, p-Nrf2, SOD2, and HO-1 were determined via Western blot. β-Actin was detected as loading control. * P < 0:05, * * P < 0:01, * * * P < 0:001; ns: not significant. 8 Oxidative Medicine and Cellular Longevity 1 and SOD2, both of which were reported deficient in vitiligo melanocytes [3,5]. Further studies using H 2 O 2 -induced vitiligo animal model [22] are needed to confirm the protective effect of FA on melanocytes from H 2 O 2 -triggered oxidation in vivo. Apoptosis is the main form of cell death for melanocytes under oxidative stress [18]. Bcl-2 family proteins including prosurvival Bcl-2 and proapoptotic BAX are the key regulators of apoptosis pathway, in which Caspase-3 acts as a classical executor protein [23]. Herein, FA exhibited potent antiapoptotic ability in human melanocytes under oxidative stress. We previously found that FA protected melanocytes from Hcy-induced apoptosis [14]. Interestingly, Li et al. and Zhou et al. observed that FA inhibited aging-induced apoptosis in astrocytes and neurocytes, respectively, suggesting that FA could suppress apoptosis in various conditions [15,24]. Additionally, our study showed the upregulation of Bax, the downregulation of Bcl2, and the inhibition of Caspase-3 activation in melanocytes undergoing oxidative stress following FA treatment, indicating that the antiapoptotic capacity of FA may result from decreasing cleaved Caspase-3 through modulating the ratio of Bax to Bcl-2 expression in oxidative injured melanocytes.
Nrf2 is a vital antioxidant regulator that functions as a potent transcriptional activator [4,25]. Under physiological conditions, Nrf2 is localized primarily in the cytoplasm and bound by Kelch-like ECH-associated protein 1 (Keap1) that hinders the activation of Nrf2 [26]. In response to oxidative stress, Nrf2 is disengaged from its binding to Keap1 and translocated into the nucleus, where Nrf2 potentiates the transcription of antioxidant response elements (AREs) and induces a battery of antioxidant proteins [27]. Nrf2 pathway plays a crucial role in protecting human melanocytes from oxidative stress as demonstrated by our previous studies [4,5,28,29]. Melanocytes in vitiligo are deficient of the activity of Nrf2 and thus more vulnerable to oxidative stress, and reintroduction of Nrf2 pathway is expectedly accompanied with improved survival of melanocytes under oxidative stress [5]. The activation of Nrf2 pathway is characterized by the phosphorylation of Nrf2, the nuclear translocation of Nrf2, and the expression of antioxidant proteins including SOD2 and HO-1 [30], all of which were observed in FA-treated melanocytes by our study. Further knockdown of Nrf2 abolished the protection of FA on melanocytes against oxidative damages, supporting that the antioxidant effect of FA is dependent on Nrf2 pathway in melanocytes. In parallel with our results, Cao et al. found that FA promoted the translocation of Nrf2 from cytoplasm to nucleus and elevated HO-1 expression in rats with spontaneous hypertension [31]. Consistently, the deficiency of folate in fish gills was accompanied with decreased expression of Nrf2 [32]. All of these findings indicate FA as a strong activator of Nrf2 that can be applied extensively.