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Simvastatin Protects Human Melanocytes from H2O2-Induced Oxidative Stress by Activating Nrf2

Open ArchivePublished:February 04, 2017DOI:https://doi.org/10.1016/j.jid.2017.01.020
      The prevention of hydrogen peroxide (H2O2)-induced oxidative stress has proved to be beneficial to vitiligo patients. Simvastatin possesses antioxidative capacity and has shown protective effect in various oxidative stress-related diseases. However, whether simvastatin can protect human melanocytes against oxidative stress has not been investigated. In this study, we initially found that pretreatment with 0.1 μmol/L to 1.0 μmol/L simvastatin led to increased cell viability and decreased cell apoptosis of melanocytes in response to H2O2. In addition, simvastatin was able to potentiate the activity of antioxidant enzymes and lessen intracellular reactive oxygen species accumulation. Furthermore, we found that simvastatin promoted the activation of nuclear erythroid 2-related factor (Nrf2) and that knockdown of Nrf2 abolished the protective effect of simvastatin against H2O2-induced oxidative damage. More importantly, the mutual enhancement between mitogen-activated protein kinase pathways and p62 contributed to simvastatin-induced Nrf2 activation in melanocytes. Finally, simvastatin showed more antioxidative capacity and better protective effect than aspirin in H2O2-treated melanocytes. Taken together, our results show that simvastatin protects human melanocytes from H2O2-induced oxidative stress by activating Nrf2, thus supporting simvastatin as a potential therapeutic agent for vitiligo.

      Abbreviations:

      CAT (catalase), Erk (extracellular signal-regulated kinase), H2O2 (hydrogen peroxide), JNK (c-Jun amino terminal kinase), MAPK (mitogen-activated protein kinase), Nrf2 (nuclear erythroid 2-related factor), ROS (reactive oxygen species), shRNA (short hairpin RNA), siRNA (small interfering RNA), SOD (superoxide dismutase)

      Introduction

      Vitiligo is an acquired skin disease that affects 0.5% to 2.0% of the population; it characterized by progressive depigmentation due to the destruction of epidermal melanocytes (
      • Ezzedine K.
      • Eleftheriadou V.
      • Whitton M.
      • van Geel N.
      Vitiligo.
      ,
      • Picardo M.
      • Dell'Anna M.L.
      • Ezzedine K.
      • Hamzavi I.
      • Harris J.E.
      • Parsad D.
      • et al.
      Vitiligo.
      ). Oxidative stress has been implicated in the onset and progression of vitiligo (
      • Xie H.
      • Zhou F.
      • Liu L.
      • Zhu G.
      • Li Q.
      • Li C.
      • et al.
      Vitiligo: how do oxidative stress-induced autoantigens trigger autoimmunity?.
      ). Specially, the pathogenic role of oxidative stress was supported by the accumulation of hydrogen peroxide (H2O2), the compromise of intrinsic antioxidant system, and the oxidative damage of lipids and proteins in vitiligo lesions (
      • Dell’Anna M.L.
      • Ottaviani M.
      • Albanesi V.
      • Vidolin A.P.
      • Leone G.
      • Ferraro C.
      • et al.
      Membrane lipid alterations as a possible basis for melanocyte degeneration in vitiligo.
      ,
      • Liu L.
      • Li C.
      • Gao J.
      • Li K.
      • Zhang R.
      • Li C.
      • et al.
      Promoter variant in the catalase gene is associated with vitiligo in Chinese people.
      ). Therefore, ameliorating oxidative stress with antioxidative agents could be a potential therapeutic approach for treating vitiligo.
      Simvastatin, one of the most common competitive inhibitors of HMGCR, is widely used for the treatment of dyslipidemia and cardiovascular diseases (
      • Zhou Q.
      • Liao J.K.
      Statins and cardiovascular diseases: from cholesterol lowering to pleiotropy.
      ). In addition to its lipid-lowering effect, simvastatin has pleiotropic effects in regulating the inflammatory process, immune response, and oxidative stress (
      • Kavalipati N.
      • Shah J.
      • Ramakrishan A.
      • Vasnawala H.
      Pleiotropic effects of statins.
      ). A patient with both vitiligo and hypercholesterolemia was reported to gain rapid repigmentation of skin after receiving high-dose simvastatin, suggesting simvastatin treatment as a potential therapy for vitiligo (
      • Noël M.
      • Gagné C.
      • Bergeron J.
      • Jobin J.
      • Poirier P.
      Positive pleiotropic effects of HMG-CoA reductase inhibitor on vitiligo.
      ). A recent study using a mouse model of vitiligo showed that simvastatin prevents and reverses depigmentation by reducing the number of infiltrating autoreactive CD8+ T cells in the skin and inhibiting their proliferation and the production of IFN-γ (
      • Agarwal P.
      • Rashighi M.
      • Essien K.I.
      • Richmond J.M.
      • Randall L.
      • Pazoki-Toroudi H.
      • et al.
      Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
      ). However, that study focused mainly on the effect of simvastatin on autoreactive CD8+ T cells rather than on the potential impact of the drug on melanocytes themselves. The therapeutic effect of simvastatin in other diseases has been attributed to its antioxidative property through the direct scavenging of superoxide and hydroxyl radicals (
      • Chartoumpekis D.
      • Ziros P.G.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Papavassiliou A.G.
      • Habeos I.G.
      Simvastatin lowers reactive oxygen species level by Nrf2 activation via PI3K/Akt pathway.
      ,
      • Cunha V.
      • Santos M.M.
      • Moradas-Ferreira P.
      • Ferreira M.
      Simvastatin effects on detoxification mechanisms in Danio rerio embryos.
      ), indicating its great potential as an effective antioxidative agent. Nevertheless, whether simvastatin can protect melanocytes from oxidative stress-induced damage in vitiligo patients is still unknown.
      The transcription factor nuclear erythroid 2-related factor (Nrf2) is a master regulator that governs redox balance of the cell (
      • Kobayashi M.
      • Yamamoto M.
      Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species.
      ,
      • Osburn W.O.
      • Kensler T.W.
      Nrf2 signaling: an adaptive response pathway for protection against environmental toxic insults.
      ). Upon exposure to oxidative stress, Nrf2 translocates to the nucleus after disassociation from Keap1 and then promotes the expression of antioxidant genes by binding to their antioxidant response element in the promoter region (
      • Lacher S.E.
      • Lee J.S.
      • Wang X.
      • Campbell M.R.
      • Bell D.A.
      • Slattery M.
      Beyond antioxidant genes in the ancient Nrf2 regulatory network.
      ). Deficiency in the Nrf2-antioxidant response element pathway contributes to the oxidative damage of melanocytes in vitiligo (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ,
      • Jian Z.
      • Li K.
      • Song P.
      • Zhu G.
      • Zhu L.
      • Cui T.
      • et al.
      Impaired activation of the Nrf2-ARE signaling pathway undermines H2O2-induced oxidative stress response: a possible mechanism for melanocyte degeneration in vitiligo.
      ). Thus, enhancing the Nrf2-antioxidant response element signaling pathway in melanocytes could be an effective strategy for vitiligo therapy. Recently, Nrf2 signaling has been proven to be responsible for the antioxidant effect of simvastatin in different tissues and cells (
      • Chartoumpekis D.
      • Ziros P.G.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Papavassiliou A.G.
      • Habeos I.G.
      Simvastatin lowers reactive oxygen species level by Nrf2 activation via PI3K/Akt pathway.
      ,
      • Habeos I.G.
      • Ziros P.G.
      • Chartoumpekis D.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Papavassiliou A.G.
      Simvastatin activates Keap1/Nrf2 signaling in rat liver.
      ). Thus, we hypothesized that simvastatin protects human melanocytes from oxidative stress by activating Nrf2.

      Results

      Simvastatin ameliorates H2O2-induced oxidative damage in human melanocytes

      Doses for use in experiments were selected after the toxicity of simvastatin (see Supplementary Figure S1a online) was determined with proliferation and viability assay. Concentrations of 0.1 μmol/L to 1.0 μmol/L simvastatin were selected for dosing because they resulted in no significant alterations of cell proliferation and viability (Figure 1a and b). To administer oxidative stress in melanocytes, we chose 1.0 mmol/L H2O2 as the stimulus, because this dose was optimal in inducing a sufficient cytotoxic effect on melanocytes (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ). The dendrites of melanocytes shortened or disappeared after treatment with H2O2 for 24 hours, and cell viability was also significantly dampened. Nevertheless, pretreatment with simvastatin reversed H2O2-induced abnormal morphology, as represented by prolonged dendrites. Moreover, simvastatin increased the cell viability of H2O2-treated melanocytes in a dose-dependent manner (Figure 1c and d). Similar to previous studies, we found that after H2O2 treatment, the average percentage of apoptotic cells increased markedly to almost 37%, whereas pretreatment of simvastatin increasingly attenuated apoptosis as the concentration gradually rose (Figure 1e). Taken together, these results show that simvastatin was able to ameliorate H2O2-induced oxidative damage of human melanocytes.
      Figure 1
      Figure 1Simvastatin ameliorates H2O2-induced oxidative damage in human melanocytes. (a, b) Melanocytes were treated with different concentrations of simvastatin for indicated times. (a) Cell proliferation and (b) viability were determined by CCK8 assay. (c–e) Melanocytes were pretreated with different concentrations of simvastatin for 24 hours and then exposed to 1.0 mmol/L H2O2 for another 24 hours. The morphological features of melanocytes were detected by microscope. Each field shown is a representative image of at least nine similar fields from three independent experiments, Scale bar = 200 μm in c. (d) Cell viability and (e) apoptosis determined by CCK-8 and flow cytometry assay, respectively. All data are presented as the mean ± standard deviation across three independent experiments. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. h, hour; H2O2, hydrogen peroxide; M, mol/L; Sim, simvastatin.

      Simvastatin potentiates the antioxidant capacity of human melanocytes

      Subsequently, by using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester as the fluorescent probe, we showed that simvastatin markedly reduced reactive oxygen species (ROS) level in H2O2-treated melanocytes, exhibiting a similar pattern as cell apoptosis (Figure 2a). Antioxidant enzymes, including catalase (CAT) and superoxide dismutase (SOD), are important in modulating ROS level by scavenging free radicals in melanocytes (
      • Bernardo I.
      • Bozinovski S.
      • Vlahos R.
      Targeting oxidant-dependent mechanisms for the treatment of COPD and its comorbidities.
      ). As in previous studies, H2O2 prominently impeded the activity of CAT and SOD (
      • Ray G.
      • Husain S.A.
      Oxidants, antioxidants and carcinogenesis.
      ). However, simvastatin abrogated the inhibition of CAT and SOD activity in a dose-dependent manner under oxidative stress (Figure 2b and c). Taken together, these results suggest that simvastatin potentiates the antioxidant capacity of melanocytes by lessening ROS accumulation and activating antioxidant enzymes.
      Figure 2
      Figure 2Simvastatin potentiates the antioxidant capacity of H2O2-treated human melanocytes. Melanocytes were exposed to various concentrations of simvastatin for 24 hours and were further treated with 1.0 mmol/L H2O2 for another 24 hours. (a) Intracellular ROS production determined by flow cytometry assay. The fluorescence intensity of ROS level is shown to the right. (b, c) The activity of antioxidant enzymes CAT and SOD. Error bars represent mean ± standard deviation across three independent experiments. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. CAT, catalase; DCF, dichlorodihydrofluorescein; H2O2, hydrogen peroxide; M, mol/L; ns, not significant; Sim, simvastatin; SOD, superoxide dismutase.

      Simvastatin promotes the activation of Nrf2 and its downstream genes in H2O2-treated melanocytes

      By using quantitative real-time PCR, immunoblotting, and immunofluorescence staining, we found that simvastatin significantly promotes Nrf2 expression in melanocytes in a dose-dependent manner (Figure 3a–c). We further showed that simvastatin increased the expression of nuclear Nrf2 but decreased that of cytosolic Nrf2 (Figure 3b). Consistently, immunofluorescence staining showed remarkable nuclear translocation of Nrf2 (Figure 3c). Additionally, we found that simvastatin accentuated the phosphorylation of Nrf2, which was essential for its nuclear translocation and transcriptional activity (Figure 3b and c).
      Figure 3
      Figure 3Simvastatin promotes the activation of Nrf2 and the expression of its downstream genes in H2O2-treated melanocytes. Melanocytes were exposed to different concentrations of simvastatin for 24 hours and were further treated with 1.0 mmol/L H2O2 for another 24 hours. (a) Nrf2 mRNA level analyzed by qRT-PCR. (b) Western blots of total, nuclear, and cytoplasmic fractions of Nrf2. The intensity of each band was quantified by densitometry analysis. The ratio of nuclear/cytosolic of Nrf2 is shown to the right. (c) The expression and nuclear translocation of phosphorylated Nrf2 determined by immunofluorescence. Scale bar = 10 μm. (d) The mRNA levels of HO-1 and NQO1 analyzed by qRT-PCR. (e) Western blots of HO-1 and NQO1. All data are expressed as mean ± standard deviation across three independent cultures. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. H2O2, hydrogen peroxide; M, mol/L; ns, not significant; p-, phosphorylated; qRT-PCR, quantitative real-time PCR; Sim, simvastatin.
      HO-1 and NQO1 are the main target genes of Nrf2 in protecting cells from oxidative stress (
      • Motohashi H.
      • Yamamoto M.
      Nrf2-Keap1 defines a physiologically important stress response mechanism.
      ). We thus assessed the expression of these two genes and found that simvastatin dramatically increased the expression of HO-1 and NQO1 at both the mRNA and protein levels, indicating functional activation of Nrf2 (Figure 3d and e). These results suggest that simvastatin possibly exerted its antioxidative effect in melanocytes by promoting the activation of the Nrf2 signaling pathway.

      Nrf2 activation is required for the protective effect of simvastatin on H2O2-treated melanocytes

      To test whether Nrf2 is required for the effects of simvastatin protecting melanocytes against oxidative stress, we selected short hairpin RNA (shRNA)-Nrf2#2 (see Supplementary Figure S1b and c) to silence Nrf2 for further experiments. Nrf2 deficiency markedly abrogated the up-regulation of HO-1 and NQO1 in simvastatin-treated melanocytes under oxidative stress (Figure 4a). In addition, simvastatin-induced activation of CAT and SOD was significantly dampened with Nrf2 silencing (Figure 4b and c). Consistently, the intracellular ROS level was dramatically increased in simvastatin-treated melanocytes with Nrf2 deficiency (Figure 4d). Furthermore, Nrf2 silencing abolished the up-regulation of cell viability caused by simvastatin pretreatment (Figure 4e) and the protection of simvastatin against H2O2-induced apoptosis (Figure 4f). These results suggest that Nrf2 activation is essential for the antioxidative capacity of simvastatin in melanocytes.
      Figure 4
      Figure 4Nrf2 activation is required for the protective effect of simvastatin on H2O2-treated melanocytes. Melanocytes were transfected with shRNA against Nrf2 or control shRNA for 24 hours and were then treated with 1.0 μmol/L simvastatin for 24 hours followed by 1.0 mmol/L H2O2 for another 24 hours. (a) Western blot analysis of Nrf2, HO-1, and NQO1. (b, c) The activity of CAT and SOD. (d) Intracellular ROS production analyzed by flow cytometry. The fluorescence intensity of ROS level is reported to the right. (e) Cell viability determined by CCK-8 assay. (f) The level of apoptosis detected by flow cytometry. Bar graphs represent the mean values of three independent flow cytometry data. Data are presented as the mean ± standard deviation. ∗∗P < 0.01, ∗∗∗P < 0.001. H2O2, hydrogen peroxide; M, mol/L; NC, normal control; shRNA, short hairpin RNA; Sim, simvastatin.

      Mutual enhancement between the mitogen-activated protein kinase pathway and p62 contributes to the activation of Nrf2 by simvastatin

      Previous studies showed that mitogen-activated protein kinases (MAPKs) are important upstream regulators of Nrf2 (
      • DeNicola G.M.
      • Karreth F.A.
      • Humpton T.J.
      • Gopinathan A.
      • Wei C.
      • Frese K.
      • et al.
      Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.
      ,
      • Xu C.
      • Yuan X.
      • Pan Z.
      • Shen G.
      • Kim J.H.
      • Yu S.
      • et al.
      Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2.
      ,
      • Yao P.
      • Nussler A.
      • Liu L.
      • Hao L.
      • Song F.
      • Schirmeier A.
      • et al.
      Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways.
      ). Thus, we examined the alterations of the main MAPK pathways, including extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK), and p38 signaling in simvastatin-treated melanocytes. We found that simvastatin potentiates the phosphorylation of ERK and JNK, but not p38, under oxidative stress (Figure 5a). The pretreatment of either the ERK inhibitor PD98059 or JNK inhibitor SP600125 markedly impaired the expression and phosphorylation of Nrf2 (Figure 5b and c, and see Supplementary Figure S1d and e). Therefore, ERK and JNK pathways mediated the activation of Nrf2 in simvastatin-treated melanocytes.
      Figure 5
      Figure 5Mutual enhancement between MAPK pathway and p62 contributes to the activation of Nrf2 by simvastatin. (a) Western blots of the MAPK pathway. (b, c) Western blots of Nrf2 and p-Nrf2 in melanocytes pretreated with (b) PD98059 and (c) SP600125 and then treated with simvastatin followed by H2O2. (d) Protein level of p62 in simvastatin pretreated H2O2-treated melanocytes. (e) Western blots of Nrf2 in melanocytes transfected with siRNA-p62 for 24 hours and then treated with simvastatin followed by H2O2. (f, g) Western blots of p62 in melanocytes pretreated with (f) PD98059 and (g) SP600125 and then treated with simvastatin and H2O2. (h) Western blots of p-Erk and p-JNK in melanocytes transfected with siRNA-p62 and then treated with simvastatin and H2O2. Erk, extracellular signal-regulated kinase; H2O2, hydrogen peroxide; JNK, c-Jun N-terminal kinase; M, mol/L; NC, normal control; p-, phosphorylated; Sim, simvastatin; siRNA, small interfering RNA.
      In addition to the MAPK pathways, p62 is another key modulator of Nrf2, which acts by forming a complex with Keap1 (
      • Copple I.M.
      • Lister A.
      • Obeng A.D.
      • Kitteringham N.R.
      • Jenkins R.E.
      • Layfield R.
      • et al.
      Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway.
      ). We found that simvastatin was able to increase the expression of p62 in a dose-dependent manner (Figure 5d). Next, the knockdown of p62 by small interfering RNA (siRNA)-p62#3 (see Supplementary Figure S1f and g) abolished Nrf2 activation by simvastatin in stressed melanocytes, showing that p62 was also involved in simvastatin-induced Nrf2 activation (Figure 5e). We then wondered whether there was crosstalk between MAPK and p62. As shown in Figure 5f and g, the expression of p62 was significantly decreased in melanocytes treated with PD98059 or SP600125. We also found that simvastatin increased the autophagy marker LC3II/LC3I expression ratio in a dose-dependent manner (Figure S1h), a situation in which p62 expression is supposed to be decreased (
      • Klionsky D.J.
      • Abdalla F.C.
      • Abeliovich H.
      • Abraham R.T.
      • Acevedo-Arozena A.
      • Adeli K.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy.
      ). This result suggests that the increased p62 expression may not depend on autophagy but on ERK/JNK activation in simvastatin-treated melanocytes. In addition, the knockdown of p62 also impairs the phosphorylation of both ERK and JNK (Figure 5h). These results show a mutual enhancement between the MAPK pathway and p62 in simvastatin-induced Nrf2 activation.

      Simvastatin promotes Nrf2 activation in a cholesterol-independent but mevalonate-dependent manner

      Because simvastatin is an inhibitor of HMG-CoA, we went on to examine the activity and expression of HMG-CoA in simvastatin-treated melanocytes. The activity of HMGCR was remarkably decreased after simvastatin treatment, whereas the expression of HMGCR was unaffected (see Supplementary Figure S2a and b). Because simvastatin can inhibit mevalonate production and subsequent cholesterol synthesis (
      • Davies J.T.
      • Delfino S.F.
      • Feinberg C.E.
      • Johnson M.F.
      • Nappi V.L.
      • Olinger J.T.
      • et al.
      Current and emerging uses of statins in clinical therapeutics: a review.
      ), we examined the cholesterol level in simvastatin-treated melanocytes via immunofluorescence. We observed a mild decline of cell membrane cholesterol concentration in melanocytes in simvastatin-treated groups (Figure S2c). However, addition of cholesterol in simvastatin-treated melanocytes had minimal effect on the phosphorylation of either ERK or JNK or on Nrf2 signaling (see Supplementary Figure S2d). Supplementing simvastatin-treated melanocytes with mevalonate impaired the activation of both the Nrf2 and MAPK signaling pathways (see Supplementary Figure S2d). These results indicate that simvastatin may promote Nrf2 activation in a cholesterol-independent but mevalonate-dependent manner.

      Simvastatin has better antioxidative capacity than aspirin in H2O2-treated melanocytes

      Recently, aspirin has been proven to protect melanocytes against H2O2-induced oxidative stress through the activation of Nrf2, resembling simvastatin in our study (
      • Jian Z.
      • Tang L.
      • Yi X.
      • Liu B.
      • Zhang Q.
      • Zhu G.
      • et al.
      Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2 -induced oxidative stress.
      ). We thus compared the effects of these two agents to confirm which one is more efficient and whether the combination of the two is better than simvastatin alone. We found that pretreatment with simvastatin alone prevented H2O2-induced apoptosis more efficiently than aspirin. In addition, the combination of simvastatin and aspirin showed no further improvement of cell survival compared with simvastatin alone (Figure 6a). Further detection of cell viability and intracellular ROS level displayed the same pattern as the apoptosis of melanocytes (Figure 6b and c). Furthermore, simvastatin recovered more CAT and SOD activity than aspirin, and there was no addictive or synergistic effect of simvastatin and aspirin on the activity of these two enzymes (Figure 6d and e). Finally, we showed that the expression and phosphorylation of Nrf2, as well as the expression of HO-1, were all higher in the simvastatin-treated group (Figure 6f). Taken together, our results show that simvastatin has more antioxidative capacity and better protective effect than aspirin in H2O2-treated melanocytes.
      Figure 6
      Figure 6Simvastatin has better antioxidative capacity than aspirin in H2O2-treated melanocytes. Melanocytes were exposed to either simvastatin (1.0 μmol/L) or aspirin (90.0 μmol/L) for 24 hours and were then treated with 1.0 mmol/L H2O2 for another 24 hours. (a) Cell apoptosis analyzed by flow cytometry. (b) Cell viability determined by CCK-8 assay. (c) Intracellular ROS production determined by flow cytometry. The fluorescence intensity of ROS level is shown to the right. (d, e) The activity of CAT and SOD. (f) Western blots of Nrf2, p-Nrf2, and HO-1 in melanocytes pretreated with either simvastatin or aspirin alone or together and then treated with H2O2. All data are presented as the mean ± standard deviation of three independent experiments. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ASA, aspirin; DCF, dichlorodihydrofluorescein; H2O2, hydrogen peroxide; M, mol/L; ns, not significant; p-, phosphorylated; Sim, simvastatin.

      Discussion

      In this study, we initially found that simvastatin was able to ameliorate H2O2-induced oxidative damage in human melanocytes. In parallel, simvastatin lessened the accumulation of intracellular ROS by potentiating the activity of antioxidant enzymes. Furthermore, we showed that simvastatin protected melanocytes against oxidative stress by activating Nrf2 and that the mutual enhancement between the MAPK pathways and p62 contributed to simvastatin-induced Nrf2 activation. Finally, we proved that simvastatin had better antioxidative capacity and protective effect than aspirin in H2O2-treated melanocytes. Our study shows that simvastatin is a potential antioxidative agent for the treatment of vitiligo.
      Oxidative stress plays a key role in the onset and progression of vitiligo because it can directly induce melanocyte apoptosis and also initiate the autoimmune response that subsequently causes melanocyte destruction (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ,
      • Jian Z.
      • Li K.
      • Song P.
      • Zhu G.
      • Zhu L.
      • Cui T.
      • et al.
      Impaired activation of the Nrf2-ARE signaling pathway undermines H2O2-induced oxidative stress response: a possible mechanism for melanocyte degeneration in vitiligo.
      ,
      • Li S.
      • Zhu G.
      • Yang Y.
      • Guo S.
      • Dai W.
      • Wang G.
      • et al.
      Oxidative stress-induced chemokine production mediates CD8+T cell skin trafficking in vitiligo.
      ,
      • Li S.
      • Zhu G.
      • Yang Y.
      • Jian Z.
      • Guo S.
      • Dai W.
      • et al.
      Oxidative stress drives CD8+T cells skin trafficking in vitiligo via CXCL16 upregulation by activating the unfolded protein response in keratinocytes.
      ). Previous studies have shown that in response to various stressful conditions, simvastatin protects neuronal cells, liver cells, and embryonic fibroblasts by lowering ROS level, which reflects its antioxidant capacity (
      • Chartoumpekis D.
      • Ziros P.G.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Papavassiliou A.G.
      • Habeos I.G.
      Simvastatin lowers reactive oxygen species level by Nrf2 activation via PI3K/Akt pathway.
      ,
      • Hsieh C.H.
      • Rau C.S.
      • Hsieh M.W.
      • Chen Y.C.
      • Jeng S.F.
      • Lu T.H.
      • et al.
      Simvastatin-induced heme oxygenase-1 increases apoptosis of Neuro 2A cells in response to glucose deprivation.
      ). In this study, we further showed that in melanocytes, simvastatin was able to protect cells from H2O2-induced cell apoptosis and ROS accumulation, highlighting the therapeutic potential of simvastatin in oxidative stress-related vitiligo.
      It has been reported that a vitiligo patient had rapid skin repigmentation after high-dose simvastatin treatment, and the therapeutic effect has been ascribed to its inhibition on the melanocyte-specific CD8+ T-cell skin accumulation (
      • Noël M.
      • Gagné C.
      • Bergeron J.
      • Jobin J.
      • Poirier P.
      Positive pleiotropic effects of HMG-CoA reductase inhibitor on vitiligo.
      ,
      • Agarwal P.
      • Rashighi M.
      • Essien K.I.
      • Richmond J.M.
      • Randall L.
      • Pazoki-Toroudi H.
      • et al.
      Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
      ). In this study, we showed that in addition to the immunomodulatory effect, simvastatin protected melanocytes against oxidative stress-induced damage by activating Nrf2. Thus, we reasoned that the direct effect of simvastatin on melanocytes could also contribute to its positive therapeutic response in vitiligo.
      Our previous study showed that defective Nrf2 activation in melanocytes increases their intolerance to oxidative stress and contributes to the pathogenesis of vitiligo (
      • Jian Z.
      • Li K.
      • Liu L.
      • Zhang Y.
      • Zhou Z.
      • Li C.
      • et al.
      Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway.
      ,
      • Jian Z.
      • Li K.
      • Song P.
      • Zhu G.
      • Zhu L.
      • Cui T.
      • et al.
      Impaired activation of the Nrf2-ARE signaling pathway undermines H2O2-induced oxidative stress response: a possible mechanism for melanocyte degeneration in vitiligo.
      ). Several studies have proven that Nrf2 activation is essential for the protective effect of simvastatin in various cells and tissues (
      • Chartoumpekis D.
      • Ziros P.G.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Papavassiliou A.G.
      • Habeos I.G.
      Simvastatin lowers reactive oxygen species level by Nrf2 activation via PI3K/Akt pathway.
      ,
      • Habeos I.G.
      • Ziros P.G.
      • Chartoumpekis D.
      • Psyrogiannis A.
      • Kyriazopoulou V.
      • Papavassiliou A.G.
      Simvastatin activates Keap1/Nrf2 signaling in rat liver.
      ). Because it is a transcriptional factor, the phosphorylation and nuclear translocation of Nrf2 are critical for its dissociation from Keap1 and binding to the antioxidant response element in the promoter region of downstream antioxidant genes (
      • Huang H.C.
      • Nguyen T.
      • Pickett C.B.
      Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription.
      ,
      • Lu M.C.
      • Ji J.A.
      • Jiang Z.Y.
      • You Q.D.
      The Keap1-Nrf2-ARE pathway as a potential preventive and therapeutic target: an update.
      ). We found that in addition to the up-regulation of Nrf2 expression, simvastatin also promoted the nuclear translocation and phosphorylation of Nrf2, indicating that the modulatory effect occurred at both the translational and posttranslational levels.
      Previous studies have shown that MAPK pathways, including ERK, JNK, and p38, are crucial signaling pathways in regulating Nrf2 expression and activity (
      • DeNicola G.M.
      • Karreth F.A.
      • Humpton T.J.
      • Gopinathan A.
      • Wei C.
      • Frese K.
      • et al.
      Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.
      ,
      • Xu C.
      • Yuan X.
      • Pan Z.
      • Shen G.
      • Kim J.H.
      • Yu S.
      • et al.
      Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2.
      ,
      • Yao P.
      • Nussler A.
      • Liu L.
      • Hao L.
      • Song F.
      • Schirmeier A.
      • et al.
      Quercetin protects human hepatocytes from ethanol-derived oxidative stress by inducing heme oxygenase-1 via the MAPK/Nrf2 pathways.
      ). Our results showed that simvastatin promoted the phosphorylation of ERK and JNK, but not p38, in the presence of H2O2. In addition, pharmacological inhibition of either ERK or JNK abolished simvastatin-induced up-regulation and phosphorylation of Nrf2, showing that these two pathways mediated Nrf2 activation in simvastatin-treated melanocytes. Furthermore, simvastatin also activated Nrf2 by increasing the expression of p62. Simvastatin increased the LC3II/LC3I expression ratio in a dose-dependent manner, suggesting the significant activation of autophagy. In the process of autophagy, p62 serves as a link between LC3 and ubiquitinated substrates and is degraded in autolysosomes with autophagy activation (
      • Klionsky D.J.
      • Abdalla F.C.
      • Abeliovich H.
      • Abraham R.T.
      • Acevedo-Arozena A.
      • Adeli K.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy.
      ). Therefore, the facilitative effect of simvastatin on p62 expression may be modulated in an autophagy-independent way. Recently, it has been reported that ERK and JNK induce p62 expression at the transcriptional level (
      • Kim J.H.
      • Hong S.K.
      • Wu P.K.
      • Richards A.L.
      • Jackson W.T.
      • Park J.I.
      Raf/MEK/ERK can regulate cellular levels of LC3B and P62 at expression levels.
      ,
      • Klionsky D.J.
      • Abdalla F.C.
      • Abeliovich H.
      • Abraham R.T.
      • Acevedo-Arozena A.
      • Adeli K.
      • et al.
      Guidelines for the use and interpretation of assays for monitoring autophagy.
      ,
      • Puissant A.
      • Robert G.
      • Fenouille N.
      • Luciano F.
      • Cassuto J.P.
      • Raynaud S.
      • et al.
      Resveratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62/SQSTM1 expression and AMPK activation.
      ) and that p62 is also able to promote the activation of both ERK and JNK (
      • Choe J.Y.
      • Jung H.Y.
      • Park K.Y.
      • Kim S.K.
      Enhanced p62 expression through impaired proteasomal degradation is involved in caspase-1 activation in monosodium urate crystal-induced interleukin-1b expression.
      ). Consistent with this, we found that ERK/JNK and p62 are mutually activated in simvastatin-treated melanocytes. These results indicate that, after the treatment with simvastatin, ERK/JNK and p62 are primarily activated and then form a positive feedback loop with each other to reach sufficient activation of Nrf2 for it to have an antioxidative effect in melanocytes.
      Aspirin, another agent that is widely used in the treatment of cardiovascular disease, can also protect melanocytes against H2O2-induced oxidative stress by activating Nrf2 (
      • Jian Z.
      • Tang L.
      • Yi X.
      • Liu B.
      • Zhang Q.
      • Zhu G.
      • et al.
      Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2 -induced oxidative stress.
      ). In this study, we first confirmed that simvastatin had better antioxidative capacity than aspirin in melanocytes. Moreover, the combination of simvastatin and aspirin showed no more benefit than simvastatin alone. Therefore, simvastatin may be more efficient than aspirin in vitiligo therapy, because it induces the full activation of Nrf2 and optimizes the antioxidative function.
      The dosage of simvastatin that led to remarkable regimentation in vitiligo patients was 80 mg/day (
      • Noël M.
      • Gagné C.
      • Bergeron J.
      • Jobin J.
      • Poirier P.
      Positive pleiotropic effects of HMG-CoA reductase inhibitor on vitiligo.
      ), and that used for in vivo studies in vitiligo mice was up to 40 mg/kg (
      • Agarwal P.
      • Rashighi M.
      • Essien K.I.
      • Richmond J.M.
      • Randall L.
      • Pazoki-Toroudi H.
      • et al.
      Simvastatin prevents and reverses depigmentation in a mouse model of vitiligo.
      ), but the exact working concentration of simvastatin accumulated in the epidermal melanocytes was unknown. Because simvastatin is metabolized and concentrated in the liver, resulting in low levels of pharmacologically active fraction in the blood and skin, high doses of oral simvastatin may be required to target skin melanocytes for vitiligo treatment (
      • Serajuddin A.T.
      • Ranadive S.A.
      • Mahoney E.M.
      Relative lipophilicities, solubilities, and structure-pharmacological considerations of 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase inhibitors pravastatin, lovastatin, mevastatin, and simvastatin.
      ,
      • Stamm J.A.
      • Ornstein D.L.
      The role of statins in cancer prevention and treatment.
      ). However, high doses of simvastatin could increase the risk of adverse effects such as myopathy and rhabdomyolysis (
      • Desai C.S.
      • Martin S.S.
      • Blumenthal R.S.
      Non-cardiovascular effects associated with statins.
      ), limiting the use of simvastatin for vitiligo therapy. Several studies have indicated that simvastatin can be used as a topical ointment at a concentration of approximately 1.0 μmol/L for skin disease treatment (
      • Adami M.
      • Prudente Ada S.
      • Mendes D.A.
      • Horinouchi C.D.
      • Cabrini D.A.
      • Otuki M.F.
      Simvastatin ointment, a new treatment for skin inflammatory conditions.
      ,
      • Otuki M.F.
      • Pietrovski E.F.
      • Cabrini D.A.
      Topical simvastatin: preclinical evidence for a treatment of skin inflammatory conditions.
      ); simvastatin used topically is a promising therapeutic approach for treating vitiligo that avoids the risks associated with simvastatin administered orally at high does.
      In conclusion, our study shows that simvastatin protects melanocytes from oxidative damage by activation of Nrf2. Additional studies using vitiligo mice and human skin xenografts are needed to confirm the therapeutic potential of simvastatin for vitiligo.

      Materials and Methods

      Cell culture and treatment

      Primary human melanocytes were extracted from human foreskin specimens obtained during circumcision surgery and cultured in Medium 254 (Gibco, Grand Island, NY) supplemented with human melanocyte growth supplement (Gibco) at 37 °C in the presence of 5% CO2, as previously reported (
      • Shi Q.
      • Zhang W.
      • Guo S.
      • Jian Z.
      • Li S.
      • Li K.
      • et al.
      Oxidative stress-induced overexpression of miR-25: the mechanism underlying the degeneration of melanocytes in vitiligo.
      ). The second- through fourth-passage melanocytes were used in all experiments. Each experiment was repeatedly performed in primary human melanocytes from at least from three different sources. All samples were collected with the approval of the Institutional Review Board of Fourth Military Medical University, Xi’an, China. Written informed consent was obtained from all donors, according to the Declaration of Helsinki. Oxidative stress in primary human melanocytes was induced by treatment with 1.0 mmol/L H2O2 (Sigma-Aldrich, St. Louis, MO) for 24 hours. Simvastatin (Calbiochem, San Diego, CA), cholesterol (Sigma), and mevalonate (Sigma) were added at indicated concentrations and times. To determine whether JNK and Erk pathways play a role in simvastatin-induced Nrf2 activation, primary melanocytes were pretreated with 25 mmol/L JNK inhibitor (SP600125, ApexBio, Hsinchu, Taiwan) and 50 mmol/L Erk inhibitor (PD98059, ApexBio) for 30 minutes. Aspirin (Sigma) was used at 90 μmol/L.

      Determination of cell viability by CCK-8 assay

      CCK-8 reduction assay kits (Beyotime Biotechnology, Shanghai, China) were used as a qualitative index of cell viability according to the manufacturer’s instructions (
      • Ge R.
      • Liu L.
      • Dai W.
      • Zhang W.
      • Yang Y.
      • Wang H.
      • et al.
      Xeroderma pigmentosum group A promotes autophagy to facilitate cisplatin resistance in melanoma cells through the activation of PARP1.
      ). The details are described in the Supplementary Materials and Methods online.

      Annexin V-FITC/propidium iodide apoptosis assay

      Melanocytes were plated into six-well plates at a density of 2 to 3 × 105 cells/well. Cell apoptosis was detected by using kits (MaiBio, Shanghai, China). FITC and propidium iodide fluorescence were measured using flow cytometry (Beckman Coulter, Brea, CA) and analyzed with Expo32 software (
      • Jian Z.
      • Tang L.
      • Yi X.
      • Liu B.
      • Zhang Q.
      • Zhu G.
      • et al.
      Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2 -induced oxidative stress.
      ).

      Determination of intracellular ROS

      Melanocytes were seeded into six-well plates at the density of 2 to 3 × 105 cells/well. After drug treatment, the intracellular ROS level was assessed by the fluorescent probe CM-H2DCFDA (Invitrogen, Waltham, MA); details have been described previously (
      • Jian Z.
      • Tang L.
      • Yi X.
      • Liu B.
      • Zhang Q.
      • Zhu G.
      • et al.
      Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2 -induced oxidative stress.
      ).

      Detection of SOD and CAT activity

      After cells were lysed, the total protein was extracted to detect the activity of SOD and CAT by using the kits (Total Superoxide Dismutase Assay Kit with WST-8, Total Catalase Analysis Kit, Beyotime Biotechnology) (
      • Zhou J.
      • Li Y.
      • Yan G.
      • Bu Q.
      • Lv L.
      • Yang Y.
      • et al.
      Protective role of taurine against morphine-induced neurotoxicity in C6 cells via inhibition of oxidative stress.
      ). More details are given in the Supplementary Materials and Methods.

      RNA isolation and quantitative real-time PCR

      Total RNA was isolated using Trizol reagent (Invitrogen), and then reverse-transcribed to cDNA using PrimeScript RT reagentKit (TaKarRa, Ohtsu, Japan). Quantitative real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa) with the iQ5 PCR Detection System (Bio-Rad, Hercules, CA) as described in Supplementary Materials and Methods.

      Western blot

      Nuclear-cytoplasmic fractionation was conducted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Fisher Scientific, Waltham, MA). Proteins were extracted from cells and quantified using BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were separated by 10% SDS-PAGE (Bio-Rad) and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). After blocking in a solution of 5% nonfat dry milk diluted in Tris-buffered saline, the membranes were incubated with primary antibodies (see the Supplementary Material and Supplementary Table S1 online) and then with horseradish peroxidase-conjugated secondary antibodies. Bound antibodies were detected using the ECL Western blotting detection system (Millipore).

      Immunofluorescence

      Melanocytes were grown and treated on single-layer glass slides, as previously reported (
      • Jian Z.
      • Tang L.
      • Yi X.
      • Liu B.
      • Zhang Q.
      • Zhu G.
      • et al.
      Aspirin induces Nrf2-mediated transcriptional activation of haem oxygenase-1 in protection of human melanocytes from H2O2 -induced oxidative stress.
      ). After they were washed and fixed, cells were incubated with primary antibodies (see Supplementary Material and Supplementary Table S1) overnight. The cells were subsequently incubated with the secondary antibodies (Cy3-tagged rabbit anti-goat, FITC-tagged mouse anti-goat) for 1 hour and the nuclear dye (DAPI) for 15 minutes at room temperature. Fluorescent images were obtained by an FV-1000/ES confocal microscope (Olympus, Tokyo, Japan).

      RNA interference

      Cells were seeded at 2 × 105 cells per well for 24 hours before transfection. Cells were transfected with Nrf2 shRNA and irrelevant shRNA control (GenePharma, China), or with p62 siRNA or control siRNA (Sangon Biotech, Shanghai, China) with Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. More details are described in the Supplementary Materials and Methods.

      Statistical analyses

      Data analysis was performed using GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA). Dual comparisons were made with two-tailed Student unpaired t test. Groups of three or more were analyzed by one-way analysis of variance with Dunnett posttests. P-values less than 0.05 were considered significant. Data represent the mean ± standard deviation for at least three independent experiments.
      Further details are available in Supplementary Materials and Methods.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by National Natural Science Foundation of China (no. 81625020, no. 81472863, no. 81172749, no. 81602764, no. 81402599, no. 81472893).

      Supplementary Material

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