Ginkgo biloba extract protects human melanocytes from H2O2‐induced oxidative stress by activating Nrf2

Abstract Vitiligo is a common skin depigmenting disorder characterized by the loss of functional melanocytes. Its pathogenesis is complicated and oxidative stress plays a critical role in the development of vitiligo. Thus, antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of depigmentation. Ginkgo biloba extract EGb761 has been confirmed to have protective effects on neurons against oxidative stress. Notably, several clinical trials have shown that patients with stable vitiligo achieved repigmentation after taking EGb761. However, the exact mechanism underlying the protective effects of EGb761 on melanocytes against oxidative stress has not been fully elucidated. In the present study, we found that EGb761 effectively protected melanocytes against oxidative stress‐induced apoptosis and alleviated the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation by enhancing the activity of antioxidative enzymes. Furthermore, the antioxidative effect of EGb761 was achieved by activating Nrf2 and its downstream antioxidative genes. In addition, interfering Nrf2 with siRNA abolished the protective effects of EGb761 on melanocytes against oxidative damage. In conclusion, our study proves that EGb761 could protect melanocytes from H2O2‐induced oxidative stress by activating Nrf2. Therefore, EGb761 is supposed to be a potential therapeutic agent for vitiligo.

extract EGb761 has been confirmed to have protective effects on neurons against oxidative stress. Notably, several clinical trials have shown that patients with stable vitiligo achieved repigmentation after taking EGb761. However, the exact mechanism underlying the protective effects of EGb761 on melanocytes against oxidative stress has not been fully elucidated. In the present study, we found that EGb761 effectively protected melanocytes against oxidative stress-induced apoptosis and alleviated the excessive accumulation of reactive oxygen species (ROS) and lipid peroxidation by enhancing the activity of antioxidative enzymes. Furthermore, the antioxidative effect of EGb761 was achieved by activating Nrf2 and its downstream antioxidative genes.
In addition, interfering Nrf2 with siRNA abolished the protective effects of EGb761 on melanocytes against oxidative damage. In conclusion, our study proves that EGb761 could protect melanocytes from H 2 O 2 -induced oxidative stress by activating Nrf2. Therefore, EGb761 is supposed to be a potential therapeutic agent for vitiligo.

K E Y W O R D S
EGb761, Melanocyte, Nrf2, Oxidative stress, Vitiligo EGb761 could prevent hippocampus neurons from oxidative damage by enhancing antioxidative ability of neurons, improving the microenvironment for nerve regeneration and promoting the proliferation of nerve cells. 4,6,7 It is known that melanocytes originate from neural crest, so it has raised concerns about how EGb761 affects the growth of melanocyte cell lines in recent years. One study reported that EGb761 could protect melanocytes against oxidative stress by activating antioxidase and inhibiting endoplasmic reticulum stress. 8 However, some other studies have controversial findings that Ginkgo biloba or its principal components could induce the apoptosis of melanoma cells. 9,10 Notably, several clinical trials have illustrated that the application of Ginkgo biloba extract can prevent the onset and the progression of vitiligo, indicating its therapeutic potential in treating vitiligo. 11 However, the exact mechanism underlying the effect of EGb761 on vitiligo remains unclear.
The transcription factor nuclear erythroid 2-related factor (Nrf2), which governs the natural function of phase II detoxification and the expression of antioxidant enzymes, is a master regulator of cellular defence against oxidative stress. 12  is produced as a cell line with unlimited growth potential and normal melanocytic properties. 17 Moreover, our previous studies have demonstrated that PIG1 cells have similar biological characteristics as primary normal human melanocytes under oxidative stress. 18,19 In the present study, we made use of PIG1 cells to evaluate the protective effects of EGb761 on melanocytes under oxidative stress and testify our hypothesis.

| Experimental reagents and antibodies
The following reagents were obtained from the indicated commer-

| MTT assay
PIG1 cells were cultured in 96-well microtitre plates for 24 hours at 37℃ and 5% CO 2 . After different treatments, the supernatant was flicked off and washed once. Twenty μL of MTT (5 mg/mL) in PBS solution was then added to each well, and the plates were further incubated for 4 hours. After this, 150 μL of DMSO was added into the wells to solubilize the crystals. Finally, the optical density (OD) was measured at a wavelength of 490 nm. Cell survival rates were expressed as folds of that of untreated group (control).

| Measurement of MDA levels
Malondialdehyde in cells was measured using commercial kits according to the manufacturer's recommendations. The absorbance of the supernatant was determined at 532 and 600 nm. Malondialdehyde levels were expressed as nmol/mg protein.

| Measurement of intracellular reactive oxygen species
Reactive oxygen species was monitored using dihydrodichlorofluo-

| Protein extraction and Western blot analysis
After the completion of respective treatments, PIG1 cells were harvested and cellular protein lysate was prepared using lysis buffer [20 mmol/L Tris-HCl, 120 mmol/L NaCl, 1.0% Triton X 100, pH 7.5, 10% glycerol, 2 mmol/L EDTA and protease inhibitor cocktail (Roche GmbH, Germany)] at 4°C for 30 min. After centrifugation at 10 000 g for 20 minutes, the supernatant was separated and stored at −80°C until use. Protein concentrations were determined using 1× Bradford protein assay kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) as a standard.
For Western blot, equal amounts of protein lysates were separated in 10% sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gel electrophoresis. The gels were blotted onto a nitrocellulose membrane and incubated with the indicated antibodies. Blots were developed by enhanced chemiluminescence (ECL) according to the manufacturer's instructions. Actin was used as a loading control.

| Quantitative polymerase chain reaction
Total RNA was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA). RNA concentrations and purities were determined by measuring the absorbance at 260 nm. First-strand cDNA was generated using SuperScript III first-strand synthesis system as

| Statistical analysis
The statistical analyses were performed using spss 16.0 (SPSS Inc, Chicago, IL). All the values were presented as mean ± standard deviation (SD). Statistical analysis was performed by two-tailed Student's t test or ANOVAs, followed by Bonferroni/Dunn post hoc comparisons. P < 0.05 was regarded statistically significant.

| EGb761 improves cell viability and inhibits the production of ROS and lipid peroxidation in melanocytes under oxidative stress
To examine whether EGb761 could protect melanocytes under oxidative stress, we built the oxidative stress model of melanocytes based on our previous studies, which was made by treating PIG1 cells with

| EGb761 restores the activity of SOD and GSH-Px in melanocytes under oxidative stress
Previous studies have indicated that the imbalance in redox state is involved in the pathogenesis of vitiligo. 14,20,21 As EGb761 effectively reduced ROS level in H 2 O 2 -treated PIG1 cells, we speculated that the effect of EGb761 could enhance the activity of antioxidative enzymes in melanocytes. To testify this, the activity of two main antioxidative enzymes including SOD and GSH-Px were measured in H 2 O 2 -treated melanocytes. As expected, H 2 O 2 -treated melanocytes displayed impaired activity of SOD and GSH-Px by 59% and 57%, respectively, while the pretreatment with EGb761 restored almost half of the inhibited activity of the two enzymes caused by H 2 O 2 (Figure 2A,B).

| EGb761 activates Nrf2-ARE signaling pathway in melanocytes under oxidative stress
To further identify whether the protective effect of EGb761 on melanocytes under oxidative stress is dependent on Nrf2-ARE pathway, the mRNA and protein levels of Nrf2 and its downstream genes were detected in PIG1 cells treated with H 2 O 2 by qPCR and Western blot respectively. As is illustrated in Figure 3B

| Nrf2 knockdown diminishes the protective effect of EGb761 on melanocytes under oxidative stress
To further demonstrate the involvement of Nrf2 on the protective effect of EGb761 on melanocytes under oxidative stress, we established PIG1 cell lines with Nrf2 knockdown by using siRNA transfection. After the transfection with si-Nrf2, the protein levels of
We further found that the pretreatment with EGb761 effectively increased the protein levels of Nrf2, SOD2, HO-1 and NQO1 in PIG1 cells transfected with scrambled siRNA (si-NC), which, however, could not be observed in the cells with Nrf2 knockdown ( Figure 4A,D). Subsequently, the viability of PIG1 cells was measured by MTT assay after the transfection with si-Nrf2 or si-NC. Three repeated experiments were made, and paired t test was used to make statistics. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 P = 0.184) in Nrf2 knockdown group ( Figure 4B).The inhibitory effect of EGb761 on intracellular ROS level in PIG1 cells treated with H 2 O 2 was also diminished after the transfection with si-Nrf2 ( Figure 4C). Collectively, these data obviously indicate that the cytoprotective effect of EGb761 on melanocytes under oxidative stress is achieved by activating Nrf2.

| D ISCUSS I ON
In the present study, we primarily found that EGb761 could prevent  24 Another study found that 60 mg of Ginkgo biloba BID could decrease the disease severity in vitiligo patients. 25 F I G U R E 3 Effect of EGb761 on Nrf2 and HO-1 protein and gene expression. Cells were pretreated with EGb761 for 48 h, and then incubated with 1.0 mmol/L H 2 O 2 for 24 h. The mRNA levels of NQO1, SOD2, Nrf2 and HO-1 were semi-quantified by RT-PCR analyses (A). Actin was used as a loading control. The mRNA expressions of Nrf-2, NQO1, SOD2 and HO-1 in EGb761 pretreatment group were significantly increased compared with single H 2 O 2 treatment group (the increased folds as 0.7, 1.2, 0.4 and 0.4, respectively; P = 0.037, 0.008, 0.033 and 0.007 respectively). Total cell lysates were prepared and subjected to Western blot analysis to monitor the expression levels of antioxidant proteins including NQO1, SOD2, Nrf2 and HO-1 (B and C). Actin served as an internal control. The protein expression levels of Nrf2 and its downstream antioxidative genes including NQO1, SOD2 and HO-1 increased slightly in H 2 O 2 -treated group, and they were further increased significantly in EGb761-pretreated group (the increased folds compared with single H 2 O 2 -treated group as 1.2-fold, 1.3-fold, 0.5-fold and 0.3-fold, respectively; P = 0.029, 0.03, 0.04 and 0.027 respectively). Three repeated experiments were made, and paired t test was used to make statistics. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01 In the present study, we initially found that EGb761 showed no toxic effect on PIG1 cells (data not shown), which was consistent with previous studies. 10 The patients with vitiligo have inferior capacity against oxidative stress due to the lower activity of SOD and GSH-Px. 20 Our previous studies have confirmed that defective Nrf2-ARE activation leads to impaired ability of melanocytes to resist oxidative stress damage, and enhancing Nrf2 activity is a potential therapeutic strategy for the treatment of vitiligo. 14 In the present study, EGb761 effectively up-regulated Nrf2 and its downstream antioxidative genes at both transcriptional and translational level. Besides, the knockdown of Nrf2 diminished the protective effect of EGb761 on melanocytes against oxidative stress. Collectively, these data strongly prove that the antioxidant effect of EGb761 on melanocytes is achieved by activating Nrf2.
EGb761 is widely used in treating cardiovascular, neurological, endocrinological and immunological disorders. 4-7 Furthermore, its safety and effectiveness in clinical use have been well recognized and its adverse effects are rare. 26 A clinical study reported that vitiligo patients achieved repigmentation by using 40 mg of Ginkgo biloba extract TID with slight adverse effects like mild nausea. 24 In the present study, 100 μg/ml of EGb761, which was lower than the concentration of EGb761 for treating other diseases, could effectively protect melanocytes against H 2 O 2 -induced oxidative damage. Therefore, the application of this concentration would be safe to vitiligo patients.
In conclusion, EGb761 can protect melanocytes against oxidative stress damage through the activation of Nrf2-ARE signaling pathway. Our study provides a new strategy in treating vitiligo with traditional Chinese medicine. As EGb761 is widely used in the treatment of oxidative stress-related diseases with remarkable safety and tolerance, its application in the treatment of vitiligo is prospective.

ACK N OWLED G EM ENTS
This work was supported by National Natural Science Foundation of China (No. 91742201, No. 81602750), Key Research and F I G U R E 4 Silencing Nrf2 gene abolished the prevention made by EGb761. PIG1 cells were transfected with siRNA or empty vector for 36 h, followed by 48 h pretreatment with EGb761 at 100 μg/ml, and then subjected to H 2 O 2 (1.0 mmol/L). The expressions of Nrf2, SOD2, HO-1 and NQO1 were measured by Western blotting (A and D). The pretreatment with EGb761 increased the levels of Nrf2, SOD2, HO-1 and NQO1 in si-NC group, which was not observed in si-Nrf2 group. The cell viability (B) and ROS level (C) of cells were determined respectively. Compared with single H 2 O 2 -treated cells, the pretreatment with EGb761 significantly increased the number of viable cells in the si-NC group, whereas the percentage of viable cells was further decreased and it failed to reverse in si-Nrf2 group. The ability of EGb761 to inhibit ROS levels was also diminished in si-Nrf2 group. Three repeated experiments were made, and paired t test was used to make statistics. Data are presented as the mean ± SD, *P < 0.05, **P < 0.01