Abstract: Jin Rong, Zhu Yiping, Lin Fuquan, Liu Dongyin, Fu Lifang, Xu Ai′e Department of Dermatology, Hangzhou Third People′s Hospital, Hangzhou 310009, China Corresponding author: Xu Ai′e, Email: xuaiehz@msn.com 【Abstract】 Objective To evaluate the protective effect of WSY6 （a caffeic acid derivative） on hydrogen peroxide （H2O2）-induced oxidative stress injury in melanocytes, and to explore its potential molecular mechanism. Methods In vitro cultured human primary melanocytes were divided into 5 groups: control group receiving no treatment, H2O2 group treated with 1 mmol/L H2O2 , 6.25, 12.5, 25 μmol/L WSY6 groups pretreated with 6.25, 12.5, 25 μmol/L WSY6 respectively followed by 1-hour treatment with 1 mmol/L H2O2 . After 24-hour treatment, MTS assay was performed to determine the survival rate of melanocytes, and the lactate dehydrogenase（LDH）kit was used to detect the LDH leakage level. Some melanocytes were divided into 2 groups: inhibitor group pretreated with the p38 inhibitor for 1 hour followed by 1-hour treatment with 1 mmol/L H2O2 , and H2O2 group treated with 1 mmol/L H2O2 for 1 hour. After 24-hour treatment, the LDH kit was used to detect the LDH leakage level. Some other melanocytes were pretreated with 25 μmol/L WSY6 for 1, 2, 4 hours separately, followed by 1-hour treatment with H2O2 . Then, flow cytometry was conducted to detect the level of intracellular reactive oxygen species （ROS）. Some melanocytes were treated with 6.25, 12.5, 25 μmol/L WSY6 separately for 1 hour, followed by 1-hour treatment with H2O2 . Then, Western bolt analysis was performed to determine the protein expression of cytochrome c （cyto-c）, caspase-3, caspase-9, phosphorylated（p）-p38 MAPK, p-ERK and p-JNK. Results Compared with the control group, the H2O2 group showed significantly decreased survival rate of melanocytes （29.22% ± 1.31%, P < 0.05）, but significantly increased intracellular LDH leakage level （47.19% ± 4.85%, P < 0.05）, elevated intracellular ROS level （18.37 ± 1.59, P < 0.05）, and increased expression of caspase-3 and caspase-9. Compared with the H2O2 group, the 6.25, 12.5, 25 μmol/L WSY6 groups showed significantly increased cell survival rate （52.48% ± 1.17%, 60.21% ± 0.25%, 78.32% ± 1.73%, P < 0.05）, but significantly decreased LDH leakage level （21.99% ± 0.22%, 15.38% ± 0.45%, 13.18% ± 0.38%, P < 0.05）, and the intracellular ROS level was significantly decreased in the 25 μmol/L WSY6 group after 1, 2, 4 hours of treatment（7.59 ± 1.00, 6.22 ± 0.52, 5.1 ± 0.48, P < 0.05）. The LDH leakage level in melanocytes was significantly lower in the p38 inhibitor group than in the H2O2 group （P < 0.05）. Western blot analysis revealed that after the pretreatment with 6.25, 12.5, 25 μmol/L WSY6 separately, the WSY6 groups all showed obviously decreased expression of caspase-3, caspase-9 and p-p38 compared with the H2O2 group. However, there was no obvious difference in the expression of p-ERK and p-JNK between the WSY6 groups and the H2O2 group. Besides, the WSY6 groups showed decreased expression of p-p53 （a downstream product of p38 MAPK）, which decreased along with the increase in the concentration of WSY6. Conclusion WSY6 shows a markedly protective effect on H2O2-induced oxidative stress injury in melanocytes, likely through the p38 MAPK pathway.

Key words: Vitiligo, Melanocytes, Oxidative stress, Caffeic acid, Lactate dehydrogenases, Extracellular signal?regulated MAP kinases