Protective effect of WSY6, a caffeic acid derivative, on oxidative stress injury in melanocytes and its mechanism

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