Protective effect of WSY6, a caffeic acid derivative, on oxidative stress injury in melanocytes and its mechanism / 中华皮肤科杂志

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.