Abstract:

Ning Weixuan*, Wang Suiquan, Hong Weisong, Liu Dongyin, Xu Ai′e. *Department of Dermatology, Guangxing Hospital Affiliated to Zhejiang Chinese Medical University, Hangzhou 310053, China Corresponding author: Xu Ai′e, Email: xuaiehz@msn.com 【Abstract】 Objective To investigate the protective effect of acetylated epigallocatechin gallate （AcEGCG） against H2O2-induced oxidative damage to human epidermal melanocytes, and to explore its possible mechanism. Methods Human epidermal melanocytes were isolated and cultured in vitro. Some melanocytes were classified into a H2O2 group induced by H2O2 only, EGCG groups and AcEGCG groups induced by H2O2 after pretreatment with different concentrations of EGCG and AcEGCG, respectively. Three concentrations （10, 20 and 40 μmol/L） of EGCG or AcEGCG were used to treat melanocytes for 1 hour in MTS assay and lactate dehydrogenase （LDH） leakage assay and for 2 hours in Western blot assay, while only one concentration （40 μmol/L） was used to treat melanocytes for 0.5, 1, 2 and 4 hours respectively in flow cytometry assay. Some melanocytes treated with only culture medium and 0.1% dimethyl sulphoxide （DMSO） served as the control group. After additional culture, MTS assay was performed to determine cell survival rate, flow cytometry to detect the level of reactive oxygen species （ROS） in melanocytes, Western blot to measure the expressions of caspase-9 and caspase-3 proteins. Lactate dehydrogenase （LDH） kit was used to detect the leakage of LDH to culture medium. Statistical analysis was carried out by using one-way analysis of variance for comparisons of multiple group means followed by Student-Newman-Keuls-q （SNK-q） test for multiple comparisons. Results Compared with the control group, the H2O2 group showed significantly decreased cell survival rate （22.99% ± 0.53%, P < 0.01）, but increased LDH leakage level （36.58% ± 0.73%, P < 0.01）, intracellular ROS level （19.08 ± 0.57, P < 0.01）, as well as caspase-9 （2.65 ± 0.079, P < 0.01） and caspase-3 （2.36 ± 0.057, P < 0.01） expressions. In comparison with the H2O2 group, the cell survival rate was significantly higher in the 10-, 20- and 40-μmol/L AcEGCG groups （79.50% ± 3.62%, 86.52% ± 5.13%, 97.81% ± 5.21%, respectively, all P < 0.01） and EGCG groups （43.19% ± 1.68%, 63.34% ± 3.60%, 70.82% ± 2.1%, respectively, all P < 0.01）. However, the 10-, 20- and 40-μmol/L AcEGCG groups and EGCG groups all showed a significant decrease in the expression levels of caspase-9 （AcEGCG groups: 1.44 ± 0.067, 1.26 ± 0.059 and 1.10 ± 0.072 respectively; EGCG groups: 2.31 ± 0.085, 2.13 ± 0.091 and 1.35 ± 0.064 respectively, all P < 0.05） and caspase-3 （AcEGCG groups: 1.70 ± 0.053, 1.57 ± 0.057 and 1.24 ± 0.068 respectively, all P < 0.05; EGCG groups: 2.09 ± 0.076, 1.98 ± 0.093 and 1.79 ± 0.056 respectively, all P < 0.05） compared with the H2O2 group. Similarly, a significant reduction was observed in the leakage level of LDH in these AcEGCG and EGCG groups （all P < 0.01） and in ROS levels in the 40-μmol/L AcEGCG and EGCG groups when compared with the H2O2 group. Conclusions AcEGCG has a stronger protective effect against H2O2-induced oxidative damage to human epidermal melanocytes compared with EGCG, which may be realized through clearance of free radicals, antioxidant effects, and decrease of caspase-9 and caspase-3 expressions.