全乙酰化表没食子儿茶素没食子酸酯对氧化应激导致黑素细胞损伤的保护作用研究

目的 研究全乙酰化表没食子儿茶素没食子酸酯（AcEGCG）对H2O2诱导的人表皮黑素细胞损伤的保护作用，探讨其可能的作用机制。 方法 体外培养人表皮黑素细胞，采用H2O2诱导细胞损伤模型。实验分对照组、H2O2组、不同浓度表没食子儿茶素没食子酸酯（EGCG）组和AcEGCG组。MTS法测定细胞存活率，LDH测试盒检测乳酸脱氢酶（LDH）的漏出量，流式细胞仪检测细胞内活性氧（ROS）的表达水平；Western印迹法检测半胱氨酸天冬氨酸蛋白酶9（caspase-9）和半胱氨酸天冬氨酸蛋白酶3（caspase-3）的表达情况。多个样本均数比较采用单因素方差分析，各样本间多重比较采用SNK-q检验。 结果 与对照组相比，H2O2处理组黑素细胞存活率明显降低（22.99% ± 0.53%，P < 0.01），细胞内LDH漏出量增加（36.58% ± 0.73%，P < 0.01），细胞内ROS水平明显升高（19.08 ± 0.57，P < 0.01），caspase-9和caspase-3表达升高（分别为2.65 ± 0.079和2.36 ± 0.057，均P < 0.01）。与H2O2组相比，细胞存活率在10、20、40 μmol/L AcEGCG组（79.50% ± 3.62%、86.52% ± 5.13%、97.81% ± 5.21%）及EGCG组（43.19% ± 1.68%、63.34% ± 3.60%、70.82% ± 2.1%）明显增加（均P < 0.01），caspase-9分别降低为1.44 ± 0.067、1.26 ± 0.059、1.10 ± 0.072及2.31 ± 0.085、2.13 ± 0.091、1.35 ± 0.064，caspase-3分别降低为1.70 ± 0.053、1.57 ± 0.057、1.24 ± 0.068及2.09 ± 0.076、1.98 ± 0.093、1.79 ± 0.056，差异均有统计学意义（P < 0.05）；LDH含量均明显下降（P < 0.01）；40 μmol/L AcEGCG或EGCG处理后，ROS含量明显下降（均P < 0.01）。 结论 AcEGCG对H2O2诱导的黑素细胞氧化应激损伤有显著的保护作用，且显著优于EGCG，可能通过清除自由基、抗氧化、降低caspase-9及caspase-3表达等途径发挥作用。

Protective effect of acetylated epigallocatechin gallate on melanocytes from oxidative stress-induced damage

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.