咖啡酸衍生物WSY6对黑素细胞氧化应激损伤的保护作用及机制研究

目的 探讨咖啡酸衍生物WSY6对过氧化氢（H2O2）诱导的黑素细胞氧化损伤的保护作用和潜在分子机制。方法 体外培养原代人表皮黑素细胞，分为对照组（不做任何处理）、H2O2组（1 mmol/L H2O2处理）和6.25、12.5、25 μmol/LWSY6组（分别用6.25、12.5、25 μmol/L WSY6预处理1 h后再用1 mmol/L H2O2处理1 h）。继续培养24 h后，用MTS法测定黑素细胞存活率，乳酸脱氢酶（LDH）试剂盒检测LDH释放量。部分黑素细胞分为抑制剂组（用p38抑制剂预处理1 h，再用1 mmol/L H2O2处理1 h）和H2O2组（直接用1 mmol/L H2O2处理1 h），处理完成后继续培养24 h，用试剂盒检测LDH的释放量。部分黑素细胞用25 μmol/L WSY6预处理1、2、4 h后，再用H2O2处理1 h，流式细胞仪检测细胞内活性氧（ROS）水平；部分黑素细胞用6.25、12.5、25 μmol/L WSY6处理1 h后，再用H2O2处理1 h，Western印迹法检测细胞色素C（cyto?c）、半胱氨酸天冬氨酸蛋白酶3（caspase?3）、caspase?9和磷酸化丝裂原活化的蛋白激酶（p?p38 MAPK）、细胞外调节蛋白激酶（p?ERK）及c?Jun氨基末端激酶（p?JNK）的表达。结果 与对照组相比，H2O2组黑素细胞存活率明显降低（29.22% ± 1.31%，P < 0.05），细胞内LDH释放量增加（47.19% ± 4.85%，P < 0.05），ROS水平明显升高（18.37 ± 1.59，P < 0.05），caspase?3和caspase?9的表达亦均升高。与H2O2组相比，6.25、12.5、25 μmol/L WSY6组细胞存活率明显增加（52.48% ± 1.17%、60.21% ± 0.25%、78.32% ± 1.73%，P < 0.05），LDH释放量明显下降（21.99% ± 0.22%、15.38% ± 0.45%、13.18% ± 0.38%，均P < 0.05），25 μmol/L WSY6处理1、2、4 h组细胞内ROS显著下降（7.59 ± 1.00、6.22 ± 0.52、5.15 ± 0.48，均P < 0.05）。同时p38抑制剂组黑素细胞LDH释放量较H2O2组显著下降（P < 0.05）。Western印迹法显示，WSY6预处理后，与H2O2组相比，caspase?3和caspase?9表达明显降低，p?p38表达下降，但p?ERK和p?JNK表达无明显变化；同时WSY6组p38 MAPK下游产物p?p53表达也下降，且WSY6浓度越高，下降越明显。结论 WSY6对H2O2诱导的黑素细胞氧化应激损伤有显著的保护作用，可能通过p38 MAPK途径发挥作用。

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

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.