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

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

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/LH2O2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1h后再用1 mmol/L H2O2处理1h).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1h,再用1 mmol/LH2O2处理1h)和H2O2组(直接用1 mmol/LH2O2处理1h),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、4h后,再用H2O2处理1h,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1h后,再用H2O2处理1h,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、25μmol/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、4h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.

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.