白藜芦醇对UVA照射HaCaT细胞的防护效应及其机制

Protective effect of resveratrol on UVA-irradiated HaCaT cells and its mechanism

采用Cell Counting Kit-8(CCK-8试剂盒)检测HaCaT细胞的增殖水平。Annexin V-FITC/PI流式细胞术检测HaCaT细胞的凋亡率和死亡率。单丹磺酰尸胺(Monodansylcadaverine,MDC)荧光染色和二氯荧光黄二乙酸酯(Dichlorodihydrofluorescein diacetate,DCFH-DA)探针标记后,用激光共聚焦显微镜进行扫描,分别检测HaCaT细胞内自噬体和活性氧(Reactive oxygen species,ROS)水平变化。Western blot检测HaCaT细胞内LC3B-II、磷酸化AKT(p-AKT)和总AKT蛋白表达变化。结果显示:经10~80 J/cm^2 UVA照射后,HaCaT细胞的增殖水平随受照剂量的增加逐渐降低(p<0.05);白藜芦醇预处理能显著促进UVA照射后HaCaT细胞增殖(p<0.01),降低其凋亡率和死亡率(p<0.05);经20 J/cm^2 UVA照射后3~24 h,HaCaT细胞内LC3B-II蛋白表达增强(p<0.01);白藜芦醇预处理能使UVA照射后HaCaT细胞内自噬增强(p<0.01),ROS水平降低(p<0.01),p-AKT蛋白表达增强(p<0.01)。提示白藜芦醇可能通过促进细胞自噬和AKT蛋白磷酸化,以及降低细胞内ROS水平,拮抗UVA导致的HaCaT细胞增殖抑制、凋亡和死亡。

The Cell Counting Kit-8 （CCK-8） was used to detect the cell survival rate of HaCaT cells. The apoptosis rate and mortality of HaCaT cells were detected using flow cytometry with the Annexin VFITC and PI double labeling method. Autophagosomes and the generation of reactive oxygen species （ROS） in HaCaT cells were monitored by laser scanning confocal microscopy using monodansylcadaverine （MDC） and dichlorodihydrofluore- scein diacetate （DCFH-DA） staining, respectively. The expression of LC3B-Ⅱ, p-AKT, and total AKT protein in HaCaT cells was determined by western blotting. The results showed that HaCaT cell proliferation was inhibited gradually （p〈0.05） after irradiation with a dose of 10~80 J/cm^2 UVA. Treatment with resveratrol enhanced the proliferation of UVA-irradiated HaCaT cells （p〈0.01） and decreased their apoptosis rate and mortality （p〈0.05）. The expression of LC3B-Ⅱ protein was increased （p〈0.01） after irradiation with 20 J/cm^2 UVA for 3~24 h. After treatment with resveratrol, autophagy was enhanced （p〈0.01）, intracellular ROS level was decreased （p〈0.01）, and p-AKT expression was significantly increased （p〈0.01） in UVA-irradiated HaCaT cells. These results indicate that resveratrol could reduce UVA-induced proliferative inhibition, apoptosis, and death in HaCaT cells by enhancing the autophagic process and p-AKT protein expression and by reducing ROS production.