乙酰化白藜芦醇对长波紫外线照射后HaCaT细胞内NRF2活化及活性氧水平的影响  

Effect of acetyl-resveratrol on NRF2 activity and reactive oxygen species level in UVA-irradiated HaCaT cells

作　　者：王春燕[1] 李辰[1] 李宁[1] 齐雪松[1] 邵帅[1] 曲功霖[1] 佟鹏[1] 苟巧[1] 

机构地区：[1]中国疾病预防控制中心辐射防护与核安全医学所辐射防护与核应急中国疾病预防控制中心重点实验室毒理学研究室,北京100088

出　　处：《中国生物制品学杂志》2017年第12期1274-1279,共6页Chinese Journal of Biologicals

摘　　要：目的探讨乙酰化白藜芦醇对长波紫外线(UVA)照射后人永生化角质形成细胞HaCaT内核转录相关因子NRF2(NF-E2-related factors 2)活化和活性氧(reactive oxygen species,ROS)水平的影响。方法用CCK-8试剂盒检测不同剂量(0、5、10、20、30、40、60、80 J/cm^2)UVA照射后24 h,及不同浓度(0、0.5、1、2μmol/L)乙酰化白藜芦醇干预24 h后再经20 J/cm^2 UVA照射后24 h HaCaT细胞的存活率;Western blot检测20 J/cm^2 UVA照射HaCaT细胞后不同时间点(0、3、6、12、24、48 h)及2μmol/L乙酰化白藜芦醇干预24 h后再经20 J/cm^2 UVA照射后6 h细胞核内NRF2蛋白表达的变化;Annexin V-FITC/PI流式细胞术检测2μmol/L乙酰化白藜芦醇干预HaCaT细胞24 h后再经20 J/cm^2 UVA照射后24 h细胞的凋亡率和死亡率;二氯荧光黄二乙酸酯(dichlorodihydrofluorescein diacetate,DCFH-DA)荧光探针标记细胞检测HaCaT细胞中ROS水平的变化。结果 10~80 J/cm^2 UVA照射后24 h,HaCaT细胞存活率随照射剂量的增加逐渐降低(P<0.05);20 J/cm^2 UVA照射HaCaT细胞后0 h,细胞核内NRF2的表达急剧增强(P<0.01),照射后3~48 h又逐渐减弱(P<0.05)。乙酰化白藜芦醇预处理能显著提高UVA照射后细胞的存活率(P<0.05),降低其凋亡率和死亡率(P<0.05),促进细胞核内NRF2蛋白的表达(P<0.01),并降低细胞内ROS水平(P<0.01)。结论乙酰化白藜芦醇能减轻UVA照射引起的HaCaT细胞增殖抑制、凋亡和死亡,其机制可能与其促进NRF2蛋白活化,降低细胞内ROS水平,从而减轻UVA照射对细胞的氧化损伤有关。Objective To investigate the effect of acetyl-resveratrol on activity of NRF2（NF-E2-related factor 2） and level of reactive oxygen species（ROS） in UVA-irradiated HaCaT cells. Methods CCK-8 Assay Kit was used to determine the survival rates of HaCaT cells irradiated by UVA at dosages of 0, 5, 10, 20, 30, 40, 60 and 80 J/cm^2 for24 h and those irradiated by UVA at a dosage of 20 J/cm^2 for 24 h after treatment with acetyl-resveratrol at concentrations of 0, 0. 5, 1 and 2 μmol/L respectively. The expressions of nuclear NRF2 protein in HaCaT cells at various time points（0, 3, 6, 12, 24 and 48 h）after irradiation by 20 J/cm^2 UVA and in those treated with 2 μmol/L acetyl-resveratrol for24 h then irradiated by 20 J/cm^2 UVA for 6 h were determiend by Western blot. The apoptosis rate and mortality of HaCaT cells treated with 2 μmol/L acetyl-resveratrol for 24 h then irradiated by 20 J/cm^2 UVA for 24 h were determined by Annexin V-FITC/PI flow cytometry. The ROS level in HaCaT cells was determined by laser scanning confocal microscopy with dichlorodihydrofluorescein diacetate（DCFH-DA）. Results After irradiation by 10 ~ 80 J/cm^2 UVA for24 h, the survival rate of HaCaT cells decreased gradually with the increasing dosage（P 0. 05）. The expression of NRF2 in nuclei of Ha CAT cells 0 h increased remarkably（P 0. 01）, while decreased gradually 3 ~ 48 h, after irradiation by 20 J/cm^2 UVA. The pre-treatment with acetyl-resveratrol increased the survival rate of HaCaT cells after irradiation by UVA significantly（P 0. 05）, while decreased the apoptosis rate and mortality（P 0. 05）, enhanced the expression of NRF2 protein in nuclei（P 0. 01）and decreased the intracellular ROS level（P 0. 01）. Conclusion Acetyl-resveratrol reduced the UVA-induced proliferative inhibition, apoptosis and death of HaCaT cells, of which the possible mechanism might be associated with enhancing the NRF2 protein activation, decreasing the intracellular ROS level thus relieving the oxidative damage of HaCaT cells c

关 键 词：乙酰化白藜芦醇 HaCaT细胞 长波紫外线 NRF2 活性氧 

分 类 号：Q256[生物学—细胞生物学]