不同剂量亚砷酸钠暴露不同时间对HaCaT细胞活性氧的影响

Dose-time effect of sodium arsenite on reactive oxygen species in HaCaT keratinocytes

目的探索不同剂量亚砷酸钠暴露不同时间对诱导人永生化角质形成(HaCaT)细胞活性氧(ROS)产生的影响以及低剂量亚砷酸钠预处理HaCaT细胞对诱导ROS水平改变的时间效应。方法设未预处理组[分别加入终浓度为0(对照)、0.15、0.6、2.5、10μmol/L的亚砷酸钠染毒8、24、72 h]和预处理8 h组(加入0.15μmol/L亚砷酸钠预处理8 h后,加入10μmol/L亚砷酸钠染毒8 h)及预处理24 h组(加入0.15μmol/L亚砷酸钠预处理24 h后,加入10μmol/L亚砷酸钠染毒8 h)。利用2’,7’-二乙酰二氯荧光素(DCFH-DA)通过流式细胞仪检测细胞内ROS水平。结果与对照组比较,各浓度亚砷酸钠染毒8 h和0.6、2.5、10μmol/L亚砷酸钠染毒24 h及0.6、2.5μmol/L亚砷酸钠染毒72 h后HaCaT细胞内ROS水平均较高,而0.15、10μmol/L亚砷酸钠染毒72 h后HaCaT细胞内ROS水平均较低,差异有统计学意义(P<0.05)。亚砷酸钠染毒8 h时,HaCaT细胞内ROS水平达到峰值;之后,低剂量(0.15、0.6μmol/L)亚砷酸钠染毒组HaCaT细胞内ROS水平于24 h下降,72 h有所回升,而高剂量(≥2.5μmol/L)亚砷酸钠染毒组HaCaT细胞内ROS水平则呈持续下降。预处理8 h组HaCaT细胞内ROS水平高于预处理24 h组,差异均有统计学意义(P<0.05);与10μmol/L亚砷酸钠染毒未预处理HaCaT细胞8 h组比较,预处理8 h组HaCaT细胞内ROS水平较高,预处理24 h组HaCaT细胞内ROS水平较低,差异均有统计学意义(P<0.05)。结论 HaCaT细胞急性暴露于亚砷酸钠后能够引起HaCaT细胞内ROS产生增加,而不同剂量亚砷酸钠暴露诱导HaCaT细胞ROS水平的改变与暴露时间密切相关,且低剂量亚砷酸钠暴露对HaCaT细胞产生的适应性反应可能取决于其作用时间。

Objective To explore the dose-time effect of sodium arsenite on reactive oxygen species （ROS） in HaCaT ceils. Methods HaCaT keratinocytes were divided into non-pretreated group（exposure to a final concentration of 0（control）, 0.15,0.6,2.5 and 10 μmol/L sodium arsenite for 8,24,72 h）,8 h-pretreatment group （exposure to 10 μmol/L of sodium arsenite for 8 h after pretreatment by 0.15 μmol/L sodium arsenite for 8 h） ,and 24 h-pretreatment group（exposure to 10 μmol/L of sodium arsenite for 8 h after pretreatment by 0.15 μmol/L sodium arsenite for 24 h）. The ROS level induced by sodium arsenite in HaCaT cells was detected by FCM （flow cytometry） with DCFH-DA. Results Compared with control group,the intracellular ROS levels in HaCaT cells, treated with various concentrations of sodium arsenite for 8 h and 0.6,2.5,10 μmol/L sodium arsenite for 24 h and 0.6,2.5 μmol/L sodium arsenite for 72 h,were higher,while the ROS levels in HaCaT cells treated with 0.15,10 μmol/L sodium arsenite for 72 h were lower,the difference was statistically significant （P〈0.05）. With sodium arsenite exposure time prolonged,the intracellular ROS levels in HaCaT cells kept higher,reached peak at the 8th hour,then decreased. But after 24 h-treatment,the ROS level in HaCaT cells treated with 0.15 μmol/L and 0.6 μmol/L sodium arsenite increased again. In 8 h-pretreatment group,the ROS level in HaCaT cells were higher than that in non-pretreated group and 24 h-pretreatment group,whereas the ROS level in HaCaT cells in 24 h-pretreatment group were lower than that in non-pretreated group,the differences were statistically significant （P〈0.05）. Conclusion Acute sodium arsenite exposure may increase the level of ROS in HaCaT cells with dose-time dependent manner.