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目的研究硒对砷诱导小鼠皮肤JB6-CI41细胞核因子κB(nuclear factorκB,NF-κB)的DNA结合活性和细胞凋亡的作用。方法调整JB6-CI41细胞密度为1×105/孔,分别设立对照(生理盐水)组,不同浓度的亚砷酸(3.125、6.25、12.5μmol/L)和亚硒酸钠(2.5μmol/L)单独染毒组,亚硒酸钠(2.5μmol/L)+不同浓度的亚砷酸(3.125、6.25和12.5μmol/L)联合染毒组,NF-κB特异化学抑制剂二乙基二硫代氨基甲酸盐(diethyldithiocarbamate,DTTC,100μmol/L,提前染毒2 h)+亚砷酸(6.25μmol/L)联合染毒组。采用凝胶阻滞电泳法(electrophoretic mobility shift assay,EMSA)检测NF-κB的DNA结合活性,采用流式细胞术检测细胞凋亡情况。结果 2.5μmol/L亚硒酸钠对小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性和细胞凋亡无显著影响(P>0.05)。6.25和12.5μmol/L的亚砷酸能够增强小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性(P<0.01),具有明显剂量-效应关系(r=0.942 6,P<0.01)。3.125、6.25和12.5μmol/L亚砷酸诱导小鼠皮肤JB6-CI41细胞凋亡(P<0.01),呈现显著剂量-反应关系(r=0.991 8,P<0.01)。NF-κB的DNA结合活性与细胞凋亡率呈显著正相关(r=0.977 5,P<0.01)。2.5μmol/L亚硒酸钠能抑制6.25和12.5μmol/L亚砷酸作用下的小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性(P<0.01),对3.125、6.25和12.5μmol/L的亚砷酸引起的小鼠皮肤JB6-CI41细胞凋亡率也表现出显著抑制作用(P<0.01)。100μmol/L的DTTC对6.25μmol/L亚砷酸引起的小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性和细胞凋亡率具有显著抑制作用(P<0.01)。结论在本实验中,一定剂量(6.25～12.5μmol/L)的砷能够使小鼠皮肤JB6-CI41细胞NF-κB的DNA结合活性显著增高并诱导细胞凋亡,2.5μmol/L的硒能抑制砷引起的NF-κB的活化和细胞凋亡。
Objective To study the effect of selenium on DNA binding activity and apoptosis of nuclear factor κB (NF-κB) induced by arsenic in mouse skin JB6-CI41 cells. Methods The density of JB6-CI41 cells was adjusted to 1 × 10 5 / well and the control group (normal saline group), different concentrations of arsenious acid (3.125,6.25,12.5μmol / L) and sodium selenite (2.5μmol / The effects of sodium selenite (2.5μmol / L) and different concentrations of arsenious acid (3.125, 6.25 and 12.5μmol / L) Carbamate (DTTC, 100μmol / L, pretreatment 2h) + arsenious acid (6.25μmol / L) in combination group. DNA binding activity of NF-κB was detected by electrophoretic mobility shift assay (EMSA), and apoptosis was detected by flow cytometry. Results 2.5 μmol / L sodium selenite had no significant effect on DNA-binding activity and apoptosis of NF-κB in mouse skin JB6-CI41 cells (P> 0.05). 6.25 and 12.5μmol / L of arsenite increased the DNA-binding activity of NF-κB in mouse skin JB6-CI41 cells (P <0.01), with a significant dose-effect relationship (r = 0.9426, P <0.01). Apoptosis of mouse JB6-CI41 cells induced by arsenic trioxide at doses of 3.125,6.25 and 12.5μmol / L (P <0.01) showed significant dose-response relationship (r = 0.991 8, P <0.01). The DNA binding activity of NF-κB was significantly and positively correlated with the apoptosis rate (r = 0.977 5, P <0.01). 2.5μmol / L sodium selenite could inhibit the DNA-binding activity of NF-κB in mouse skin JB6-CI41 cells treated with 6.25 and 12.5μmol / L arsenic trioxide (P <0.01) L of arsenic trioxide induced apoptosis in mouse skin JB6-CI41 cells also showed a significant inhibitory effect (P <0.01). 100μmol / L DTTC could significantly inhibit the DNA-binding activity and apoptosis rate of NF-κB induced by 6.25μmol / L arsenite in mouse skin JB6-CI41 cells (P <0.01). Conclusion Arsenic at a dose of 6.25 ~ 12.5μmol / L could significantly increase the DNA-binding activity of NF-κB in mouse skin JB6-CI41 cells and induce apoptosis. Selenium at 2.5μmol / L inhibited Arsenic-induced NF-κB activation and apoptosis.