摘要
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 explore the effects of selenium on arsenic-induced DNA binding activity of nuclear factor κB （NF-KB） and apoptosis in mouse skin JB6-CI41 cells in vitro. Methods Mouse skin JB6-CI41 cells（cell density was 1×10^5/well） were exposed to selenium and arsenic alone as well as joint action of selenium and arsenic in vitro. The experiment included nine groups, included one control group treated with physical saline, one selenium treatment group at the dose of 2.5 μmol/L sodium selenite, three arsenic treatment groups at the doses of 3.125,6.25 and 12.5 μmol/L arsenous acid respectively, and three joint action groups of selenium and arsenic combined selenium at the dose of 2.5μmol/L sodium selenite with arsenic at the doses of 3.125,6.25 and 12.5 μmol/L arsenous acid. Meanwhile, diethyldithiocarbamate （DTTC, 100 /μmol/L）,a specific inhibitor of NF-KB, was used to treat mouse skin JB6-CI41 cells 2 h previously ,and then treated with arsenous acid （6.25 μmol/L） together in order to further investigate the relationship between arsenic-induced DNA binding activity of NF-KB and apoptosis. The DNA binding activity of NF-KB was analyzed by electrophoretic mobility shift assay （EMSA） and apoptosis was measured by flow cytometry. Results Sodium selenite （2.5 μmol/L） showed no obvious effects on DNA binding activity of NF-κB and apoptosis in mouse skin JB6-CI41 cells. Arsenous acid at 6.25 and 12.5 μmol/L increased DNA binding activity of NF-KB significantly （P〈0.01） with dose-dependent manner （relative coefficient was 0.942 6）. Arsenous acid at the doses of 3.125,6.25 and 12.5 μmol/L could significantly induced apoptosis （relative coefficient was 0.991 8）. DNA binding activity of NF-KB had close positive relationship with apoptosis treated by arsenic in mouse skin JB6-CI41 cells,relative coefficient was0.977 5 （P〈0.01）. Compared with corresponding dose of arsenic groups,2.5 μmol/L sodium selenite co-exposed with 6.25 and 12.5 μmol/L arsenous acid to mouse skin JB6-CI41 cells,the DNA binding activity of NF-KB was decreased significantly （P〈 0.01 ）. 2.5 μmol/L sodium selenite combined with arsenous acid at the doses of 3.125,6.25 and 12.5 μmol/L,apoptosis rates were lower than those of corresponding arsenic group （P〈0.01）. Mouse skin JB6-CI41 cells co-exposed to 100 μmol/L DTTC and 6.25 μmol/L arsenous acid,DNA binding activity of NF-κB and apoptosis were decreased compared with corresponding arsenic group（P〈0.01 ）. Conclusion Arsenic at doses of 6.25-12.5 μmol/L arsenous acid may promote DNA binding activity of NF-KB and induce apoptosis in mouse skin JB6-CI41 cells,selenium at the dose of 2.5 μmol/L sodium selenite have inhibitive effects on DNA binding activity of NF-KB and apoptosis caused by arsenic in vitro.
出处
《环境与健康杂志》
CAS
CSCD
北大核心
2012年第1期26-29,共4页
Journal of Environment and Health
基金
广东药学院师资队伍建设经费资助(广药[2008]154号)
国家自然科学基金(30471500)
关键词
硒
砷
核因子κB
细胞凋亡
Selenium
Arsenic
Nuclear factor κB （NF- κB ）
Apoptosis
作者简介
作者简介：余日安（1964-），男，研究员，博士，从事环境与职业毒理学研究。
通讯作者：鲁文清，E-mail：luwq@mails．tjmu．edu．cn
