亚砷酸钠暴露对SVEC4-10细胞Nrf2信号通路的影响  

Effect of sodium arsenite exposure on nuclear factor E2-related factor 2 signaling pathway in SVEC4-10 cells

作　　者：夏应驰 吕航 刘志远 刘翠杰 王惠惠 皮静波 Xia Yingchi;Lyu Hang;Liu Zhiyuan;Liu Cuijie;Wang Huihui;Pi Jingbo(Program of Environment Toxicology,School of Public Health,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学公共卫生学院环境毒理学研究室,沈阳110122

出　　处：《中华地方病学杂志》2020年第1期16-21,共6页Chinese Journal of Endemiology

基　　金：国家自然科学基金(81830099、81573106)。

摘　　要：目的探讨亚砷酸钠(NaAsO2)暴露对小鼠淋巴结血管内皮细胞系SVEC4-10细胞中核因子E2相关因子2(Nrf2)信号通路转录活性的影响。方法采用细胞体外培养方法,分别以不同剂量NaAsO2[0(对照)、2、5、10、20、50、100、150μmol/L]处理SVEC4-10细胞24 h,四唑化合物(MTS)法检测细胞活性。时间-效应关系研究分别以5μmol/L NaAsO2处理SVEC4-10细胞0(对照)、2、6、12 h。剂量-效应关系研究分别以0(对照)、2、5、10μmol/L NaAsO2处理SVEC4-10细胞6 h。采用实时荧光定量PCR(RT-qPCR)法检测Nrf2信号通路相关基因Nrf2、谷氨酰半胱氨酸连接酶催化亚单位(Gclc)、谷氨酰半胱氨酸连接酶修饰亚单位(Gclm)、醌氧化还原酶1(Nqo1)、金属硫蛋白1(Mt1)mRNA水平。采用SVEC4-10细胞建立Nrf2基因稳转沉默(Nrf2-KD)细胞,分别以0(对照)、10、20μmol/L NaAsO2处理干扰对照(scramble,SCR)细胞和Nrf2-KD细胞16 h,流式细胞仪检测细胞凋亡情况。结果MTS检测结果显示,对照组,2、5、10、20、50、100、150μmol/L NaAsO2处理组细胞活性分别为(100.00±19.53)%、(98.18±9.85)%、(96.09±30.04)%、(90.64±8.74)%、(59.75±12.09)%、(35.43±8.58)%、(26.35±5.89)%、(17.54±4.48)%,不同剂量组间细胞活性比较差异有统计学意义(F=18.30,P<0.05);且20、50、100、150μmol/L NaAsO2处理组细胞活性显著低于对照组(P均<0.05)。时间-效应关系研究结果显示,对照组,2、6、12 h处理组组间Nrf2、Gclc、Gclm、Nqo1、Mt1 mRNA水平比较差异有统计学意义(F=56.69、85.28、90.82、80.46、758.60,P均<0.05);随着砷暴露时间的延长,Nrf2、Gclc、Gclm、Mt1 mRNA水平先上升后下降,Nqo1 mRNA水平不断上升;其中,Nrf2 mRNA水平在2 h达到峰值,Gclc、Gclm、Mt1 mRNA水平在6 h达到峰值,Nqo1 mRNA水平在12 h达到峰值。剂量-效应关系研究结果显示,对照组,2、5、10μmol/L NaAsO2处理组组间Nrf2、Gclc、Gclm、Nqo1、Mt1 mRNA水平比较差异有统计学意义(F=68.39、72.26、30.41、397.00�Objective To investigate the effect of sodium arsenite(NaAsO2)on transcriptional activity of nuclear factor E2-related factor 2(Nrf2)signaling pathway in mouse lymph node vascular endothelial cell line(SVEC4-10).Methods In vitro cell culture method was used to treat SVEC4-10 cells for 24 h with different doses of NaAsO2[0(control),2,5,10,20,50,100,150μmol/L],and the cell viability was detected by tetrazole compound(MTS)method.The time-response relationship was studied with SVEC4-10 cells treated with 5μmol/L NaAsO2 for 0(control),2,6 and 12 h;the dose-response relationship was studied with SVEC4-10 cells treated with 0(control),2,5 and 10μmol/L NaAsO2 for 6 h;real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the mRNA expression of Nrf2 and its downstream genes glutamate-cysteine ligase catalytic subunit(Gclc),glutamate-cysteine ligase modifier subunit(Gclm),NAD(P)H dehydrogenase quinone 1(Nqo1)and metallothionein 1(Mt1).Establishment of Nrf2 gene stably silenced(Nrf2-KD)cells using SVEC4-10 cells,the interference control(scramble,SCR)cells and Nrf2-KD cells were treated with 0(control),10 and 20μmol/L NaAsO2 for 16 h,and apoptosis was detected by flow cytometry.Results MTS test results showed that the cell viability of the control,2,5,10,20,50,100,150μmol/L NaAsO2 treatment groups was(100.00±19.53)%,(98.18±9.85)%,(96.09±30.04)%,(90.64±8.74)%,(59.75±12.09)%,(35.43±8.58)%,(26.35±5.89)%and(17.54±4.48)%,respectivily.There was statistically significant difference in cell viability between different dose groups(F=18.30,P<0.05);and the cell viability of the 20,50,100,150μmol/L NaAsO2 treatment groups was significantly lower than that of the control group(P<0.05).The time-response relationship results showed that there were statistically significant differences in Nrf2,Gclc,Gclm,Nqo1 and Mt1 mRNA level between control,2,6 and 12 h treatment groups(F=56.69,85.28,90.82,80.46,758.60,P<0.05);with extension of arsenic exposure time,the mRNA level of Nrf2,Gclc,Gclm and Mt1 first increased and the

关 键 词：亚砷酸盐类 淋巴结血管内皮细胞系 核因子E2相关因子2 

分 类 号：R1[医药卫生—公共卫生与预防医学]