Nrf2信号通路在锰致TM3细胞氧化损伤中的作用

Role of Nrf2 signaling pathway in manganese-induced oxidative damage in TM3 cells

目的在锰所导致TM3细胞毒性中,探究锰是否介导Nrf2信号通路调控发挥抗氧化损伤的作用。方法染锰剂量设置低(100μmol/L)、中(200μmol/L)、高(300μmol/L)剂量,染锰时间均为24 h。该实验抑制剂为ATRA,激动剂为TBHQ,终浓度10μmol/L。采用倒置相差显微镜观察TM3细胞形态,MTT法测细胞活力AnnnexinV/PI双染色法测凋亡率,DCFH-DA探针测ROS水平,TBA法测细胞内MDA含量,Western blot及RT-qPCR技术测细胞内Nrf2、HO-1、GSTpi、SOD、NQO1等蛋白和基因的表达。结果TM3细胞的染锰IC 50为300μmol/L。随着染锰剂量的增加,细胞存活率降低,且在一定剂量范围内,染锰浓度越高,ROS、MDA产生越多。锰中毒可导致TM3细胞中氧化应激相关蛋白HO-1、NQO1、SOD、GSTpi及Nrf2蛋白表达降低,从而引起细胞氧化损伤。Western Blot及RT-qPCR技术所得结果显示一致,均表明,Nrf2被抑制后,在低中高剂量组中,ROS生成有一定增高,具有剂量效应关系,反之当Nrf2被激活后,ROS、MDA生成有一定降低,也具有剂量效应关系。结论采用MTT实验,Western blot等3种方法观察不同剂量Mn对TM3细胞的影响,3种结果均一致,即在一定剂量范围内,随染锰浓度增加,细胞损伤亦增加。且在同一染锰浓度下,经ATRA预处理后的细胞损伤程度更大,经TBHQ预处理后的细胞损伤程度减轻,提示锰可诱导TM3细胞氧化损伤,且Nrf2信号通路在TM3细胞中具有一定抗氧化作用。

Objective To explore whether manganese(Mn)mediated the regulation of Nrf2 signal pathway to playan anti-oxidative effect in the TM3 cell.Methods Efficacy of low,medium and high doses of Mn were included.The experimental inhibitor was ATRA and the agonist was TBHQ,at a final concentration of 10μmol/L.The cell experiment was divided into 12 groups,namely:high,medium and low concentration of Mn groups(100,200,300μmol/L);agonist with high,medium and low concentration of Mn groups;inhibitor with high,medium and low concentration of Mn groups;blank control group,agonist control Group and inhibitor control group.The Mn exposure time was 24 h.In this experiment,the cell apoptosis rate,cell ROS level,MDA content and other oxidative stress indicators were detected.Western blot and RT-qPCR assays were used to measure the expression of Nrf2,HO-1,GSTpi,SOD,NQO1 and other oxidative stress-related proteins and genes.Results The IC 50 of Mn in TM3 cells was 300μmol/L.With the dose of Mn increased,the survival rate of cells was decreased.The higher concentration of Mn produced,more ROS and MDA.The expressions of oxidative stress-related proteins of HO-1,NQO1,SOD,GSTpi and Nrf2 were decreased after exposure to Mn,which caused cell oxidative damage.Consistent results were shown in western blot and RT-qPCR assays.After Nrf2 was inhibited,ROS generation was increased in all doses of Mn groups,with dose-effect relationship.On the contrary,when Nrf2 was activated,ROS and MDA were increased.Conclusion Mn could damage TM3 cell with a dose-dependent relationship and one of the underlying mechanism might be related to oxidative damage,and activation of Nrf2 signaling pathway might play anti-oxidant role in the cell damage.