突变SOD1导致星形胶质细胞对氧化应激的易损性

Primary astrocytes with mutant SOD1G93A are susceptible to oxidative stress

目的研究突变SOD1G93A星形胶质细胞是否表现了对氧化应激的易损性。方法原代突变SOD1星形胶质细胞和野生型SOD1星形胶质细胞体外培养。实验细胞分为突变型组和野生型组;突变组和野生型组又分为对照组、血清剥夺组和EGCG干预组;使用5和10μmol/L EGCG分别干预;用MTT检测细胞的增殖能力;激光共聚焦检测ROS;用Western blot检测细胞中Nrf2及其介导的HO1和NQO1的表达。结果与正常星形胶质细胞相比,含有突变SOD1星形胶质细胞MTT显著下降(P<0.01)。细胞中Nrf2表达下降了44%(P<0.01)。Nrf2介导的HO1和NQO1的表达水平也分别下降了43%(P<0.01)和40%(P<0.05)。5和10μmol/L EGCG使NQO1的表达水平分别升高了1.5和2.5倍(P<0.05),也升高了Nrf2的表达并促进了Nrf2向细胞核中转移。结论突变SOD1造成了星形胶质细胞对氧化应激的易受损性。

Objective To study the vulnerability of astrocytes bearing mutant SOD1 under the oxidative stress.Methods The cytotoxicity of the serum deprived astrocytes was measured by MTT.The level of ROS was shown by fluorescence of DCF through confocal microscopy.The expression of Nrf2,HO1 and NQO1 in the different cells was detected by Western blot.Results The level of cellular toxicity was higher in the astrocytes bearing mutant SOD1 exposed to the oxidative stress than the astrocytes bearing wild type SOD1.In the astrocytes bearing mutant SOD1,the expression of Nrf2,HO1 and NQO1 decreased.In the presence of mutant SOD1,an unexpected 44 percent decrease of Nrf2 was detected.This was associated with a decreases in multiple downstream phase Ⅱ detoxifying enzymes and antioxidant enzymes,known as NQO1 and HO1.Furthermore,our results showed that the expression of NQO1 increased 1.5 and 2.5-fold by EGCG at 5 and 10 μmol/L.EGCG also elevated the expression of total Nrf2.Confocal microscopy showed that EGCG caused Nrf2 translocation from the cytoplasm to the nucleus.Conclusion Decrease in Nrf2 expression is the mechanism to explain the vulnerability of astrocytes bearing mutant SOD1 and EGCG strengthened antioxidation function by upregulating the activity of Nrf2.