G1通过核因子E2相关因子2减轻神经元氧糖剥夺损伤

G1 attenuates neuronal injury induced by oxygen glucose deprivation through nuclear factor-E2-related factor 2

目的探讨G1对原代神经元氧糖剥夺损伤后氧化应激指标的影响及核因子E2相关因子2(nuclear factor-E2-related factor 2,Nrf2)在其中的作用。方法将培养7 d的C57BL/6胎鼠皮质原代神经元细胞按随机数字表法分为6组(每组6孔):对照组(C组)、氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)组、溶剂组、G1组、G1+对照病毒组和G1+Nrf2慢病毒组。C组,不做任何处理;OGD/R组,OGD 2 h后恢复氧糖;溶剂组,OGD 2 h后在培养液中加入适量溶剂,继续培养22 h;G1组,行OGD/R模型,复氧复糖后的正常培养液中加入终浓度为5μmol/L的G1,继续培养22 h;G1+对照病毒组,感染对照慢病毒的原代神经元细胞行OGD/R模型,其余处理同G1组;G1+Nrf2慢病毒组,感染了Nrf2 shRNA慢病毒的原代神经元细胞行OGD/R模型,其余处理同G1组。利用分光光度法检测乳酸脱氢酶(lactate dehydrogenase,LDH)含量,应用细胞计数试剂盒(cell counting kit-8,CCK-8)法检测细胞活力,采用ELISA法检测活性氧(reactive oxygen species,ROS)、丙二醛(malonaldehyde,MDA)、超氧化物歧化酶(superoxide,SOD)和总抗氧化能力(total antioxidant capacity,T-AOC)的含量,采用Western blot和免疫荧光法检测Nrf2的表达。结果与C组比较,OGD/R组和溶剂组LDH、MDA和ROS含量升高,细胞活力、SOD和T-AOC含量降低,Nrf2总蛋白水平和核内蛋白水平均升高(P<0.05);与OGD/R组比较,G1组LDH、MDA和ROS含量降低,细胞活力、SOD和T-AOC含量升高,Nrf2总蛋白水平和核内蛋白水平升高(P<0.05)。与G1组及G1+对照病毒组比较,G1+Nrf2慢病毒组细胞活力、SOD和T-AOC含量降低,LDH、ROS及MDA含量升高(P<0.05);G1组与G1+对照病毒组比较,各指标差异均无统计学意义(P>0.05)。结论G1可以通过Nrf2通路增加抗氧化酶活性减轻原代神经元OGD/R损伤。

Objective To explore the effects of G1 on oxidative stress index in primary cortical neurons after oxygen glucose deprivation and the role of nuclear factor related factor 2(Nrf2)during the process.Methods Primary cortical neurons from C57BL/6 embryonic mice were cultured for seven days and divided into six groups according to the random number table method(n=6):a control(C)group,an oxygen glucose deprivation/reoxygenation(OGD/R)group,a solvent group,a G1 group,a G1+control virus group and a G1+Nrf2 lentiviral group.The C group did not receive any treatment,while the OGD/R group was deprived of oxygen and glucose for 2 h before recovery.The solvent group was added with a proper amount of solvent 2 h after OGD followed by cultivation over 22 h.For the G1 group,OGD/R modeled neurons were used,and G1 was added into normal medium to reach a final concentration of 5μmol/L to treat over 22 h.Primary neurons in the G1+control virus group were infected with control lentivirus to establish the model of OGD/R,before receiving the same treatment as the G1 group.Primary neurons in the G1+Nrf2 lentivirus group were infected with Nrf2 shRNA lentivirus to establish the model of OGD/R,before receiving the same treatment as the G1 group.The release of lactate dehydrogenase(LDH)was detected by spectrophotometry,and the cell survival was measured by cell counting kit-8(CCK-8)assay.Then,the contents of reactive oxygen species(ROS),malonaldehyde(MDA),superoxide(SOD)and total antioxidant capacity(T-AOC)were quantified by enzyme-linked immunosorbent assay(ELISA).The levels of Nrf2 were examined by Western blot and immunofluorescence.Results Compared with the C group,both the OGD/R group and the solvent group presented remarkable increases in LDH,MOA and ROS contents,decreases in cell survival,SOD and T-AOC contents,and increases in total Nrf2 and nuclear protein levels(P<0.05).Compared with the OGD/R group,the G1 group presented remarkable decreases in LDH,ROS and MDA contents,increases in cell survival,SOD and T-AOC contents,and increases in total Nrf2 and nuclear protein levels(P<0.05).Compared with the G1 group and the G1+control virus group,the G1+Nrf2 lentivirus virus group produced decreases in cell survival,SOD and T-AOC contents,and increases in LDH,ROS and MDA contents(P<0.05).There were no statistical differences in each indicator between the G1 group and the G1+contrl virus group(P>0.05).Conclusions G1 can alleviate OGD/R injury in primary neurons through increasing the activity of antioxidant enzymes via the Nrf2 pathway.