金雀异黄酮调节Nrf2/HO-1信号通路对海马神经元缺氧/复氧损伤的影响

Effect of Genistein on hypoxia/reoxygenation injury of hippocampal neurons by regulating Nrf2/HO-1 signaling pathway

目的探讨金雀异黄酮(Genistein,Gen)调节核因子E2相关因子2/血红素加氧酶1(Nuclear factor E2 related factor 2/heme oxygenase-1,Nrf2/HO-1)信号通路对海马神经元缺氧/复氧(Hypoxia/reoxygenation,H/R)损伤的影响。方法原代分离并体外培养SD(Sprague Dawley)大鼠胎鼠(妊娠18 d)海马神经元,设正常组、模型组、Gen(12.5μmol/L)组、Gen(12.5μmol/L)+Nrf2激动剂叔丁基对苯二酚(Tert butyl hydroquinone,TBHQ)(10μmol/L)组和Gen(12.5μmol/L)+Nrf2抑制剂(Nrf2 inhibitor,ML385)(10μmol/L)组;采用缺氧4 h复氧24 h的方法构建海马神经元H/R损伤模型,各组均于造模前2 h给药;噻唑蓝四氮唑(Thiazolyl blue tetrazolium,MTT)法、膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-fluorescein isothiocyanate/propyl iodide,Annexin V-FITC/PI)双染法检测海马神经元活力和凋亡率,分光光度法检测活性氧(Reactive oxygen species,ROS)、丙二醛(Malondialdehyde,MDA)水平和超氧化物歧化酶(Superoxide dismutase,SOD)、过氧化氢酶(Catalase,CAT)活性,Western blot法检测海马神经元Nrf2/HO-1信号通路相关蛋白[Nrf2,HO-1、B淋巴细胞瘤-2基因(B-cell lymphoma-2 gene,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、半胱氨酸蛋白酶-3(Cysteine proteinase-3,Caspase-3)、激活型Caspase-3(Cleaved Caspase-3)]表达水平。结果与模型组比较,Gen组海马神经元活力显著升高、凋亡率显著降低(P<0.05);ROS,MDA水平显著降低,SOD,CAT活性显著升高(P<0.05);Nrf2,HO-1,Bcl-2表达水平显著升高,Bax,Cleaved Caspase-3表达水平及Bax/Bcl-2、Cleaved Caspase-3/Caspase-3比值显著降低(P<0.05)。TBHQ可明显增强Gen对H/R损伤海马神经元活力、凋亡率、氧化应激以及Nrf2/HO-1信号通路相关蛋白表达水平的调控作用;ML385则明显逆转Gen对H/R损伤海马神经元的上述调控作用。结论Gen对海马神经元H/R损伤具有保护作用,其机制可能与Gen上调Nrf2/HO-1信号通路,进而抑制氧化应激和神经元凋亡有关。

Objective To investigate the effect Genistein(Gen)on hypoxia/reoxygenation(H/R)injury of hippocampal neurons and its potential mechanism based on the nuclear factor E2 related factor 2/heme oxygenase-1(Nrf2/HO-1)signaling pathway.Methods The primary hippocampal neurons of SD(Sprague Dawley)fetal rats(18 d gestation)were isolated and cultured in vitro,and the normal group,model group,Gen(12.5μmol/L)group,Gen(12.5μmol/L)+Nrf2 agonis tert butyl hydroquinone(TBHQ)(10μmol/L)group,Gen(12.5μmol/L)+Nrf2 inhibitor ML385(10μmol/L)group were set up.The H/R injury model of hippocampal neurons was established by hypoxia for 4 h and reoxygenation for 24 h.2 h before modeling,the hippocampal neurons in each group were intervened.The viability and apoptosis rate of hippocampal neurons were detected by thiazolyl blue tetrazolium(MTT)or Annexin V-fluorescein isothiocyanate/propyl iodide(Annexin V-FITC/PI)double dyeing.The content of reactive oxygen species(ROS),malondialdehyde(MDA)and the activity of superoxide dismutase(SOD),catalase(CAT)were detected by spectrophotometry.The related protein expression of Nrf2/HO-1 signaling pathway[Nrf2,HO-1,B-lymphoblastoma-2 gene(Bcl-2),Bcl-2 associated X protein(Bax),Cysteine proteinase-3(Caspase-3),Cleaved Caspase-3]were detected by Western blot.Results Compared with the model group,the viability of hippocampal neurons in Gen group was significantly increased,while the apoptosis rate was significantly decreased(P<0.05).The content of ROS,MDA were significantly decreased and the activity of SOD,CAT was significantly increased(P<0.05).The expression of Nrf2,HO-1,Bcl-2 were significantly increased,the expression of Bax,Cleaved Caspase-3 and the ratio of Bax/Bcl-2,Cleaved Caspase-3/Caspase-3 were significantly decreased(P<0.05).TBHQ could significantly enhance the regulatory effects of Gen on the viability,apoptosis rate,oxidative stress and Nrf2/HO-1 signaling pathway related protein expression of H/R damaged hippocampal neurons.ML385 significantly reversed the regulatory effects of Gen on H/R damaged hippocampal neurons.Conclusion Gen has protective effect on H/R injury of hippocampal neurons through the up-regulation of Nrf2/HO-1 signaling pathway and inhibition of oxidative stress and neuronal apoptosis.