摘要
目的研究金雀异黄酮(GEN)对皮质神经元低氧/复氧(H/R)损伤的影响,并基于核因子E2相关因子2/血红素加氧酶1(Nrf2/HO-1)信号通路探讨其机制。方法分离并体外培养C57BL/6J小鼠胎鼠(妊娠15 d)皮质神经元,设正常对照(Normal)组、模型(H/R)组、GEN(12.5μmol·L^(-1))组、TBHQ(Nrf2激动剂,10μmol·L^(-1))组、GEN(12.5μmol·L^(-1))+TBHQ(10μmol·L^(-1))组。除正常对照组外,其他组采用低氧(5%CO_(2)+95%N_(2))4 h后复氧(5%CO_(2)+95%空气)24 h的方法制备H/R损伤皮质神经元模型,各组分别于造模前2h给药干预。采用CCK-8法、流式细胞术检测神经元活力和凋亡率,DCFH-DA荧光探针法检测神经元活性氧(ROS)含量,分光光度法检测神经元中丙二醛(MDA)含量和抗氧化酶[超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)]活性,RT-PCR法检测神经元Nrf2、HO-1 mRNA表达,Western blot法检测神经元Nrf2、HO-1、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、激活型Caspase-3(cleaved Caspase-3)蛋白表达。结果与H/R组比较,GEN组、TBHQ组和GEN+TBHQ组皮质神经元活力明显升高、凋亡率明显降低(P<0.05);神经元中ROS、MDA含量明显降低,SOD、GSH-Px活性明显升高(P<0.05);Nrf2、HO-1 mRNA表达量明显升高(P<0.05);Nrf2、HO-1、Bcl-2蛋白表达量及Bcl-2/Bax比值明显升高,Bax、cleaved Caspase-3蛋白表达量明显降低(P<0.05)。GEN+TBHQ组对H/R损伤皮质神经元活力、凋亡率、氧化应激指标、Nrf2/HO-1信号通路相关mRNA和蛋白表达的调控作用均明显优于GEN组和TBHQ组(P<0.05)。结论GEN可通过促进Nrf2/HO-1信号通路活化抑制氧化应激损伤和神经元凋亡,对皮质神经元H/R损伤起到保护作用。
Objective To investigate the effects of Genistein(GEN)on hypoxia/reoxygenation(H/R)injury of cortical neurons,and explore its mechanism based on nuclear E2 related factor 2/heme oxygenase-1(Nrf2/HO-1)signaling pathway.Methods The fetal cortical neurons of C57BL/6J mice(15 d gestation)were isolated and cultured in vitro,and the normal group,model(H/R)group,GEN(12.5μmol·L^(-1))group,TBHQ(Nrf2 agonist,10μmol·L^(-1))group and GEN(12.5μmol·L^(-1))+TBHQ(10μmol·L^(-1))group were set up.Except for the normal control group,the H/R damaged cortical neuron model was prepared in other groups by hypoxia(5%CO_(2)+95%N_(2))for 4 h and then reoxygenation(5%C02+95%air)for 24 h.2 h before modling,the cortical neurons in each group were intervened.The activity and apoptosis rate of neurons were detected by CCK-8 method or flow cytometry.The content of reactive oxygen species(ROS)in neurons was detected by DCFH-DA fluorescent probe.The content of malondialdehyde(MDA)and the activity of antioxidant enzymes[superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)]in neurons were detected by spectrophotometry.The mRNA expression of Nrf2 and HO-1 in neurons was detected by RT-PCR.The protein expression of Nrf2,HO-1,B-lymphoblastoma-2 gene(Bcl-2),Bcl-2 associated X protein(Bax),cleaved Caspase-3 were detected by Western blot.Results Compared with the H/R group,the activity of cortical neurons in GEN group,TBHQ group and GEN+TBHQ group was significantly increased,while the apoptosis rate was significantly decreased(P<0.05).The contents of ROS,MDA in neurons were significantly decreased,while the activities of SOD,GSH-Px were significantly increased(P<0.05).The mRNA expressions of Nrr,HO-1 were significantly increased(P<0.05).The protein expression of Nrf2,HO-1,Bcl-2 and the ratio of Bcl-2/Bax were significantly increased,and the protein expression of Bax,cleaved Caspase-3 were significantly decreased(P<0.05).The effects of GEN+TBHQ group on the activity,apoptosis rate,oxidative stress index,Nrf2/HO-1 signaling pathway related mRNA and protein expression of H/R damaged cortical neurons were significantly better than those of GEN group and TBHQ group(P<0.05).Conclusion GEN can inhibit oxidative stress injury and neuronal apoptosis through promoting the activation of Nrf2/HO-1 signaling pathway,which play a protective role in H/R injury of cortical neurons.
作者
李慧
刘少军
霍好利
邢瑞敏
樊璐洁
Li Hui;Liu Shaojun;Huo Haoli;Xing Ruimin;Fan Lujie(Department of Pharmacy,Handan Central Hospital,Hebei 056008,China)
出处
《脑与神经疾病杂志》
CAS
2024年第4期251-257,共7页
Journal of Brain and Nervous Diseases
基金
河北省医学科学研究课题(20220570)。
