基于Nrf2信号通路探讨桃叶珊瑚苷对神经元氧糖剥夺再复氧损伤的保护作用

Protective effect of Aucubin against oxygen-glucose deprivation/reoxygenation-induced neuronal injury based on Nrf2 signaling pathway

目的 探讨桃叶珊瑚苷(Aucubin,AU)对氧糖剥夺再复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导神经元损伤的影响和分子调控机制。方法 采用小鼠海马神经元细胞HT22构建OGD/R模型。HT22神经元细胞分为常氧组、OGD/R组、OGD/R+50μmol/L AU组和OGD/R+100μmol/L AU组。采用Calcein AM细胞活性试剂盒检测神经元存活率。采用原位缺口末端标记(TdT-mediated dUTP nick-end labeling,TUNEL)实验检测神经元凋亡。采用活性氧(reactive oxygen species,ROS)试剂盒检测ROS水平。采用Western blot检测Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)、B细胞淋巴瘤因子2(B-cell leukemia/lymphoma 2,Bcl-2)、核转录因子E2相关因子2(nuclear factor-erythroid 2-related factor 2,Nrf2)、醌氧化还原酶1(quinone oxidoreductase 1,Nqo-1)和血红素加氧酶1(heme oxygenase 1,Hmox-1)的蛋白表达水平。采用荧光素酶报告基因实验检测Nrf2的转录活性。采用Nrf2抑制剂ML385验证AU是否通过激活Nrf2通路减轻OGD/R神经元损伤。将HT22神经元细胞分为常氧组、OGD/R组、OGD/R+100μmol/L AU组和OGD/R+100μmol/L AU+5μmol/L ML385组,采用荧光素酶报告基因实验检测Nrf2的转录活性,采用TUNEL实验检测神经元凋亡,采用ROS试剂盒检测ROS水平。结果 与常氧组比较,OGD/R组神经元存活率显著降低,凋亡神经元数量显著增加,Bcl-2蛋白表达显著下调,Bax蛋白表达显著上调,ROS水平显著升高(均P<0.01)。与OGD/R组比较,OGD/R+50μmol/L AU组和OGD/R+100μmol/L AU组神经元存活率显著提高,凋亡神经元数量显著减少,Bcl-2蛋白表达显著上调,Bax蛋白表达显著下调,ROS水平显著降低(均P<0.05)。与常氧组比较,OGD/R组Nrf2转录活性显著提高,Nrf2、Nqo-1和Hmox-1的蛋白表达水平显著增加(均P<0.05)。与OGD/R组比较,OGD/R+50μmol/L AU组和OGD/R+100μmol/L AU组Nrf2转录活性提高,Nrf2、Nqo-1和Hmox-1的蛋白表达水平增加(均P<0.05)。与OGD/R+100μmol/L AU组比较,OGD/R+100μmol/L AU+5μmol/L ML385组Nrf2转录活性显著降低,凋亡神经元数量显著增加,ROS水平显著升高(均P<0.01)。结论 AU通过激活Nrf2信号通路减轻OGD/R诱导的神经元损伤。

Objective To explore the effect of Aucubin(AU)on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced neuronal injury and its molecular mechanism.Methods OGD/R model was constructed using mouse hippocampal neurons HT22.HT22 neurons were divided into normoxic group,OGD/R group,OGD/R+50μmol/L AU group,and OGD/R+100μmol/L AU group.The survival of neurons was detected by Calcein AM cell viability assay kit.Neuronal apoptosis was measured by TdT-mediated dUTP nick-end labeling(TUNEL)assay.The level of reactive oxygen species(ROS)was determined by ROS assay kit.The protein levels of Bcl-2-associated X protein(Bax),B-cell leukemia/lymphoma 2(Bcl-2),quinone oxidoreductase 1(Nqo-1),heme oxygenase 1(Hmox-1)were examined by Western blot.The transcriptional activity of Nrf2 was monitored by luciferase reporter assay.Nrf2 inhibitor ML385 was utilized to verify whether AU ameliorated OGD/R-induced neuronal injury by enhancing Nrf2 signaling pathway.HT22 neurons were divided into normoxic group,OGD/R group,OGD/R+100μmol/L AU group,and OGD/R+100μmol/L AU+5μmol/L ML385 group.Nrf2 transcriptional activity was monitored by luciferase reporter assay,the neuronal apoptosis was measured by TUNEL assay,and ROS level was determined by ROS assay kit.Results Compared with normoxic group,the survival rate of neurons was significantly decreased in OGD/R group,the number of apoptotic neurons was significantly increased,Bcl-2 protein expression was significantly decreased,Bax protein expression was significantly increased,and ROS level was significantly upregulated(all P<0.01).Compared with OGD/R group,the survival rate of neurons was significantly improved,the number of apoptotic neurons was decreased,Bcl-2 protein expression was significantly increased,Bax protein expression was decreased,and ROS level was downregulated in OGD/R+50μmol/L AU group and OGD/R+100μmol/L AU group(all P<0.05).Compared with normoxic group,Nrf2 transcriptional activity was significantly increased,and the protein levels of Nrf2,Nqo-1 and Hmox-1 were significantly upregulated in OGD/R group(P<0.05).Compared with OGD/R group,Nrf2 transcriptional activity was further upregulated,and the protein levels of Nrf2,Nqo-1 and Hmox-1 were further increased in OGD/R+50μmol/L AU group and OGD/R+100μmol/L AU group(P<0.05).Compared with OGD/R+100μmol/L AU group,Nrf2 transcriptional activity was significantly decreased,the number of apoptotic neurons was significantly increased,and ROS level was significantly upregulated in OGD/R+100μmol/L AU+5μmol/L ML385 group(all P<0.01).Conclusion AU can ameliorate OGD/R-induced neuronal injury by enhancing the activation of Nrf2 signaling pathway.