神经干细胞联合依达拉奉对氧糖剥夺-再灌注皮层神经元的保护作用及机制探讨

Protective role and mechanism of neural stem cells combined with edaravone in cortical neurons after oxygen-glucose deprivation/reperfusion

目的探讨神经干细胞联合依达拉奉治疗对氧糖剥夺-再灌注(OGD-R)皮层神经元的保护作用及可能机制。方法(1)体外培养胎龄14~16 d的SD胎鼠脑组织神经干细胞,采用免疫荧光染色检测巢蛋白、神经胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)的表达进行鉴定。原代培养新生24 h内SD大鼠皮层神经元,采用免疫荧光染色检测神经元特异性核蛋白(NeuN)、β-微管蛋白(β-Tubulin)的表达进行鉴定。(2)将原代培养的皮层神经元分为正常组(正常培养)、OGD-R模型组(缺氧处理1.5 h后再复氧培养24 h)、OGD-R+神经干细胞组(缺氧处理1.5 h后再复氧培养时,加入神经干细胞与皮层神经元共培养24 h)、OGD-R+依达拉奉组(缺氧处理前加入100μmol/L依达拉奉,缺氧处理1.5 h后再复氧培养24 h)、OGD-R+神经干细胞组+依达拉奉组(缺氧处理前加入100μmol/L依达拉奉,缺氧处理1.5 h后再复氧培养时,加入神经干细胞与皮层神经元共培养24 h),24 h后采用细胞计数试剂盒-8(CCK-8)检测各组神经元的增殖,流式细胞仪检测神经元凋亡率,酶联免疫吸附实验(ELISA)检测神经元上清中白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)含量,实时荧光定量聚合酶链反应(qRT-PCR)、Western blotting实验分别检测神经元Notch1、Hes1、Hes5 mRNA及蛋白的表达。结果(1)免疫荧光染色检测结果显示神经干细胞球巢蛋白、GFAP、MAP2阳性,皮层神经元NeuN及β-Tubulin阳性,均鉴定成功。(2)与正常组比较,OGD-R模型组、OGD-R+神经干细胞组、OGD-R+依达拉奉组神经元细胞存活率减低、凋亡率增加、上清中IL-1β及TNF-α含量升高、Notch1 mRNA及蛋白的表达升高,差异均有统计学意义(P<0.05)。与OGD-R模型组比较,OGD-R+神经干细胞组+依达拉奉组神经元细胞存活率升高、凋亡率减少、上清中IL-1β及TNF-α含量减低、Hes1和Hes5 mRNA及蛋白的表达均降低,OGD-R+依达拉奉组、OGD-R+神经干细胞+依达拉奉组神经元Notch1 mRNA及蛋白表达降低,差异均有统计学意义(P<0.05)。结论神经干细胞联合依达拉奉治疗可以逆转OGD-R导致的神经元损伤,可能是通过抑制炎性因子和Notch信号通路上关键信号分子Notch1、Hes1、Hes5的表达来发挥作用。

Objective To explore the protective role and possible mechanism of neural stem cells combined with edaravone in cortical neurons after oxygen-glucose deprivation/reperfusion(OGD-R).Methods(1)Neural stem cells from brain tissues of SD fetal rats aged 14-16 d were cultured in vitro,and identified with Nestin,glial fibrillary acidic protein(GFAP),and microtubule-associated protein 2(MAP2)immunofluorescent staining.Expressions of neuron-specific nuclear protein(NeuN)andβ-Tubulin were detected by immunofluorescent staining in primary cortical neurons from SD rats born within 24 h.(2)Primary cortical neurons were divided into normal group(normal culture),OGD-R model group(re-oxygenated culture for 24 h after hypoxia for 1.5 h),OGD-R+neural stem cells group(re-oxygenated co-culture with cortical neurons and neural stem cells for 24 h after hypoxia for 1.5 h),OGD-R+edaravone group(re-oxygenated culture for 24 h after hypoxia for 1.5 h;100μmol/L edaravone before hypoxia),OGD-R+neural stem cells+edaravone group(re-oxygenated co-culture with cortical neurons and neural stem cells for 24 h after hypoxia for 1.5 h;100μmol/L edaravone before hypoxia);24 h after each treatment,neuron proliferation in each group was detected by cell counting Kit 8(CCK8),apoptosis rate was detected by flow cytometry,contents of interleukin-1β(IL-1β),and tumor necrosis factorα(TNF-α)in neuronal supernatant were detected by enzyme-linked immunosorbent assay(ELISA),and real-time quantitative fluorescent polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the mRNA and protein expressions of Notch1,Hes1 and Hes5,respectively.Results(1)Immunofluorescent staining results showed that neural stem cells were positive for Nestin,GFAP and MAP2,and cortical neurons were positive for NeuN andβ-Tubulin;all of them were successfully identified.(2)Compared with normal group,OGD-R model group,OGD-R+neural stem cell group and OGD-R+edaravone group had decreased neuron viability,increased apoptosis,increased supernatant IL-1βand TNF-αcontents,and increased Notch1 mRNA and protein expressions,with significant differences(P<0.05).Compared with OGD-R model group,OGD-R+neural stem cells+edaravone group had increased neuron viability,decreased apoptosis,decreased supernatant IL-1βand TNF-αcontents,and decreased mRNA and protein expressions of Hes1 and Hes5,with significant differences(P<0.05).Compared with the OGD-R model group,OGD-R+edaravone group and OGD-R+neural stem cell+edaravone group had significantly decreased Notch1 mRNA and protein expressions(P<0.05).Conclusion Combination of edaravone and neural stem cell therapy can reverse the neuronal damage caused by OGD-R,whose mechanism may be by inhibiting the expressions of inflammatory factors and key signaling molecules in Notch signaling pathway,such as Notch1,Hes1,and Hes5.