基于BDNF/TrkB/CREB途径探讨缺氧缺血性新生幼鼠海马神经元凋亡及脑发育损伤的机制研究

Exploring the mechanism of neuronal apoptosis and brain developmental damage in the hippocampus of hypoxic-ischemic neonatal rats based on BDNF/TrkB/CREB pathway

目的:基于BDNF/TrkB/CREB途径探讨缺氧缺血性新生幼鼠海马神经元凋亡及脑发育损伤发生的相关作用机制。方法:Wistar幼鼠结扎一侧颈总动脉,置8%氧气、92%氮气缺氧箱内2 h,制备缺氧缺血性脑损伤(hypoxic ischemic brain injury,HIBI)模型,健康大鼠作为对照组,取对照组、模型组幼鼠各10只,Y迷宫测试学习记忆能力;2,3,5-氯化三苯基四氮唑(TTC)染色检测脑组织损伤情况;蛋白质免疫印迹测定海马组织中脑源性神经营养因子(brain derived neurotrophic factor,BDNF)、酪氨酸蛋白激酶B(TrKB)、环磷腺苷效应元件结合蛋白(cAMP-response element binding protein,CREB)的表达。另取对照组15只,模型组幼鼠60只分为阴性对照(negative control,NC)组、BDNF过表达(LV-BDNF)组、TrkB过表达(LV-TrkB)组、CREB过表达(LV-CREB)组,尾静脉注射空白载体、BDNF、TrkB、CREB腺病毒过表达载体。Y迷宫测试学习记忆能力;TTC染色法检测脑组织损伤情况;原位末端转移酶标记法检测海马区神经元凋亡水平;Western blot检测海马组织中凋亡相关蛋白B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、BCL2-Associated X的蛋白质(Bcl-2 Assaciated X,Bax)及核因子κB(nuclear factor kappa-B,NF-κB)的表达。结果:模型组幼鼠学习记忆能力明显降低,脑组织梗死体积明显增大,海马组织中BDNF、TrkB蛋白的表达明显升高,CREB蛋白表达明显降低,差异均具有统计学意义(P<0.05);过表达BDNF/TrkB/CREB后,LV-BDNF、LV-TrkB、LV-CREB组幼鼠学习记忆能力明显提高,脑组织梗死体积明显缩小,海马区神经元凋亡明显减少,Bax、NF-κB蛋白表达明显降低,Bcl-2蛋白表达明显增强,差异均具有统计学意义(P<0.05)。结论:HIBI模型幼鼠存在BDNF/TrkB/CREB表达异常,过表达BDNF/TrkB/CREB可以改善幼鼠学习记忆能力,修复脑组织损伤,抑制神经元凋亡,因此,HIBI的发生机制可能与BDNF/TrkB/CREB途径有关。

Objective:Based on the BDNF/TrkB/CREB pathway,to explore the mechanism of neuronal apoptosis and brain developmental injury in the hippocampus of hypoxic-ischemic neonatal rats.Methods:Wistar young rats were ligated on one side of the common carotid artery and placed in an 8%oxygen and 92%nitrogen hypoxia box for 2 h to prepare hypoxic-ischemic brain injury models.Healthy rats were used as the control group.Control group and model group were selected,with 10 rats in each group,and the learning and memory ability was tested by Y-maze;2,3,5-triphenyltetrazolium chloride(TTC)staining was used to detect brain tissue damage;Western blot was performed to determine the expression of brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrKB)and cAMP-response element binding protein(CREB)in hippocampal tissue.Another 15 mice in the control group and 60 mice in the model group were divided into negative control group(NC),BDNF overexpression group(LV-BDNF),TrkB overexpression group(LV-TrkB),and CREB overexpression group(LV-CREB),blank vector,BDNF,TrkB,CREB adenovirus overexpression vector was injected into the tail vein.Y-maze test for learning and memory ability;TTC staining method to detect brain tissue damage;neuronal apoptosis in the hippocampus were detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling;Western blot to detect the level of neuronal apoptosis in the hippocampus.Apoptosis-related protein B-cell lymphoma-2(Bcl-2),BCL2-associated X protein(Bcl-2 Assaciated X,Bax)and nuclear factor kappa-B(NF-κB)expression.Results:The learning and memory ability of the young mice in the model group was significantly reduced,the brain infarct volume was significantly increased,the expressions of BDNF and TrkB proteins in the hippocampus were significantly increased,and the expression of CREB proteins was significantly decreased;After overexpression of BDNF and TrkB CREB,in the LV-BDNF,LV-TrkB,and LV-CREB group,the learning and memory ability of young mice were significantly improved,the brain infarct volume were significantly reduced,the hippocampal neuronal apoptosis were significantly reduced,The protein expression of Bax and NF-κB were significantly decreased and the protein expression of Bcl-2 were significantly enhanced.Conclusion:The expression of BDNF/TrkB/CREB is abnormal in HIBI model young mice.Overexpression of BDNF/TrkB/CREB can improve the learning and memory ability of young mice,repair brain tissue damage,and inhibit neuronal apoptosis.Therefore,the mechanism of HIBI may be related to BDNF/TrkB/CREB pathways.