程序性坏死RIPK1-RIPK3复合体介导中枢神经系统发育期七氟醚神经毒性

Programmed Necrosis RIPK1-RIPK3 Complex Mediates Sevoflurane-induced Developmental Neurotoxicity

目的探究RIPK1-RIPK3复合体介导的程序性坏死机制是否参与中枢神经系统发育期七氟醚神经毒性过程。方法对离体培养7 d的海马神经细胞及出生后7 d的SD幼鼠进行七氟醚处理(3%,6 h),制备七氟醚神经毒性模型。应用基因芯片检测海马脑区相关目的基因的mRNA变化;应用免疫共沉淀及免疫印迹检测目的蛋白的结合及表达变化;应用TUNEL染色检测七氟醚处理后神经细胞死亡状况;应用水迷宫检测七氟醚处理后实验动物学习记忆的行为学改变。结果七氟醚处理(3%,6h)可使Ripk1、Ripk3、Mlkl、Daxx的mRNA表达水平显著升高,同时应用免疫印迹在蛋白水平进行了验证。七氟醚处理导致神经细胞TUNEL的阳性细胞数量明显增加。应用RIPK1特异性抑制剂Nec-1可明显降低TUNEL阳性数量。动物行为学结果显示,七氟醚处理可导致实验动物空间学习记忆能力降低,而应用Nec-1可明显改善动物的空间学习记忆能力。结论在中枢神经系统发育期,程序性坏死机制可能参与了七氟醚神经毒性过程。其机制可能与RIPK1-RIPK3复合体介导MLKL、Daxx信号通路有关。

OBJECTIVE To examine that programmed necrosis RIPK1-RIPK3 complex participates in the mechanism of developmental sevoflurane neurotoxicity.METHODS The developmental sevoflurane neurotoxicity model was established by exposure of primary hippocampal neurons(7 d in vitro)and SD rat pups(7 postnatal days)with sevoflurane(3%,6 h).The gene chip was used to detect the mRNA levels of the targeted genes.The CO-immunoprecipitation and immunoblotting were performed to explore the combination and expression differences of targeted proteins.The developmental sevoflurane neurotoxicity was determined by TUNEL-staining.The spatial learning and memory functions were examined by Morris Water Maze.RESULTS Sevoflurane exposure(3%,6 h)significantly enhanced the mRNA levels of Ripk1,Ripk3,Mlkl and Daxx.These results were confirmed by Western blot in protein level.Sevoflurane treatment dramatically increased TUNEL-positive neurons.In addition,administration of RIPK1-RIPK3-specific inhibitor Nec-1 significantly decreased TUNEL-positive neurons.Furthermore,treatment with Nec-1 significantly improved the spatial cognitive disorders which was decreased by postnatal sevoflurane exposure.CONCLUSION These data suggest that programmed necrosis participates in developmental sevoflurane neurotoxicity,and its mechanism may be related to the RIPK1-RIPK3 complex mediating MLKL and Daxx signaling pathways.