BI-1调控内质网应激对蛛网膜下腔出血后早期脑损伤的保护作用及机制研究

It has been considered that the primary cause of death in subarachnoid hemorrhage (SAH) is early brain injury(EBI), and the pathomechanisms remain unclear. Our study had demonstrated that the inhibition of JNK, an endoplasmic reticulum (ER) stress associated protein, can offer some protection by reduce hippocampal neuronal apoptosis, but the related mechanisms were unclear. Bax inhibitor 1 (BI-1), a conservative inhibitor of apoptosis protein, which is closely related to ER stress and locate in neurons. Our preliminary experiment also showed that the upregulation of BI-1 can reduce hippocampal neuronal apoptosis in 72h after SAH. Accordingly, we hypothesized that BI-1 protects from EBI by reducing celluar apoptosis through the inhibition of JNK. With the application of behavioral assessment, RT-PCR, immunoblotting, RNAi and gene overexpression on the established model of SAH, we will investigate whether BI-1 protects from EBI following SAH by modulating ER stress and the exact roles of BI-1 on modulation of several ER stress associated molecules. The aim of the study is to explore the molecular mechanism of BI-1 on ameliorating EBI after SAH , which may reveal new therapeutic avenues that can be exploited to serve as combination therapy way on EBI in the future.

早期脑损伤是蛛网膜下腔出血（SAH）患者死亡的首要原因，其病理机制不详。我们研究发现，抑制内质网应激相关蛋白JNK的表达，能减少海马神经元凋亡，改善SAH后早期脑损伤，但具体调控机制尚不清楚。BI-1是存在于神经元，与内质网应激密切相关的保守性细胞凋亡抑制因子。在预实验中我们发现，上调BI-1的表达，能明显减少SAH后72h内海马神经元的凋亡。因此，我们推测BI-1可能通过调控内质网应激，减少JNK的表达，进而减少神经元凋亡，对早期脑损伤发挥保护作用。本项目通过构建大鼠SAH模型，应用RNAi和基因过表达、免疫印迹、神经功能学检测等方法，探讨BI-1是否通过内质网应激参与了SAH后早期脑损伤，以及BI-1通过调控内质网应激相关分子，发挥脑保护作用的相关机制。本项目旨在深入探索SAH后早期脑保护的治疗新靶点，为临床治疗SAH，降低其病死率和病残率提供理论依据。

早期脑损伤（early brain injury，EBI）是蛛网膜下腔出血（subarachnoid hemorrhage，SAH）患者死亡的首要原因，其病理机制不详。本项目通过构建大鼠SAH模型，应用RNAi和基因过表达、免疫印迹、神经功能学检测等方法，探讨BI-1是否通过内质网应激参与了SAH后早期脑损伤，以及BI-1通过调控内质网应激相关分子，发挥脑保护作用的相关机制。研究结果发现：（1）BI-1基因的过表达可以降低SAH后脑水肿程度,减少海马神经元的凋亡。（2）SAH可激活内质网应激相关蛋白CHOP、GRP78的表达；BI-1的过表达可抑制SAH后内质网应激相关蛋白CHOP、GRP78的表达；说明BI-1过表达可抑制SAH后内质网应激的过度激活。（3）干预BI-1过表达及阻断IRE1-JNK信号通路对海马神经元凋亡情况的影响：与SAH组相比，SAH+BI-1 over组和JNK抑制剂SP600125组神经元凋亡明显减少，且SAH+BI-1 over组、SP600125组与SAH+BI-1 over+SP600125组相比，三组神经元凋亡均无显著性差异；说明干预BI-1过表达及阻断IRE1-JNK信号通路均可减少SAH后大鼠海马神经元的凋亡。（4）干预BI-1的表达对IRE1-JNK通路信号分子影响：SAH组、SAH+NC组、SAH+BI-1 shRNA组、SAH+BI-1 over组与sham组相比，GPR78、CHOP、ASK1、IRE1、JNK和TRAF2的表达均明显增高； SAH+BI-1 shRNA组与SAH组相比，以上分子表达更高；SAH+BI-1 over组与SAH组相比，以上分子的表达均明显降低，说明干预BI-1的表达可以调控SAH后内质网应激，该过程可能是通过调节IRE1-JNK信号通路而实现的。综合以上结果，我们认为BI-1参与了SAH后早期脑损伤；上调BI-1的表达，可通过负性调节IRE1-JNK通路，降低JNK的表达，减轻内质网应激，进而减少神经元凋亡，改善SAH后EBI；为临床治疗SAH，降低其病死率和致残率提供了理论依据。