RACK1调控内质网应激-自噬途径对创伤性脑损伤的保护机制研究

Apoptosis is the main cause of high mortality and disability after traumatic brain injury (TBI). However, its pathogenesis remains largely unknown and treatment options are very limited. Recent publications suggest that endoplasmic reticulum stress-mediated autophagy, which serves as a repair mechanism post-injury, plays a key role in regulating apoptosis. Based on our previous research which found that RACK1 upregulation could effectively inhibit neuronal apoptosis after TBI, we further observed that RACK1 could induce PERK and IRE1 activation and promote neuronal autophagy. Therefore, we proposed the following hypothesis: RACK1 activates PERK and IRE1 pathways of endoplasmic reticulum stress to increase autophagy, thus reduces neuronal apoptosis and eventually ameliorates secondary brain injury after TBI. Based on our previous work, this study plans to further explore the regulatory relationship between RACK1 and neuronal autophagy, and its effect on secondary brain injury after TBI using siRNA interference/overexpression techniques; then, the role and mechanism of endoplasmic reticulum stress in RACK1-mediated regulation of neuronal autophagy will be also explored with PERK and IRE1 signaling pathways as the breakthrough point. This study will help to reveal the endogenous protective mechanism against neuronal apoptosis after TBI, and provide a new idea and theoretical basis for the prevention and treatment of TBI.

细胞凋亡是造成创伤性脑损伤（TBI）高死残率的主要原因，其发生机制尚未阐明，治疗手段短缺。研究表明，内质网应激介导的自噬作为一种损伤后修复机制，在调控细胞凋亡中发挥重要作用。申请人在前期研究证实支架蛋白RACK1过表达可有效抑制TBI后神经元凋亡的基础上，进一步观察到RACK1能够诱导内质网应激信号PERK和IRE1活化，并促进神经元自噬。因此，我们提出假说：TBI后，RACK1激活内质网应激途径中PERK和IRE1通路，并通过级联反应上调自噬水平，继而减轻神经元凋亡，改善继发性脑损伤。本项目在前期工作基础上拟运用干扰/过表达等技术，探讨RACK1与神经元自噬的调控关系，及其对TBI后继发性脑损伤的影响；并以PERK和IRE1信号通路为切入点，探究内质网应激在RACK1调控神经元自噬中的作用和机制。本项目将有助于揭示TBI后神经元凋亡的内源性防御机制，为TBI的防治提供新思路和理论依据。

创伤性脑损伤（TBI）是全球最突出的健康问题之一，常导致患者严重的神经功能障碍。近年来研究发现继发性脑损伤是TBI患者伤情预后及转归的主要决定因素，但至今对其发生机制仍未完全阐明，使得针对性治疗未取得突破性进展。我们在前期研究证实支架蛋白RACK1过表达可有效抑制TBI后神经元凋亡。进一步研究我们发现，在TBI后RACK1高表达的脑区，伴随自噬相关蛋白LC3、beclin1、Atg5表达增强，而P62表达降低；并且免疫荧光染色观察到RACK1与LC3在TBI大鼠脑组织中共定位，且两者荧光强度呈正相关。进一步我们运用干扰/过表达技术证实RACK1参与诱导了TBI后神经元自噬的发生。随后，在过表达RACK1的大鼠TBI模型中，我们发现通过侧脑室注射自噬抑制剂3-MA、Si-Beclin1或Si-Atg5抑制自噬激活后，能够显著翻转过表达RACK1的抗凋亡和神经功能保护作用。此外，通过侧脑室注射PERK抑制剂GSK2606414或Si-ATF4抑制PERK通路的激活，能够显著逆转过表达RACK1对神经元自噬的诱导，提示PERK通路很可能参与介导了RACK1对TBI后神经元自噬的调控。因此，RACK1很可能通过激活PERK-自噬途径在TBI中发挥内源性防御作用，有助于为临床治疗TBI 提供新的潜在靶点。