GRP94/HAX-1在脊髓损伤修复过程中的作用及其对内质网应激的调控

Spinal cord injury seriously influences the quality of patients' lives with high disability, and regulating neurons from apoptosis is helpful to the repair of spinal cord injury. Endoplasmic reticulum stress induced by spinal cord injury palys a vital role in the secondary neuronal apoptosis, but the mechanisms are not clearly understood. Our preliminary experiments found that the expression GRP94 which is a marker protein of endoplasmic reticulum stress was up-regulated after spinal cord injury, meanwhile, the expression of inhibitor apoptosis protein of HAX-1 also detected a significant increase. Besides, HAX-1 could interact with GRP94 and it could regulate the expression of IRE-1 apoptosis signal, which suggested that GRP94/HAX-1 involved in reparative processes after spinal cord injury. Research group firstly detects the spatiotemporal changes of GRP94 and HAX-1 as well as related proteins to explore their correlation with neuronal apoptosis in spinal cord injury model. Furthermore, we study the detailed pathways and molecular mechanisms by which GRP94/HAX-1 regulate the expression of IRE-1 and affect the neuronal apoptosis in vitro. Finally, we interrupt the expression of GRP94/HAX-1 to regulate the IRE-1 expression and thus clarifying their roles in functional recovery after spinal cord injury in vivo. This study is helpful to clarify the effect of endoplasmic reticulum stress on reparative processes in spinal cord injury and regulatory mechanisms, which can provide a new targets for clinical neurological rehabilitation.

脊髓损伤致残率高，严重影响患者的生存质量，调控神经元免于凋亡有利于脊髓损伤的修复。内质网应激在脊髓损伤后继发性神经元凋亡中发挥重要作用，但其相关机制尚不清楚。预实验发现内质网应激标志物GRP94和凋亡抑制蛋白HAX-1在脊髓损伤后表达明显上调，且存在相互作用，HAX-1可以调控内质网应激诱导的肌醇需酶1凋亡信号的表达，提示GRP94/HAX-1参与脊髓损伤的修复过程。本课题拟先在脊髓损伤模型中检测GRP94/HAX-1及相关蛋白时空表达变化，探知其与神经元凋亡的相关性；其次在细胞模型中探究GRP94/HAX-1调节肌醇需酶1信号通路影响神经元凋亡的具体分子机制，探讨其对内质网应激的调控；最后体内干预GRP94/HAX-1，调节肌醇需酶1的表达，明确其在脊髓损伤修复过程中的作用。本研究有助于阐明内质网应激在脊髓损伤修复过程中的作用及调控机制，为临床功能康复提供新的治疗靶点。

工伤、交通事故等导致的脊髓损伤(spinal cord injury, SCI)发病率逐年增高，成为临床常见的疾病，而SCI的治疗和临床康复仍然是世界性的难题。调控神经元免于凋亡有利于SCI的修复。内质网应激在SCI后继发性神经元凋亡中发挥重要作用。我们首先在大鼠SCI模型中发现SCI后凋亡抑制蛋白HAX-1、内质网应激的标志物GRP94和凋亡信号IRE-1均发生明显表达变化，这种变化与SCI后神经元的凋亡密切相关；免疫共沉淀发现GRP94在脊髓组织中与HAX-1和IRE-1相互作用，且损伤后两者作用增强。随后我们在细胞凋亡模型中发现HAX-1、GRP94、IRE-1以及IRE-1下游Caspase12、pJNK和CHOP的表达明显增加，与大鼠SCI模型结果相一致。显微镜下观察和TUNEL均证实过表达HAX-1可以减少PC12细胞的凋亡，而下调HAX-1的表达将失去这种保护作用。RT-PCR和Western blot结果显示过表达HAX-1后，GRP94的表达也显著增加，Caspase12和CHOP表达减少，IRE-1的表达没有变化，但pIRE-1的表达减少；下调HAX-1的表达后，GRP94的表达也下降，Caspase12和CHOP表达增加，IRE-1仍没有变化，pIRE-1的表达增加。提示HAX-1可能通过与GRP94相互作用抑制IRE-1的磷酸化，降低了pIRE-1的表达，从而促进神经元的存活。最后我们用腺病毒转染干预大鼠脊髓HAX-1的表达，并以同样的方法制作SCI模型。过表达HAX-1后，大鼠后肢运动功能明显改善，BBB评分增加，反之干扰HAX-1表达后, 后肢运动功能恢复受限，BBB评分降低。HE染色也表明，HAX-1可以保护SCI期间的神经元免于凋亡。这一结果显示HAX-1在大鼠SCI后的修复过程中发挥了重要作用，体内干预HAX-1的表达将影响大鼠神经功能的恢复。总体而言，HAX-1调节SCI后神经功能的修复可能是通过与GRP94相互作用抑制IRE-1的磷酸化，降低了pIRE-1的表达，减少了IRE-1下游CHOP基因通路、JNK通路、Caspase-12通路激活，从而起到抗神经元凋亡作用，促进神经功能康复。我们课题研究发现了HAX-1调控内质网应激在SCI后神经元凋亡过程中的作用及其相关机制，HAX-1可能是临床治疗SCI的一个潜在的靶点。