脑脊液外泌体内转录因子SP1调控长链非编码RNATUG1参与七氟烷保护脊髓缺血再灌注损伤的机制研究

Spinal cord ischemia-reperfusion injury (SCIRI) is one of the most serious complications of thoracic abdominal aneurysm surgery. Sevoflurane has a protective effect on SCIRI but the mechanism is unclear. In the early stage, it was found that TUG1 increased and miR-29 decreased in spinal cord tissue suffered by SCIRI,，while sevoflurane inhibited TUG1 and increased miR-29. Previous experiments confirmed that TUG1 could bind to miR-29 and negatively regulate miR-29, and predicted that SP1 might be related to TUG1 ，combined with positive regulation of TUG1 changes. Cerebrospinal fluid contains a variety of cell-derived exosomes, which may be involved in SCIRI by carrying SP1-mediated spinal neurons injury. Based on a series of preliminary research, we speculated that sevoflurane suppressed the expression of SP1 in exosomes of cerebrospinal fluid and TUG1 while increased miR-29 in spinal cord neurons suffered by SCIRI, further inhibited the pathway of TRAF3-Smac/DIABLO-caspase3, BMF-Bax-caspase3 and TRIL-TLR4-IL-1 to protect spinal neurons. This study was first to confirm the binding and regulation of SP1 in exosomes of cerebrospinal fluid and TUG1 in spinal cord. Secondly，to confirm the direct effect and binding site of miR-29 and TRAF3, BMF and TRIL, We also investigated the expression and function of SP1 in exosomes of cerebrospinal fluid 、TUG1, miR-29 and TRAF3, BMF and TRIL in spinal cord on the pretreatment of sevoflurane in the oxygen glucose deprivation of models of neurons，In the end，we tend to explore the protective effects of sevoflurane, SP1 inhibitor, TUG1 inhibitor, miR-29 alone or combination in SCIRI.

脊髓缺血再灌注损伤是胸腹主动脉瘤手术的严重并发症，七氟烷对SCIRI有保护作用。前期发现SCIRI时脊髓TUG1增加，miR-29降低，七氟烷抑制TUG1，增加miR-29，TUG1可结合并负性调控miR-29，推测脑脊液exosomes内SP1结合并正向调控脊髓TUG1。推测SCIRI时七氟烷抑制SP1高表达和TUG1高表达，增加miR-29，负性调控TRAF3-Smac/DIABLO-caspase3、BMF-Bax-caspase3和TRIL-TLR4-IL-1β通路保护脊髓神经元。本项目先验证SP1与TUG1结合作用；再验证miR-29与TRAF3、BMF和TRIL结合作用，再研究七氟烷预处理对氧糖剥夺神经元和SCIRI时SP1、TUG1、miR-29及TRAF3、BMF和TRIL基因表达和功能影响，最后探讨七氟烷、SP1抑制剂、TUG1抑制剂、miR-29对SCIRI的保护作用。

脊髓缺血再灌注损伤是胸腹主动脉瘤手术的严重并发症，七氟烷对SCIRI有保护作用。前期发现SCIRI大鼠脊髓SP1高表达，TUG1高表达，miR-29b-1-5p低表达，TRIL/TLR4高表达, BMF高表达,TUG1通过调控miR-29b-1-5p/MTDH和TRIL/TLR4炎症通路发挥神经保护，并发现了七氟烷通过作用于一系列miRNA发挥神经保护作用,与课题预期结果一致。经过探索，发现了一系列非编码RNA和SCIRI相关。miR-128-3p 通过靶向结合SP1参与 SCIRI大鼠神经炎症和凋亡; 七氟烷预处理通过调控miR-29b-3p抑制凋亡改善SCIRI大鼠运动功能；七氟烷通过miR-199a-5p参与调控凋亡机制改善SCIRI大鼠下肢运动功能;GAS5通过抑制MMP-7减轻SCIRI大鼠神经凋亡和炎症反应发挥神经保护作用;发现MPO，IL-6，IL-15炎症机制在SCIRI发挥重要作用；激动MiR-187-3p抑制小鼠P2X7R和IL-1β释放缓解小鼠SCIRI诱导的疼痛超敏反应；miR-137-3p/CAPN-2通过调节p35切割和Caspase-8过度激活参与SCIRI诱导的神经元凋亡；缺氧预处理的VSC4.1神经元中富含mir-126-3p的胞外囊泡通过调节pik3r2减轻SCIRI诱导的疼痛超敏反应；抑制AIM2炎性小体的激活和caspase-1和IL-1β的释放可以抑制SCIRI诱导的脊髓神经元焦亡；发现SCIRI大鼠脊髓铁死亡相关指标变化，沉默EphA4通过减少运动神经元铁死亡，改善BSCB通透性，降低炎症反应，参与神经保护。EphA4可能通过激活ERK1/2,增加C-myc的表达增加TFR1的表达促进铁死亡的发生。课题组后续探索发现低氧预处理BMSC来源的外泌体内lncRNA LOC108351028表达明显上调，大鼠脊髓LOC108351028在IR后明显下调,通过双荧光素酶报告基因进一步证实 LOC108351028 可以和 miR-1224 靶向结合。发现鞘内注射低氧预处理BMSC外泌体通过抑制细胞焦亡减轻SCIRI，为课题组后续探索神经保护方法提供了良好的基础。