GIT1通过thioredoxin保护继发性脊髓损伤的机制研究

Spinal cord injury is a kind of damage to the quality of life, but its mechanism is still unknown. Previous studies have showed that the oxidative stress in the spinal cord is the key factor to determine the process of secondary spinal cord injury; at the same time, the G-protein coupled receptor kinase-interacting protein 1 (GIT1) can protect nerve cells from apoptosis after spinal cord injury (Yin G: JCI, 2001; BRB, 2013; CBP & AHM, 2016 ). We will investigate the interaction of the LTDRL amino acid in SHD domain of GIT1 and the 31W in WCGPC domain of thioredoxin, as well as the S419 amino acid in CC2 domain of GIT1 and the CC domain of ASK1 domain, and subsequently detect the effect on the activity of Apoptosis signal-regulating kinase-1 (ASK1) by decreasing the phosphorylation of threonine 845 site in ASK1 with reduced secondary spinal cord injury, by immunoblotting, co-immunoprecipitation, immunofluorescence and luciferase report gene assay in GIT1 wild type (GIT1 WT)/ GIT1 knock out (GIT1 KO) and ASK1 wild type (ASK1 WT)/ ASK1 knock out (ASK1 KO) mice. To explore the function of GIT1 and thioredoxin in secondary spinal cord injury, not only can solve the problem about a lack of research on its mechanism in China and fill the gaps in international research, but also can provide a solid theoretical basis in protection and treatment in spinal cord injury.

脊髓损伤严重损害生存质量，至今机制不清。既往研究显示氧化应激是决定继发性脊髓损伤的关键因素，同时GIT1蛋白可以抑制脊髓损伤后神经细胞的凋亡(Yin G: JCI, 2001; BRB, 2013; CBP & AHM, 2016)。本研究将通过国内唯一具有的GIT1和ASK1基因敲除小鼠建立脊髓缺血再灌注模型，运用免疫杂交、免疫共沉淀、免疫组化、免疫荧光和荧光素酶报告基因检测等方法，探讨GIT1内SHD结构域的LTDRL氨基酸序列和thioredoxin内WCGPC结构域内的31W，以及GIT1内CC2结构域S419位点和ASK1内CC结构域相互结合，从而抑制ASK1 T845位点的磷酸化，减少继发性脊髓损伤。阐明GIT1和thioredoxin在继发性脊髓损伤中的作用，不仅解决国内对其机制研究的缺乏问题，还能填补国际研究邻域的空白，同时为临床更好地预防和治疗脊髓损伤提供坚实的理论基础。

脊髓损伤严重损害患者生存质量，至今机制不清。既往研究显示氧化应激（ROS）是决定继发性脊髓损伤的关键因素，同时GIT1蛋白可以抑制脊髓损伤后神经细胞的凋亡。该项目主要研究内容包括：确立GIT1与thioredoxin结合的关键氨基酸序列和位点、GTI1 CC2结构域中S419位点对其与ASK1内CC结构域结合的作用以及GIT1和thioredoxin结合对ASK1 T845位点的磷酸化的影响，最终阐明GIT1对脊髓损伤调控的具体机制。在本项目资助下，我们明确了GIT1通过CC2结构域与ASK1 CC结构域发生结合，通过竞争性结合机制抑制由CC结构域介导的ASK1二聚化的形成，抑制ASK1的T845位点的磷酸化，从而下调ROS对ASK1/JNK/p38信号轴的激活，抑制神经元凋亡；明确GIT1可通过促进Beclin1 Thr119位点磷酸化，以依赖于Pink1的方式正向调控Parkin线粒体膜转位，最终诱导神经元线粒体自噬发生；明确GIT1 通过与PGK1的结合，从而调节NRF2/THR，保护神经元抵抗ROS，减少脊髓缺血再灌注中神经元的损伤。阐明GIT1在继发性脊髓损伤中调控致病关键因子ROS的作用，不仅解决国内对其机制研究的缺乏问题，还能填补国际研究邻域的空白，同时为临床更好地预防和治疗脊髓损伤提供坚实的理论基础。