SIRT1/NF-κB介导CEPO调控活性氧耐受促进创伤后脑微血管内皮细胞修复的实验研究

One of the most important reasons for Blood brain barrier injury after traumatic brain injury(TBI) was the damage of cerebral microvascular endothelial cells . triggered by Reactive oxygen species(ROS) after TBI, and the damage of cerebral microvascular endothelial cells(CMEC) could be significantly alleviated after ROS tolerance (ROST) induced in CMECs. CMECs are injuryed by ROS through many sophisticated signal transduction pathways. Recent studies have demonstrated that changes on chromatin of specific ROS signal transduction proteins caused by nuclear receptors provided the major regulatory mechanisms for ROS induction. NF-kB was important nuclear receptors to activate CMECs caused by ROS, and receptor-interacting protein. Silent information regulation 1(SIRT1) mainly expressed in brain was one of the most important co-activator for NF-κB. The up regulation of SIRT1 promotes ROST in brain. Our previous study found that CEPO can activate SIRT1 expression to inhibit downstream molecular activity of NF-kB, promoting the repair of microvascular endothelial cells after traumatic brain injury..We put forward that if we can confirm that CEPO is a key regulatory factor to SIRT1, SIRT1 effectively blocked the damage of active oxygen on blood brain barrier. This project further study on the protection mechanism of CEPO -SIRT1- NF-κB signal transduction pathways and molecular targets on the blood-brain barrier, provides the theory for the development of new drugs to protect blood brain barrier after trauma.

研究发现活性氧触发脑微血管内皮细胞损伤是导致颅脑创伤后血脑屏障损伤重要原因之一，诱导脑微血管内皮细胞对活性氧耐受（ROST)可显著减轻血脑屏障损伤。调控核受体活性,使促活性氧生成的相关传导因子染色质发生改变是诱导脑微血管内皮细胞对活性氧耐受的主要机制。而富集表达于神经系统的SIRT1又是调控核受体NF-kB 转录活性的关键性抑制因子，我们前期研究发现CEPO可激活SIRT1表达从而抑制下游分子NF-kB活性，促进创伤后脑微血管内皮细胞修复。.我们提出，如能证实CEPO是SIRT1关键调控因子，那促进SIRT1表达将从调控核受体NF-kB活性角度有效阻断活性氧对颅脑创伤微血管内皮细胞损害。本项目进一步研究CEPO—SIRT1—NF-κB信号通路及分子靶点对血脑屏障的保护机制，为创伤后血脑屏障保护新药开发提供理论依据。

本课题围绕TBI后发生氧化应激的分子机制，以C57BL小鼠、bEnd3小鼠血管内皮细胞、HT22小鼠海马神经元细胞为研究对象，建立了体内的小鼠的TBI模型及细胞中Hemin、H2O2干预致氧化应激损伤的体外细胞模型。我们采用第二代转录组测序及分析、基因沉默、基因过表达、RNA pull-down、双荧光素酶报告基因等技术研究EPO减轻TBI后氧化应激的主要作用机制，阐明了其中涉及的部分分子机制，并提供了EPO在促进TBI后神经功能恢复的实验依据。我们发现EPO改善了TBI小鼠学习记忆能力，也促进了神经功能的恢复并使损伤灶血管生成从而改善循环、减少损伤体积。我们通过对第二代转录组测序结果的分析发现，EPO治疗后与TBI组对比，共有367个mRNA差异表达，392个lncRNA差异表达，1774个circRNA差异表达，25个miRNA差异表达。进一步的实验证明了circLphn3/miR-185-5p/ZO1轴对TBI后组成血脑屏障的紧密连接蛋白的调控，以及通过circPtpn14/miR-351-5p/5-LOX轴对脂质过氧化反应、内质网应激及铁死亡的调控作用。该课题研究为TBI后的继发性脑损伤提出了新的分子机制，即circRNA参与的分子病理过程，从而为TBI提供了新的治疗靶点。