Nrf2-ARE通路在缓解SAH后脑血管痉挛作用机制的实验研究

Cerebral vasospasm(CVS) and delayed cerebral ischemia are almost thought as the major complication of aneurysmal subarachnoid hemorrhage (SAH). It is associated with high morbidity and mortality rates. It has been suggested that the pathogenesis of CVS is complex including oxidative stress, inflammatory damage，cell apoptosis and the accumulation of toxic metabolites. Although a great deal of experimental and clinical researches have been conducted to find ways to prevent this complication, the management of vasospasm in patients who have experienced an aneurysmal SAH remains a significant clinical problem. Nuclear factor erythroid 2-related factor 2,is a transcription factor which has been reported to be the key regulator in reducing oxidative stress, inflammatory damages and accumulation of toxic metabolites. A growing body of evidence indicates that the nuclear factor erythroid 2-related factor 2-antioxidant response element(Nrf2-ARE) pathway plays a protective role in many physiological stress processes such as inflammatory damage, oxidative stress and the accumulation of toxic metabolites, which are all involved in the development of CVS following SAH. Furthermore, Nrf2-ARE pathway has been considered to play a protective role in several central nervous system (CNS) diseases. Therefore,we suppose that Nrf2-ARE pathway may contribute to protection against CVS following SAH. To date, however, there was no study to explore the potential role of Nrf2-ARE pathway in the CVS after SAH.The purpose of this study was to investigate the activation of Nrf2-ARE pathway on CVS after SAH and determine the protection role of Nrf2-ARE pathway on CVS after SAH. Anterior circulation mouse model of SAH is used in this study. This mouse model used here may provide a closer simulation of clinical SAH and to create a mouse model that could then be used in transgenic and knockout animals in order to further knowledge of SAH and vasospasm. For creation of SAH, the head was fixed in a stereotactic frame equipped with a mouse adaptor. Stereotactic manipulators were used for holding the injection needle and a laser Doppler flow probe to measure cerebral blood flow (CBF). Nrf2 wild type (WT) or knockout (KO) mice are subjected to injection of 100 μl autologous blood over seconds. SUL(a specific Nrf2 activator) was intraperitoneal injected as the blood injection manner every 24 h from 30 min after blood injection to the last day. In control animals, the same technique was applied with injection of sterile saline instead of blood. The experimental groups consisted of sham WT, sham KO, SAH WT, SAH KO,SAH WT+SUL. All animals were recorded daily by using the behavior scores until sacrificed. After decapitated, the middle and anterior cerebral arteries are harvested for H-E staining，immunohistochemistry, western blot, real-time PCR, ELISA, EMSA and flow cytometry analysis.We could conclude whether Nrf2-ARE patway have a therapeutic role for CVS after SAH.

蛛网膜下腔出血(SAH）后的脑血管痉挛(CVS)发生率较高，成为SAH后致死、致残的主要原因，其发病机制十分复杂，可能与氧化应激、炎症损伤、细胞凋亡、钙离子超载等多种因素有关。Nrf2-ARE通路是细胞内在的一种保护性调节通路，已有研究证明该通路激活后所表达的下游产物具有抗氧化应激、调节炎症损伤、抗细胞凋亡、缓解细胞内钙离子超载等多重细胞保护作用。关于该通路激活后是否能够缓解CVS的发生及其作用机制目前仍不清楚。本研究利用普通小鼠及Nrf2基因敲除小鼠制作SAH模型，给予Nrf2-ARE通路激活剂作为CVS的治疗药物，评定给药前后小鼠神经功能改变，测定脑血流、动脉管腔直径、氧化损伤、炎症、细胞凋亡、钙离子稳态等相关指标，通过比较对照分析，明确Nrf2-ARE通路在SAH后的脑血管中是否被激活及其是否具有缓解CVS的作用，并明确其具体作用机制，为今后开展针对该靶点的进一步研究提供实验基础。

蛛网膜下腔出血(SAH）后的脑血管痉挛(CVS)发生率较高，成为SAH 后致死、致残的主要原因，其发病机制十分复杂，可能与氧化应激、炎症损伤、细胞凋亡、钙离子超载等多种因素有关。Nrf2-ARE通路是细胞内在的一种保护性调节通路，已有研究证明该通路激活后所表达的下游产物具有抗氧化应激、调节炎症损伤、抗细胞凋亡、缓解细胞内钙离子超载等多重细胞保护作用。关于该通路激活后是否能够缓解CVS 的发生及其作用机制目前仍不清楚。随着研究的深入, Nrf2-ARE通路的细胞内保护作用日益受到重视。本研究通过体内及体外蛛网膜下腔出血模型证实了Nrf2-ARE通路在SAH后被激活，在此基础上利用普通小鼠及 Nrf2 基因敲除小鼠制作SAH模型，给予 Nrf2-ARE通路激活剂作为 CVS 的治疗药物，评定给药前后小鼠神经功能改变，测定脑血流、动脉管腔直径、氧化损伤、炎症、细胞凋亡、钙离子稳态等相关指标，通过比较对照分析，探讨 Nrf2-ARE通路在SAH 后是否能够缓解 CVS，并明确其具体作用机制。研究表明，SAH后Nrf2-ARE通路在SAH后的脑血管中被激活，并参与调节了SAH后CVS的多种病理生理机制，运用Nrf2-ARE通路的激动剂剂莱菔硫烷后，Nrf2-ARE通路活性明显增强，试验动物的神经功能学评分、脑血流、动脉管腔直径、氧化损伤、炎症反应、细胞凋亡、钙离子稳态等相关指标都得到了明显改善。试验结果证明Nrf2-ARE通路调节了SAH后CVS发生和发展的多种病理生理机制，该信号通路激活后有助于缓解脑血管痉挛，改善SAH后神经功能缺损，为SAH后CVS的治疗寻找性的靶点。课题组按照研究计划基本如期完成了该课题的研究工作。