苏合香对脑缺血再灌注损伤NF-κB介导炎症反应的作用及机制研究

Resuscitation agents are not only the most characteristic medicine in the Traditional Chinese Medicine(TCM), but also focus and an essential part in the modern TCM research. Storax is one of the important traditional resuscitation agents, a preliminary study to whose pharmacological effects have been conducted at home and abroad. However, no feature in-depth study has yet targeted on its effects on anti-brain injury and its preliminary mechanisms. Based on the study of resuscitation agents, this project aims to study effects of storax on anti-the cerebral ischemia-reperfusion injury , and its effect on brain injury inflammatory factors by modeling on rats' cerebral ischemia-reperfusion injury. "Neurovascular Unit" is employed in this project to establish models of oxygen and glucose deprivation / reoxygenation injury neurons, and brain microvascular endothelial cells and astrocyte cells. Focusing on the NF-κB-mediated inflammatory injury, we will observe effects of storax on inflammation factors, adhesion molecules and selectin expression, so as to study its effects on the inflammatory injury of the neurovascular unit induced by oxygen and glucose deprivation / reoxygenation. In addition, we will study the distribution of NF-κB in the neurovascular unit and its activation to explore the initial mechanisms of its function. By modeling on vitro blood-brain barrier injury induced by oxygen and glucose deprivation / reoxygenation, we also study effects of storax on anti-blood-brain barrier damage, which will enrich the scientific connotation of storax's " enlighten " function, and provide scientific basis for the development of traditional Chinese medicine in the prevention and treatment of cerebrovascular diseases.

开窍药是最具中医特色的中药，也是现代中药研究的热点和重要组成部分。苏合香是重要的传统开窍药之一，国内外学者已对其药理作用开展了初步研究。但苏合香是否具有抗脑损伤的作用及其初步机制尚未深入研究。本课题在前期开窍药研究的基础上，建立大鼠脑缺血再灌注损伤模型，研究苏合香抗脑缺血再灌注损伤作用，并探讨其对脑损伤炎症因子的影响；同时引入"神经血管单元"的研究方法，分别建立氧糖剥夺/复氧损伤的神经元、脑微血管内皮细胞和星形胶质细胞模型，围绕NF-κB介导的炎症损伤，观察苏合香对炎症因子、粘附分子、选择素表达的影响，研究其对氧糖剥夺/复氧诱导的神经血管单元炎症损伤的作用，并探讨NF-κB在神经血管单元中分布及活化水平，探讨其作用的初步机制；建立氧糖剥夺/复氧诱导的体外血脑屏障损伤模型，探讨苏合香抗血脑屏障损伤的作用，丰富苏合香"开窍"作用的科学内涵，为研发防治脑血管疾病中药提供科学依据。

主要研究内容.开窍药是具有中医特色的中药，是现代中药研究的热点和重要组成部分。苏合香是传统开窍药之一，其抗脑损伤和开窍作用机制尚未深入研究。课题通过苏合香对大鼠局灶性脑缺血的治疗作用及机制、体外对脑微血管内皮细胞bEnd.3、神经元缺氧/复氧（OGD/R）损伤后的抗炎作用及bEnd.3细胞增殖和迁移的影响三个研究，为苏合香治疗脑缺血的临床转化应用及阐明苏合香“开窍”的机制提供实验依据。.重要结果 采用线栓法建立大鼠大脑中动脉阻塞模型，观察苏合香对脑缺血再灌注损伤的影响，发现苏合香预给药能够显著降低大鼠脑缺血后脑梗死体积、半球水肿率及神经功能评分，降低缺血侧脑组织中IL-1β、TNF-α的表达，NF-κB入核量也降低；同时，苏合香能够显著降低恢复期大鼠神经功能缺损评分（mNSS）及足误步数。苏合香也可升高缺血脑组织周边血小板内皮细胞黏附分子-1（CD31）、突触素（SYP）的表达，提示苏合香通过抗脑缺血再灌注损伤大鼠炎症反应发挥脑缺血保护作用，并且对内源性神经血管重塑、长期神经功能恢复也有促进作用。围绕“神经血管单元”的研究模式，研究培养b.END3细胞，发现苏合香可不同程度的降低OGD/R所致炎症损伤的脑微血管内皮细胞中TNF-α、ICAM-1、IL-6和IL-1β的表达，并且降低I-κB蛋白磷酸化、抑制NF-κB蛋白在细胞核内表达。体外培养神经元，发现苏合香可以通过TLR4/TRAF6/IKKβ/IKBα/NF-κB途径调控相关炎症因子IL-1β、TNF-α、ICAM-1和IL-10的表达，抑制TLR4内源性配体HMGB-1从细胞核向胞外分泌从而发挥保护神经元作用。在脑缺血后脑血管网络的重建和再通方面，研究发现苏合香促进脑微血管内皮细胞增殖，改善脑微血管内皮细胞的划痕愈合能力，显著增加迁移到Transwell下室细胞的数目，其机制可能与显著提高脑微血管内皮细胞相关促血管新生因子VEGF、Ang-1和bFGF的表达与释放相关。.研究阐明了苏合香能通过NF-κB信号通路发挥抗脑缺血再灌注损伤作用，并且脑缺血恢复期苏合香可以促进神经血管重塑。.科学意义：研究丰富了苏合香“开窍”作用的科学内涵，为苏合香治疗脑缺血的临床转化应用及阐明苏合香“开窍”的机制提供实验依据。