TLR4信号通路在脑损伤后交感兴奋中的细胞和分子机制研究

The PSH following TBI increased the mortality and morbidity greatly. PVN is the key area that stimulate central sympathetic excitation, while the regulation of PSH in TBI is still unclear. Therefore it is difficult to make a breakthrough in the clinical treatment of this disease.Our research predicts that the mechanism of PSH in TBI is the up-regulation of TLR4 in the surface of microglia in PVN after TBI.The combination of pattern recognition molecules and TLR4 activates microglia to produce mess of inflammantory factors and chemotactic factors,which gather more T cells that improve the excitation of sympathetic nerves.In order to identify this mechanism,our project will use rat DAI models to research the rules of PSH after TBI and the relationship with mutiple regulatory patheways of microglia, which will adopt experimental methods such as culture in vitro, animal tests, and techniques in cell biolgy and molecular biology.1)to discuss the relationship between PSH and activation of microglia. 2)to identify the impact and mechanism of TRL4 of microglia on PSH.3)to observe the change in PSH after interfering the secretion of imflammatory factors.4) to discuss the relationship between the collection of T cells and excition of sympathetic nerves.The result of our project will provide experimental proves for the effect of TLR4 signaling pathway in PSH following TBI and explore the pathophysiological mechanism of this disease, which will provide new targets and methods for the clinical treatment research of PSH.

颅脑损伤（TBI）后交感兴奋使TBI的致死率及致残率显著上升。下丘脑室旁核（PVN）是多种病理情况下启动中枢交感兴奋的关键区域，但其在TBI后交感兴奋中的调节机制仍不清楚，因此该疾病的临床治疗难以取得突破性进展。我们的研究推测TBI后交感兴奋核心机制是TBI后PVN内小胶质细胞表面Toll样受体4(TLR4)上调，TLR4与模式识别分子结合激活小胶质细胞，产生炎性因子及趋化因子，引起T细胞聚集加重交感神经兴奋。因此，本课题拟利用大鼠弥漫性轴索损伤模型明确TBI后交感兴奋与TLR4调控途径的关系：1)探讨交感兴奋与小胶质细胞活化关系2)明确小胶质细胞TLR-4表达对交感兴奋的影响及作用机制3)通过干预TBI后小胶质细胞的炎性因子分泌观察交感兴奋变化4)对T细胞募集与交感兴奋的关系进行初步探讨。本研究结果阐明TLR4信号通路在TBI后交感兴奋中的作用，为TBI后交感兴奋的临床治疗提供新靶点。

创伤性颅脑损伤(Traumatic Brain Injury, TBI)一直是一个困扰世界的医疗问题，TBI轻重程度往往决定患者的预后情况。重型TBI患者可出现交感神经兴奋，主要表现为血压、心率和血浆儿荼酚胺水平升高，并引发严重的颅外器官功能障碍，给患者带来不良的临床结局和高昂的医疗支出。目前TBI后发生交感神经兴奋的病理生理学机制尚不清楚，为临床治疗带来极大困难。.既往研究表明，下丘脑室旁核（The Hypothalamic Paraventricular Nucleus, PVN）是中枢神经系统内调节交感神经活动的关键部位。PVN内的促炎细胞因子水平升高与交感神经兴奋的发生有重要关系。Toll样受体4（Toll Like Receptor 4, TLR4）是细胞表面的模式识别受体，能够通过与环境中的配体结合启动细胞内信号通路激活核因子-κB（Nuclear Factor Kappa-light-chain-enhancer Of Activated B cells, NF-κB）。激活的NF-κB复合物进入细胞核可以调控TNF-α、IL1-β等多种促炎细胞因子的基因转录，刺激PVN神经元引起交感神经系统兴奋。研究表明，TBI会导致小胶质细胞活化，而中枢神经系统内TLR4主要表达在小胶质细胞上。.因此提出以下假设：（1）在颅脑损伤后，PVN内活化的小胶质细胞TLR4表达升高；（2）TLR4能够激活下游的NF-κB通路，产生大量TNF-α和IL-1β等炎症因子，改变了PVN内分子环境，导致交感神经兴奋的发生。.为验证以上假设，进行了以下实验，（1）利用大鼠液压打击模型，明确血压、心率、心率变异性和血浆去甲肾上腺素的水平，并探索交感神经兴奋随时间的变化规律；（2）选取最佳时间点观察下丘脑PVN内小胶质细胞的活化以及TLR4在小胶质细胞上的共表达情况；并通过PCR、Western blot和免疫组化方法检测PVN内TLR4、NF-κB、TNFα和IL-1β的水平；（3）分别对TLR4进行抑制和激动，明确TLR4与TBI大鼠交感兴奋的关系。探索TBI后TLR4对交感神经兴奋性的调控作用，为TBI后交感兴奋的发病机制提供理论依据和新的治疗靶点。