S100A8/A9参与心肺复苏后脑缺血再灌注损伤相关信号通路机制研究

It has been shown recently that S100A8/A9 can serve as an endogenous activator of Toll-like receptor 4 (TLR4), and be highly expressed in inflamed tissues compared with other endogenous ligands of TLR4. Our latest study proved that S100A8/A9 played a significant role in the early phase of cerebral ischemia reperfusion (I/R) injury after cardiac arrest and cardiopulmonary resuscitation (CA/CPR), but its intracelluar signaling pathway remained to be investigated.It is unclear that whether S100A8/A9 takes part in cerebral I/R injury after CA/CPR via TLR4 or via both TLR4 and receptor for advanced glycation end products（RAGE) ? And when TLR4 is activated, which molecules are involved ? So the purpose of our research is to explore the mechanism of the signaling pathway of S100A8/A9 mediating cerebral I/R injury after CA/CPR. And the result of the study will establish the new theoretical basis for the treatment of cerebral injury after CA/CPR. The in vitro study will be conducted to investigate the different expressions of proinflammatory cytokines in microglial cell culture fluid with or without TLR4 antibody intervention following the S100A8/A9 stimulation. And the protein expressions both upstream and downstream in TLR4 signaling pathway also will be detected. Moreover, the molecules which are involved in TLR4 signaling pathway in this situation will be found by using different signal blocking agent. The in vivo study will be performed to check whether S100A8/A9 is binded with TLR4 or RAGE on the surface of microglia in the mouse model with cerebral I/R injury after CA/CPR through the Co-Immunoprecipitation and indirect immunofluorescence double labeling techniques.

S100A8/A9作为TLR4一种新发现的内源性配体，具有在炎症组织早期高表达的重要特性。我们最新研究发现S100A8/A9参与心脏骤停心肺复苏(CA/CPR)后早期脑缺血再灌注（I/R）损伤，但它究竟是通过全部依赖TLR4，或是部分依赖TLR4（与RAGE有关）参与其中，又将有哪些下游分子介入尚不清楚？本课题拟在前期工作基础上探讨S100A8/A9在CA/CPR后脑I/R损伤中的相关信号通路机制，为CPR后脑损伤的防治提供新的重要理论基础。本研究体外实验利用TLR4抗体等作为干预工具，观察在S100A8/A9刺激下小胶质细胞培养液中促炎因子变化以及TLR4信号通路中上下游蛋白的表达；同时利用不同信号阻断剂来检测有哪些下游分子介入其中；体内实验复制CA/CPR后脑I/R损伤小鼠模型，通过免疫共沉淀技术和间接免疫荧光双标法检测S100A8/A9是否与小胶质细胞表面的TLR4或RAGE结合。

S100A8/A9作为Toll样受体4（TLR4）一种新发现的内源性配体，具有在炎症组织早期高表达的重要特性。本课题拟在前期工作基础上探讨S100A8/A9在心肺复苏（CPR）后脑缺血再灌注（I/R）损伤中的相关信号通路机制，为CPR后脑损伤的防治提供新的重要理论基础。首先课题组成功合成符合实验要求的S100A8/A9蛋白。我们用不同浓度S100A8/A9蛋白刺激BV-2细胞，证实BV-2细胞被活化。第二，我们用TLR4抗体和（或）RAGE抗体预先封闭BV-2细胞后再给予S100A8/A9蛋白刺激，进一步证实S100A8/A9蛋白激活BV-2细胞与TLR4和RAGE双通路密切相关。我们还利用小干扰RNA技术，设计合成TLR4siRNA 和RAGEsiRNA，分别转染BV-2细胞基因沉默TLR4或RAGE表达，然后用S100A8/A9蛋白刺激，结果发现上清液中炎性因子表达均下调，进一步证实S100A8/A9蛋白刺激小胶质细胞与TLR4和RAGE有关。第三，课题组着重研究了S100A8/A9通过TLR激活BV-2细胞所涉及的相关炎症信号通路。通过用不同浓度ERK、P38、、JNK以及NF-κB阻断剂孵育BV-2细胞后给予S100A8/A9蛋白刺激，采用PCR和ELISA检测炎性产物含量变化、EMSA和细胞免疫荧光等方法检测NF-κB活性变化，初步证实S100A8/A9刺激小胶质细胞激活途径与ERK/ NF-κB 和JNK/ NF-κB通路有关。最后在体研究方面，我们采用静脉推注冷氯化钾溶液的方法成功构建心跳骤停8分钟后开始心肺复苏的小鼠模型，并且利用该模型探讨CPR早期给予米诺环素后的脑保护作用及其机制，结果发现TLR4和RAGE在心肺复苏后小鼠脑组织中表达均明显增加，且和脑组织S100A8/A9表达变化趋势一致，提示S100A8/A9与TLR4和/或RAGE关系密切，共同参与了CA/CPR后脑炎症损伤过程。而米诺环素可以有效抑制心肺复苏后脑组织内小胶质细胞的激活，减少神经元的死亡。