缺血后处理调控TLR4-MyD88-NF-κB信号通路减弱小胶质细胞炎症损伤的机制研究

Inflammatory response have been suggested in the pathogenesis of cerebral ischemia/reperfusion, toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway was found to play an important role during the process. Studies demonstrated that microglia as inherent immune response cells, which are activated immediately after acute ischemia. TLR4 is the main receptor in the central nervous system which activates the M1 microglia secreting inflammatory factors, mediating inflammatory reaction and apoptosis of nerve cells. Our previous study found that ischemic postconditioning can down-regulate the expression of TLR4, and promote endogenous neurogenesis after cerebral ischemia. We therefore put forward the central hypothesis of this project: ischemic postconditioning decreases the number of M1 microglia and down-regulates the expression of TLR4 after cerebral ischami/reperfusion injury, and consequently reduces the expression of proinflammatory factors and inhibits cell apoptosis by MyD88-NF-κB signaling pathway, and then induce endogenous neuroprotection. We will test this hypothesis with cerebral ischami/reperfusion model, using Immunohistochemical, Immunofluorescense, Flow cytometry, Western blot, and Real time PCR, to lucubrate the mechanism of ischemic postconditiong inhibiting the expression of TLR4 of M1 microglia and consequently mediating inflammatory response by TLR4-MyD88-NF-κB after cerebral ischemia. This study will provide a novel theory basis for the prevention and treatment for cerebral ischemia.

TLR4/NF-κB信号在脑缺血/再灌注损伤后炎症反应中发挥重要作用。小胶质细胞作为中枢神经系统固有的免疫应答细胞，在急性脑缺血后立即活化。TLR4是激活M1型小胶质细胞分泌炎性因子、介导炎症反应和神经细胞凋亡的主要受体。课题组研究发现：（1）缺血后处理可下调脑缺血后TLR4的表达；（2）缺血后处理可促进内源性神经再生，介导内源性保护机制。综上，我们提出本课题假说：缺血后处理可减少脑缺血/再灌注损伤后M1型小胶质细胞的数量并下调TLR4的表达，通过调控MyD88-NF-κB信号通路，减少脑缺血再灌注后促炎因子的表达和细胞凋亡，激活内源性保护机制。本课题拟采用免疫组化、免疫荧光、流式细胞技术、免疫印迹、实时定量PCR等方法，深入研究缺血后处理减弱M1型小胶质细胞TLR4的活性并调控TLR4-MyD88-NF-κB介导的炎症反应机制，为脑缺血损伤防治提供新的理论依据。

越来越多的研究表明，远隔肢体缺血后处理(remote limb ischemic postconditioning, RIPC)可通过减轻脑缺血/再灌注(cerebral ischemia/reperfusion, I/R)后炎症反应来发挥神经保护作用，且小胶质细胞的激活与极化与炎症反应密切相关。本项目采用Sprague-Dawley大鼠I/R模型，于再灌注后立即给予RIPC干预（双侧股动脉，I-10min/R-10min×3）或空白处理。通过神经行为学评估、病理学、免疫组化、ELISA、Western blot、PCR等技术评估RIPC干预的影响。结果显示，RIPC可显著改善脑缺血模型大鼠神经行为学功能、学习与记忆能力，减少脑梗死体积，减轻脑皮质和纹状体区组织损伤和神经细胞丢失，提高神经元存活数量，降低炎症细胞因子IL-β、TNF-α的表达和细胞凋亡数量，促进IL-10和Bcl-2的表达。此外，RIPC抑制了小胶质细胞的激活，促进了小胶质细胞向M2型极化，并下调了TLR4蛋白及TLR4、MyD88、IkB-α、NF-kB mRNA的表达。以上研究结果表明，RIPC可能通过抑制TLR4-MyD88-NF-kB信号通路调节小胶质细胞的激活，促进M2表型的极化，从而对脑缺血发挥神经保护作用。本项目再次证实缺血后处理对脑缺血的神经保护作用是通过调控。