TLR4-NOX4-MMP9通路参与深低温停循环下神经血管单元紧密连接蛋白功能改变的机制研究

Long period of Deep Hypothermic Circulatory Arrest (DHCA) results in post-operative neurological dysfunctions, which has currently become one of the most challenging issues in cardiac surgery. Recent studies reveal that disorder of capillary permeability in Neurovascular Unit (NVU) is one of mechanisms of brain injury during DHCA, in which malfunction of Tight Junctions Protein (TJs) plays an important role. Former studies reported that up-regulated expression of MMP9 resulted in TJs disorders, which mediated by activation of NF-ΚB and AP-1(c-FOS). Whether this mechanism involves in DHCA remains unknown. Our former study demonstrated that activation of TLR4 in DHCA mediated up-regulated expression of NF-ΚB and c-FOS, also down-regulated expression of occludin(TJs) and changes of metabolic parameters in extracellular fluid(ECF) are observed. We hypothesize that TLR4-NOX4 pathway mediates expression of MMP9 by activation of NF-ΚB and AP-1(c-FOS), which impair the function of TJs. Current investigation focus on changes of TJs in NVU during DHCA, expecting to investigate in vivo and in vitro whether activation of TLR4-NOX4-MMP9 Pathway is the underlying mechanism of TJs malfunction, as well as its role in cerebral perfusion technique SACP. Furthermore, the correlation between the metabolic changes in cortex ECF and malfunctioning TJs is also investigated.

深低温停循环技术(DHCA)导致的脑损伤一直是心外科临床研究的难点。最近发现神经血管单元(NVU)微血管通透性功能障碍是脑损伤的主要原因之一，其中紧密连接蛋白(TJs)功能改变扮演重要角色。有研究表明MMP9的表达上调会损伤TJs功能，其激活与NF-ΚB及AP-1(c-FOS)相关，目前该机制是否参与DHCA脑损伤未有报道。我们先前研究证实DHCA时TLR4激活导致NF-ΚB及c-FOS表达上调，并观察到occludin(TJs)表达下调及脑细胞外液代谢改变。由此设想TLR4-NOX4通路通过分别激活NF-ΚB及c-FOS上调MMP9表达，进而损伤TJs功能。本项目拟进一步研究DHCA对NVU中TJs功能的影响，并通过体外及体内实验阐明其机制是否由TLR4-NOX4-MMP9通路介导，进而探讨SACP的脑保护机制是否与抑制该通路激活有关，并揭示脑细胞外液代谢改变与TJs功能改变的关系。

项目的背景：.本课题旨在探讨深低温停循环（DHCA）脑损伤过程中神经血管单元（NVU）通透性改变的可能机制是否与TLR4-NOX4-MMP9通路的激活并降解连接蛋白（TJs）相关，并进一步阐明SACP的脑保护作用是否与抑制此通路激活相关。.主要研究内容、重要结果、关键数据：.课题组在成功建立大鼠DHCA模型与神经胶质细胞的低温氧葡萄糖剥夺（OGD）模型的基础上证实了：（1）DHCA脑损伤过程中存在NVU微血管通透性改变且是脑损伤的重要环节之一,而TJs表达异常在该损伤过程中扮演关键作用。（2）BV2的OGD模型结果提示DHCA脑损伤中TLR4/NOX4/MMP-9通路的激活是其关键环节之一，TLR4中和抗体可使MMP-9表达下调，连接蛋白Occludin上调，提示TLR4/NOX4、/MMP-9通路的激活会导致TJs降解进而导致NVYU微血管通透性改变（3）大鼠DHCA模型证实DHCA脑损伤机制中TJs存在表达异常，应用TLR4受体阻断剂后检测脑皮质的连接蛋白表达变化证实TLR4通路激活参与并导致DHCA后NVU微血管通透性损伤。（7）与常见脑组织热缺血不相同的是，在DHCA脑损伤中TLR4与NOX4并不是通过NF-κB与MAPKs/c-FOS这两条途径共同作用激活下游MMP-9的，而是仅仅通过NF-κB激活下游MMP-9发挥作用，期间MAPKs/c-FOS通路分子表达并未出现改变，提示TLR4-NOX4主要是通过NF-Κb发挥作用的，显示DHCA脑损伤及NVU通透性改变有着与脑热缺血不同的机制存在，值得进一步深入研究。.科学意义：. 综合目前的研究结果，我们证实DHCA脑损伤存在NVU通透性改变， 这种改变与TJs的表达异常相关，TJs表达异常的机制之一是TLR4-NOX4-MMP9通路的激活，结果同时也提示了与常见脑组织缺血性损伤机制不相同的是，在DHCA脑损伤中TLR4与NOX4不是通过NF-κB与MAPKs/c-FOS这两条途径共同作用激活下游MMP-9的，而主要是通过NF-Κb发挥作用的，该发现提示DHCA导致的脑损伤及NVU通透性改变有着与脑组织缺血性损伤不同的机制存在，值得进一步深入研究。