Hedgehog信号通路在tPA加剧脑梗塞血脑屏障损害中的作用及机制研究

The risk of hemorrhagic transformation in ischemic stroke after thrombolytic therapy limits the wide use of tPA. The disruption of blood-brain barrier (BBB) is a critical event in the pathogenesis of hemorrhagic transformation after tPA thrombolysis in acute ischemic stroke, however, the molecular mechanisms involved are not well understood. Recent report showed the Hedgehog pathway contributes to the maintenance of BBB functions, even including its immune quiescence. After brain injury and ischemia, the components of Hedgehog signaling pathway are activated whereas inhibition of Hedgehog pathway exacerbated rat ischemic damage caused by MCAO, which showed that stimulation of Hedgehog signaling could play a beneficial role in ischemia. However, there is no report about the effect of Hedgehog signal pathway on damage of neurovascular unit and BBB in ischemic stroke after tPA therapy. In our previous study, we found Shh secreted by oxygen-glucose deprivation (OGD)-activated astrocytes promotes proliferation of brain microvascular endothelial cells(BMVECs) when co-cultured with astrocytes. The inhibition of BMVECs proliferation by OGD was enhanced by tPA. Further experiments should to examine the effect of tPA on structure and function of BBB under OGD environment and the change of signal pathway molecular. Moreover, whether promotion of this pathway can attenuate breakdown of BBB and hemorrhagic transformation should be elucidated. In this study, brain microvascular endothelial cells and astrocytes will be co-cultured to established BBB model in vitro and will be treated with OGD, the Hedgehog signal involves in disruption of BBB will be examined after treated with tPA. Then, MCAO animal models will be established in wide-type and Shh knockout mice and high dosage tPA will be used to establish hemorrhagic transformation models, the brain infarction volume, water content, permeability of BBB and Hedgehog signal molecular involve in disruption of BBB will be tested. The purpose of this study is to investigate the effect of Hedgehog pathway on disruption of BBB in ischemic stroke after treated with tPA, which will hopefully provide us a new target to prevent and treat hemorrhagic transformation in ischemic stroke after thrombolysis with tPA.

tPA加剧脑梗塞血脑屏障(BBB)的损害引起出血性转化(HT)的风险限制了溶栓治疗的广泛应用。最近的研究显示，Hedgehog信号通路与BBB的完整性有关。我们前期共培养大鼠脑微血管内皮细胞(BMVEC)和星形胶质细胞，发现星形胶质细胞能分泌Shh促进BMVEC的增殖。那么，tPA加剧BBB损害是否有该信号通路参与？我们的预实验发现tPA能增强氧糖剥夺对BMVEC增殖的抑制。tPA对处于应激状态的"神经血管单元"主要成员有何影响？Hedgehog信号通路在其中有何变化？干预该信号通路能否减轻tPA加剧脑梗塞BBB的损害和预防HT？为弄清这些问题，本研究拟通过建立体内外脑梗塞tPA溶栓模型，检测tPA对处于缺血缺氧状态BBB结构和功能的影响及Hedgehog信号通路的变化，旨在探讨Hedgehog信号通路在tPA加剧脑梗塞BBB损害中的作用及机制，为脑梗塞tPA溶栓后HT的防治提供新的思路。

溶栓是治疗急性缺血性卒中最有效的方法之一。目前FDA唯一批准使用的溶栓药物组织型纤溶酶原激活剂(tPA)有发生出血转化(HT)的风险，大大限制了其临床的广泛应用。研究发现，血脑屏障（blood-brain barrier,BBB）的破坏和通透性增高是造成HT的主要原因，然而tPA引起BBB破坏和HT的具体分子机制仍不清楚。有研究报道，Hedgehog相关信号通路参与紧密连接蛋白调节，影响BBB通透性的维持，但该通路是否参与脑梗塞溶栓后tPA引起BBB损害加剧过程，尚未见相关的文献报道。为了弄清这一问题，本研究检测了该信号通路在 tPA 加剧脑梗塞 BBB 损害中的作用及机制。同时探讨了GSK-3β抑制剂TWS119对HT模型大鼠的神经保护作用及GSK-3β在短暂性脑缺血中所起的作用及机制。我们还探讨了白藜芦醇和细胞色素C抑制剂醋甲唑胺对小鼠BMVECs氧化应激损伤的保护作用，以及重组人脑源性神经生长因子（rhBDNF）对血液溶解产物诱导小鼠皮层神经元凋亡的保护作用及机制，并探讨了细胞粘附分子Vinexin和炎性因子Tollip在氧糖剥夺神经元中的表达情况及在脑缺血/再灌注损伤中的作用。研究发现，Hedgehog 相关信号通路参与脑梗塞 tPA 溶栓后HT。GSK-3β抑制剂TWS119能显著缓解大鼠神经功能损伤，减少HT的发生，并可能是通过Wnt/β-catenin信号通路发挥作用。rhBDNF能抑制血液溶解产物对神经元的损伤，减少神经元凋亡率。白藜芦醇对BMVECs氧化应激损伤具有保护作用，其机制可能通过介导PI3K/Akt信号通路和稳定细胞骨架而实现。醋甲唑胺可改善血液溶解产物对神经元的损伤，并可能是通过抑制caspase-3的激活、抑制海马神经元的凋亡来起作用的。Vinexin和Tollip在脑缺血/再灌注损伤中起促进作用。本研究结果理论上阐明了Hedgehog相关信号通路参与调节tPA加剧脑梗塞BBB损害过程。BDNF、醋甲唑胺、白藜芦醇等能抑制脑微血管内皮细胞及神经元的凋亡，阻止BBB的破坏而抑制神经功能损害。针对Hedgehog相关信号通路的治疗可能在脑缺血缺氧和BBB损害中起保护作用。研究成果为HT和脑缺血及其他神经系统疾病的防治提供了理论依据。