缺血再灌注后tPA调控自噬相关信号通路的神经保护机制与纳米靶向投递治疗

Cerebral ischemia /reperfusion (IR) injury is a severe condition which threaten human health. Tissue-type plasminogen activator (tPA) is a serine proteinase found in the intravascular space and the CNS. Whereas intravascular space tPA has been approved as a thrombolytic agent for acute ischemic stroke patients by FDA, its other biologic functions in the CNS have been under investigation. However, a growing body of evidence indicates that, besides the unquestionable benefit from its thrombolytic activity, tPA also has a neuroprotective effect on the ischemic brain. In our previous study, we found that tPA was rapidly released from neurons, and hippocampal areas of the mouse brain lacking tPA activity were more vulnerable to neuronal death following an ischemic insult. We also showed tPA can act as an endogenous neuroprotectant via the induction of the mammalian target of rapamycin (mTOR) activation during IR injury in the murine hippocampus, which can subsequently induce the HIF1α pathway activation. It has been shown that mTOR pathway activation plays an important role in autophagy functions in recent studies. In our preliminary experiments, we found that the induction of autophagy functions were associated with the neuroprotective effects of tPA. However the lower level of endogenous tPA released from neuron is insufficient to induce autophagosome clearance and the higher level of exogenous rtPA disrupt the blood - brain barrier, leading to hemorrhagic transformation resulting in the limitation of clinical use. The hypothesis we propose here is that endogenous and exogenous tPA plays an important role as a signaling molecule in protecting neurons against IR injury through the induction of the autophagy associated signal pathway and the clinical use of the targeted delivery of therapeutic rtPA nanoparticles can reduce it’s side effects. This study will further evaluate the association between the level of endogenous tPA and the induction of neuronal autophagosome clearance, and whether exogenous rtPA can enhance the neuroprotection of endogenous tPA through mTOR- HIF1α signal pathway during IR injuries. We will assess the effects of the targeted delivery of therapeutic rtPA nanoparticles induced the activation of neuronal autophagy on neuronal fate, neurite outgrowth, synapse plasticity and integrity of BBB.We will focus on the biologic effect of neuronal tPA which was independent of it’s thrombolytic activity during IR injuries. Our study will have important clinical implications not only for the prevention of neuronal death, but also for the development of an effective neuroprotective strategy for clinical use.

申请人前期发现缺血再灌注（IR）损害时释放内源性tPA激活mTOR-HIF1α信号保护神经元，机制与自噬功能增强、毒性物质清除增加有关。内源性tPA释放相对不足或常规给予外源性rtPA在脑内一过性浓度过高引起血脑屏障破坏可能是临床治疗失败的主要原因。本课题集中在“IR后tPA调控自噬相关信号通路的神经保护机制与纳米靶向投递治疗”，拟通过分析IR后神经元释放内源性tPA水平与自噬参与下的神经元自净功能之间的关系；外源性rtPA是否补充脑组织内源性tPA的相对不足、通过mTOR-HIF1α信号通路增加其神经保护作用，并利用纳米靶向投递技术建立rtPA的精确稳定与可控性的治疗投递系统用以解决临床中rtPA治疗后因为血脑屏障破坏导致的出血转化这个关键问题，以便进一步实现临床前的转化研究。本课题对神经元源性tPA的独立于血栓溶解的神经保护作用深入研究，为缺血性脑卒中的治疗提供一种新策略和治疗靶点。

组织型纤溶酶原激活剂（tissue-type plasminogen activator，tPA）是临床重要的溶栓药物。我们前期研究发现，神经元释放的内源性tPA调控Akt和mTOR在内的多种细胞信号通路发挥神经保护作用，而Akt/mTOR是否与细胞自噬相关，调控神经元凋亡值得我们进行深入研究。IR后常规给予外源性rtPA在保护神经元的同时也破坏了血脑屏障的完整性，使脑梗死后出血转化风险从0.6%上升到6.4%。IR后tPA的快速释放和快速代谢的特征，不能稳定和长程维持tPA保护作用，因此构建新型tPA靶向投递平台，持续发挥tPA的神经保护作用,降低IR后出血转化风险，这些方面的研究对临床tPA溶栓治疗具有重要意义。. 本课题组从以上三方面出发，进行了IR后神经元释放的内源性tPA对神经元自噬通路的调控研究，我们在体内和体外水平建立tPA基因敲除IR模型，首次发现tPA通过提高p-AMPK水平调控FUNDC1蛋白表达，诱导神经元线粒体的自噬活性，减轻IR后神经元凋亡。另一方面，本课题组研究了常规途径给予外源性rtPA对脑组织的作用和血脑屏障的影响。我们在实验室条件下模拟临床缺血性脑卒中的溶栓治疗，发现rtPA通过周细胞PDGFR-β/TGF-β/p-Smad2/3信号通路，介导炎症反应，加重血脑屏障损伤。本课题组同时还进行了纳米靶向投递外源性tPA的研究，利用单一蛋白纳米微粒包裹tPA,经尾静脉注射后检测IR不同时间点tPA脑组织内浓度以及体内代谢方式和速度，建立tPA脑内靶向投递平台。. 本项目研究结果具有重要的科学意义。通过IR后tPA对神经元自噬通路的调控研究，发现tPA新的神经保护作用机制，目前国内外未见相关报道，为临床IR损伤提供新的治疗靶点。研究rtPA对血脑屏障的作用，避免rtPA在临床应用中破坏血脑屏障通透性的副作用，具有重要临床转化意义。建立纳米微粒靶向投递tPA平台，有利于最大限度提高药物生物利用度，提高临床疗效。

内容获取失败，请点击重试