基于“抗氧化联合双通路抑制”策略构建的主动靶向性双药纳米纤维对脑缺血再灌注损伤的保护作用及机制

It is reported that both ROS induced oxidative stress and TGFβ1 related gliosis play important roles in cerebral ischemia reperfusion injury (IRI). TGFβ1 activates astrocytes not only through SMAD2 depended pathway but also through non SMAD2 depended（PI3K/Akt）pathway. Antioxidant can scavenge ROS and up-regulate PI3K/Akt activity, which can lead to TGFβ1 escaping through upregulated PI3K/Akt activity. Thus, antioxidant combined double inhibition of SMAD2 and PI3K/Akt pathway can reduce oxidative stress and inhibit TGFβ1 escaping. According to above strategy, resveratrol (an antioxidant) and curcumin (a double inhibitor of SMAD2 and PI3K/Akt) were chosen to perform combined therapy for cerebral IRI. Moreover, co-delivery of synergistic drugs by nanodrug delivery system will combine the advantages of nano-drug systems with the synergism. To overcome the shortcomings of polymeric carriers, a novel nanoscale drug delivery system was developed basing on the concept of “drug-self assembled peptide-functional peptide”. Curcumin was linked with RGD and FFE to construct a novel drug carrier and Res was loaded in the carriers. RGD helped the targeting of nano-system to the area of IRI. The synergistic anti-IRI effect of this novel co-drug loaded nanofiber was evaluated in vitro and in vivo to elucidate the potential mechanisms through anti-oxidative stress combined double inhibition of SMAD2 and PI3K/Akt pathway.

活性氧簇（ROS）介导的氧化应激和TGFβ1介导的胶质细胞激活均是脑缺血再灌注损伤的重要致病机制。TGFβ1通过SMAD2依赖信号途径和PI3K/Akt途径激活星形胶质细胞。清除ROS减轻氧化应激同时上调PI3K/Akt通路，可导致TGFβ1通过上调的PI3K/Akt通路逃逸，而激活胶质细胞。因此，抗氧化减轻自由基损伤同时双重抑制SMAD2通路和PI3K/Akt通路，可有效抑制TGFβ1逃逸。基于该 “抗氧化联合双通路抑制”的协同机制，我们用抗氧化剂白藜芦醇(Res)及对SMAD2通路和PI3K/Akt通路均有抑制作用的姜黄素(Cur)组成脑缺血再灌注联合治疗方案。并基于“药-自组装短肽-功能肽”构建高效低毒的主动靶向性纳米纤维Cur/Res双药递药体系，利用RGD引导双药纳米纤维主动靶向脑缺血再灌注损伤部位。最后在体内外模型上考察该靶向性纳米纤维双药递药体系协同脑保护的作用和机制。

脑缺血再灌注时，除了升高的活性氧簇（ROS）介导氧化应激外，脑内增高的TGFβ1亦通过SMAD2依赖信号途径和PI3K/Akt途径激活胶质细胞增生而加重损伤。白藜芦醇(Res)是一种天然抗氧化剂，可减轻缺血再灌注时的氧化应激。然而，清除ROS同时PI3K/Akt通路会上调，从而导致TGFβ1通过上调的PI3K/Akt通路逃逸而激活胶质细胞，减弱抗氧化治疗的效果。姜黄素(Cur)是一种天然提取物，可通过抑制SMAD2通路和PI3K/Akt通路发挥抗炎、抗增殖作用，文献证明其具有神经保护作用。Cur与Res具有协同保护缺血再灌注损伤的作用。然而，两者的低水溶性使其应用潜力受到极大限制。本课题中，我们采用纳米载药技术构建双药共载体系，以Cur本身为零载体构建纳米载药系统来解决此问题，并修饰以靶向的精氨酸-甘氨酸-天冬氨酸短肽结构（RGD），形成以Cur为骨架结构的自组装纳米纤维（Cur-FEE-RGD）。新的纳米纤维与传统高分子纳米载药系统相比，具有更高效的载药率和高水溶性。随后，我们将Res与Cur同时载入自组装体系中，形成以Cur为骨架的双药纳米纤维（C/R-NFs）。以神经细胞株为体外模型，验证了新型自组装双药纳米纤维的细胞保护效应和低毒性。缺血再灌注大鼠体内实验证明C/R-NFs治疗可显著减轻缺血再灌注损伤。体内机制分析也证实了C/R-NFs可减轻ROS介导的氧化应激同时双重抑制SMAD2通路和PI3K/Akt通路，减轻凋亡以及胶质细胞增生，表明其有效抗氧化同时抑制TGFβ1逃逸。本研究为抗氧化剂Res和中药提取物Cur在缺血再灌注治疗中的潜在应用提供了理论及实验基础。更重要的是“以药物本身为载体”的纳米共载体系在缺血再灌注治疗领域的应用提供了新的思路。本项目共发表相关SCI论文6篇，参加国际会议做口头报告1次，协助培养博士研究生1名，硕士研究生1名。