基于抑制铁跨膜转运防治脑缺血再灌注损伤机制探讨黄芪“固护玄府”的微观基础

The microscopic molecular events of cerebral ischemia reperfusion (CIR) injury may be caused by the excessive opening of “Xuan Fu”. The CIR injury is mainly due to the overproduction of free radical catalyzed by iron, and the transmembrane transport of iron plays a key role during the process. The pathway of non-transferrin/transferrin receptor (Tf/TfR) dependence absorbs iron faster, which is the main cause of free radical formation. Astragalus membranaceus has a function of scavenging free radicals, and our previous study confirmed that it played a neuroprotective role. This study is designed to observe the neuroprotective effect of astragalus membranaceus and its effective monomer components on CIR damage in CIR model rats, cultured nerve cells and endoplasmic reticulums. We further observe the expression of iron transmembrane transporters in non- Tf/TfR dependent pathway and adopt gene technology to change their expression level to clarify the targets of astragalus membranaceuson on the transmembrane transport of iron. This study will provide experimental basis for the clinical application of astragalus membranaceuson as a neuroprotective agent and afford theoretical support for the TCM treatment of "the prevention of astragalus membranaceuson on cerebral stroke".

我们认为脑缺血再灌注（CIR）损伤的微观分子事件可能是玄府开通太过所致。CIR损伤以铁催化的自由基损伤最为主要，铁跨膜转运起着关键作用。转铁蛋白/转铁蛋白受体（Tf/TfR）非依赖性途径摄入铁的速度较快，是自由基形成的主要原因。黄芪具有清除自由基的功能，我们前期实验证实黄芪甲苷具有显著的神经保护作用。本项目拟在CIR模型大鼠、体外培养神经细胞和提取内质网上，观察铁超载状态下黄芪及其有效单体成分对CIR损伤的神经保护作用；观察Tf/TfR非依赖性铁跨膜转运蛋白表达，并采用基因技术改变其表达水平观察给药前后变化，明确黄芪在铁跨膜转运中的作用靶点，论证黄芪通过调控Tf/TfR非依赖性铁跨膜转运对抗铁进入脑内发挥神经保护作用的假说。为黄芪作为神经保护剂的临床应用提供实验依据，为“黄芪防治脑中风”的中药疗法国际化提供理论支持。

本项目首先通过整体动物实验明确黄芪对脑缺血再灌注损伤的神经保护作用，发现黄芪对脑内Tf / TfR 非依赖性铁转运蛋白的具有调控作用，黄芪通过下调铁转运蛋白Fn、FHC、FLC、DMT1、LTCC、TRPC6，上调FPN1，调节铁转运蛋白的表达，发挥对脑缺血再灌注损伤的防治作用；进一步通过体外细胞实验阐明黄芪对神经细胞 Tf / TfR 非依赖性铁跨膜转运的作用，发现黄芪单体能够明显下调神经细胞DMT1、LTCC的表达，发挥神经细胞保护作用；通过获取黄芪的靶点活性成分，经整体实验和细胞实验研究发现，黄芪单体能够抑制RyR激活，显著降低实验动物脑组织内质网氧化应激相关蛋白水平和线粒体钙超载相关蛋白p-GRK2、p-PLCβ、p-PLCA、p-CaMKII、p-IP3R水平，抑制钙超载性氧化应激，发挥神经保护作用。黄芪作为神经保护剂的临床应用提供了实验依据，丰富了中医“玄府”理论内涵。