基于铁代谢失调探讨脑缺血及缺血再灌注损伤新机制及益气活血法的干预作用

Iron is an essential element in brain. It Participates in synthesizing the myelin sheath protein of oligodendrocyte in brain ,and also can compound neurotransmitter . Neuronal iron homeostasis disruption lead to oxidative stress . The increased production of reactive oxygen species and lipid peroxidation will damage neurons of brain. In the project ,iron content of cerebral cortex and hippocampus will be determined by Flame Atomic Absorption Spectrophotometer and electron microscopy after brain ischemia and ischemia reperfusion . and Methods of immunohistochemistry,PCR and WB will be used to detect the iron transporter proteins TFR, DMT1, Fpn, FLVCR and BCRP at different time points. we will reveal the dysregulation of intracellular iron and discover the new mechanism of brain ischemia and ischemia-reperfusion injury; Then we will detect how the signal pathway of PI3K/Akt and p38MAPK adjust the iron transporter proteins in vitro experiments，in order to provide the new target point and approach for medicine; In vivo and in vitro experiments ，intervened with traditional Chinese herbs Naotaifang extract which Supplementing Qi and activating blood circulation, we study the appropriate concentration of Naotaifang extract which lead to Effective treatmentand， and Whether the Naotaifang extract can pass through the blood brain barrier. Further more，we observe the effect of Naotaifang extract on iron, the iron transport proteins and signal pathway，and reveal the new mechanism of Naotaifang extract which can regulate the iron metabolism after cerebral ischemia and reperfusion .

铁是脑内必不可少的微量元素，参与神经递质及少突胶质细胞髓鞘蛋白的合成，而铁超载催化的产物自由基可导致神经元损伤。本课题采用电镜及原子吸收光谱法，检测铁在大鼠脑皮质及海马分布，免疫组化及PCR、WB法检测铁转运蛋白TFR、DMT1、Fpn、FLVCR、BCRP不同时间点的表达，阐明脑缺血及缺血再灌注后铁代谢失调导致脑皮质及海马损伤的新机制；通过体外细胞培养，检测PI3K/Akt及p38MAPK信号通路对各铁转运蛋白的调节，研究脑细胞铁跨膜转运蛋白的信号调节通路,为药物的防治提供新的靶点；体内和体外实验采用益气活血中药脑泰方提取物干预，探讨药物的血脑屏障通过情况及达到治疗作用的浓度，进而观察脑泰方提取物对铁、各铁转运蛋白及信号通路的影响，阐明益气活血法通过调节细胞内铁代谢达到抗脑缺血及缺血再灌注损伤的作用机理。

摘要： 本课题在脑缺血后谷氨酸神经细胞毒、钙超载，细胞凋亡、氧自由基及线粒体损伤等级联反应是导致神经元坏死的病理生理机制基础上，提出铁代谢失衡可能作为脑缺血及缺血再灌注损伤的新机制。并探讨益气活血中药脑泰方调节脑缺血及缺血再灌注后神经元铁代谢失衡的新机制。 围绕这一核心问题，电镜观察神经元内铁离子颗粒，采用免疫组化及PCR法检测海马CA2区各铁跨膜转运蛋白TFR、Fpn、FLVCR、BCRP不同时间点的表达，结果显示MCAO术后2h、6h、12h、24h、72h，铁跨膜输入蛋白TFR表达逐渐增加，铁跨膜输出蛋白FPn术后12h达到峰值、FLVCR及BCRP表达在术后72h降低，神经元内铁离子聚集 ；中药脑泰方干预后， 电镜观察神经元内铁离子颗粒分布，采用免疫组化及PCR、WB法检测各铁跨膜转运蛋白 。结果显示益气活血中药脑泰方降低TFR、DMT1的表达，减少神经元摄取铁离子。同时增加Fpn、FLVCR的表达，促进细胞内铁外排，抑制神经元内铁聚集，减轻脑缺血及缺血再灌注后因铁代谢失调导致神经元损伤； 离体实验选用18d胎龄SD大鼠海马神经元原代培养，中药NTE干预后，通过MTT法检测比较各组神经元的存活率， PI3K及p38MAPK抑制剂干预后，免疫荧光检测评价FLVCR和 Fpn的表达。结果是脑泰方通过PI3K通路促进Fpn和FLVCR的表达，通过P38MAKP通路促进Fpn的表达，增加脑缺血后细胞内铁外排，达到保护神经元的作用 。本研究提出了铁代谢失调是脑缺血及缺血再灌注损伤的新机制，新靶点，通过益气活血中药干预研究脑缺血及再灌新的治疗方案 。