脑缺血后胶质细胞铁代谢与炎症损伤机制研究及脑泰方干预作用

Previous studies have demonstrated that iron overload is involved in cerebral ischemic injury. On the basis, the project put forward that iron metablism of glial cells is correlate to inflammation intimately ; The iron transmembrane protein is the key target of Cerebral ischemia injury caused by iron overload , and the expression of iron transmembrane protein is regulated by inflammatory reaction ; Naotaifang can intervene imflammation and protect the neurons by increasing the expression of iron transmembrane protein and inhibiting the overload of neuronal iron in cerebral ischemia. The MCAO model rats will be detected by experimental techniques such as Immuno histochemistry, PCR, WB and so on. These methods will be used to detect and alanysis the changes of intracellular iron , iron transmembrane proteins（TFR、DMT1、Fpn、FLVCR、BCRP）, Inflammatory factors （IL-6, IL-1β, TNF-α）and the expression of signal pathways related to “inflammation--iron overload mechanism”such as BMP6/SMAD and IL6/STAT . In the external experiment,we cultivate original glial cells with cultivate basement which is lack of oxygen and glucose but full of iron ion. Then we intervened them respectively and detected the iron transmembrane proteins and signal proteins by laser confocal microscope. The topic is in order to illustrate the correlation between iron transportion and the iron transmembrane protein expression , induced by inflammation and treated with Naotaifang . and my research concentrates on effect of Naotaifang on signal transduction mechanism of the iron transmembrane protein regulation . The research Provide an important experimental basis for clinical application and material basis about Naotaifang treating ischemic brain injury.

课题组前期研究证实铁超载参与了脑缺血神经元损伤，在此基础上，本项目提出脑缺血后神经胶质细胞铁代谢失衡与炎症反应密切相关；铁跨膜转运蛋白是脑缺血铁超载损伤的关键靶点，其表达受炎症反应调控；脑泰方可通过干预炎症反应调节铁跨膜转运蛋白表达抑制脑缺血后细胞内铁超载，发挥神经元保护作用。以MCAO模型大鼠为受试对象，运用免疫组化、PCR、WB等实验技术，检测分析缺血区小胶质细胞、星型胶质细胞的铁跨膜转运蛋白表达调控及神经肽（SP）、炎症因子（IL-1、IL-6、TNF-α）、“炎症--铁超载”信号通路BMP6/SMAD 和IL-6/STAT表达；体外实验，原代培养胶质细胞, 缺氧缺糖高铁处理后分组干预，检测铁跨膜转运蛋白及通路蛋白。阐明脑泰方对炎症诱导铁跨膜转运蛋白的表达干预铁转运的相关性以及调控信号转导机制的影响，为益气活血中药脑泰方防治缺血性脑损伤的临床应用和物质基础研究提供重要的实验依据。

脑缺血后的炎症反应与脑缺血急性期及继发性脑损害密切相关。胶质细胞激活和细胞死亡触发炎症级联反应，损伤脑组织。课题开展脑缺血损伤后神经元及胶质细胞铁代谢状态与炎症反应相关性机制研究，为脑缺血的中医药防治及作用机制研究提供重要实验依据。. 本课题探讨MCAO大鼠脑缺血模型后炎症-铁代谢失衡的相关蛋白的动态变化；体内和体外实验验证脑泰方对MCAO模型大鼠炎症-铁代谢失衡的相关的干预机制。实验结果发现脑缺血24h，大脑皮质和髓质IL-6、HIF-1a、p-STAT3、Hepcidin 的表达明显增加，Fpn、BMP6表达明显减少，Smad4在12h-48h的表达明显增加。益气活血脑泰方可降低缺血后脑组织的IL-6、p-Smad、Hepcidin的表达，增加Fpn 的表达，促进脑组织细胞的铁外排，降低铁聚集和铁介导的神经损伤，减轻炎症损伤，进而发挥脑的神经保护作用。脑泰方通过抑制BMP6/Smad，调节hepcidin，增加Fpn的表达，减少铁聚集。 因BMP6/Smad竞争性抑制STAT3，则脑泰方促进STAT3的磷酸化，抑制炎症反应。由此，脑泰方通过BMP6/Smad/STAT3减少铁聚集，抑制炎症，起到脑缺血后保护作用。本课题对于益气活血中药在脑缺血后干预炎症反应调节铁代谢方面的神经保护作用的相关研究具有重要价值。.