基于细胞铁转运网络调控机制探讨缺血性中风气虚血瘀证的生物学基础

Previous studies have shown that intracellular iron overload participate in cerebral ischemia injury, Yiqi Huoxue principle had a significant effect on the inhibition of iron overload and regulation of the expression of Iron transmembrane regulator (ITTs). On the basis of the construction of the regulation model of intracellular iron transport network, the hypothesis is that the imbalance of intracellular iron transport is an important biological basis of ischemic stroke with Qi deficiency and blood stasis syndrome (IS-XY). As the research object, IS-XY rat model made by Continuous exhaustion swimming composite thread bolt method will be observed, ITTs (TFR,HCP1, DMT1, Fpn1, FLVCR, BCRP) expression at the subcortical ischemic area and the nerve cells in the hippocampus will be tested by immunohistochemistry, PCR and WB technology and the intervention of Yiqi Huoxue principle will also be studied; nerve cells model from rats made by sugar-oxygen deprivation will be observed on cell viability, iron deposition, the expression of ITTs ,Smad1/3/5 after applying different inhibitors, using laser confocal microscopy technique and the intervention effects of Yiqi Huoxue principle will also be studied. The modern biological basis of IS-XY will be clarified by the regulation of iron transport network point and positive and negative aspects .The research results provide an important basis for further understanding of the essence of IS-XY, and it is of great significance to promote the clinical application of Yiqi Huoxue principle in the prevention and treatment of ischemic stroke.

前期研究表明，细胞内铁超载参与了脑缺血损伤，益气活血法方药对抑制铁超载和调节铁跨膜转运蛋白（ITTs）表达具有显著作用。本项目在构建细胞内铁转运网络调控模式的基础上提出假说：细胞内铁转运失衡是缺血性中风气虚血瘀证（IS-XY）的重要生物学基础。以持续力竭性游泳复合线栓法制作的IS-XY大鼠模型为研究对象，运用免疫组化、PCR、WB等技术观察皮质下缺血区域及海马区神经细胞ITTs（TFR、HCP1、DMT1、Fpn1、FLVCR、BCRP）表达以及益气活血法的干预作用；以OGDI细胞模型为受试对象，运用激光共聚焦等技术，观察细胞活力、铁沉积、ITTs、Smad1/3/5表达及益气活血法的干预作用，从铁转运网络调控的角度、采用同病异证、以方测证方法阐明IS-XY的现代生物学基础。研究成果为深入理解IS-XY的实质提供重要依据，对促进益气活血法防治缺血性中风的临床应用和物质基础研究具有重要意义。

缺血性中风（ischemic stroke，IS）在中医属“中风”范畴，多责之“虚、风、火、痰、瘀”五端，而尤以“内虚邪中”为最。 IS 所有中医临床证型中气虚血瘀证占据首位，运用益气活血法为主组方临床疗效显著。深入研究缺血性中风气虚血瘀证的生物学基础，阐明其具体机制对于提高临床疗效、拓展研究领域具有重要意义。.本课题探讨缺血性中风气虚血瘀证（IS-XY）模型大鼠不同脑区铁转运蛋白表达的动态变化；补阳还五汤（BHT）对IS-XY模型大鼠脑损伤保护作用和不同脑区转铁蛋白表达的作用；BHT对IS-XY模型大鼠脑膜铁转运蛋白调控通路变化的干预作用；离体实验揭示BHT对IS-XY模型大鼠脑膜铁转运蛋白调控通路变化的作用。.结果显示，造模后48h组血瘀型中风DMT1、Fpn表达较单纯缺血性中风明显增高，FLVCR的表达明显降低；其中气虚血瘀组DMT1、Fpn表达较气滞血瘀组明显增高，FLVCR的表达明显降低；脑缺血后补阳还五汤干预，可通过TF-Hepcidin通路抑制转铁蛋白（TF）及转铁蛋白受体（TFR）的表达，减少Smad1/5/8蛋白的磷酸化，降低铁调素Hepcidin的产生， 减少Fpn内化降解现象，促进铁外排，减轻铁聚集导致的神经元损伤。 .课题从铁稳态失衡的角度首次阐明缺血中风气虚血瘀证的证候实质，为益气活血法防治缺血中风气虚血瘀证机制研究拓宽新的方向，同时也为筛选新的治疗缺血中风气虚血瘀证有效中药提供重要的分子靶点。