铁调素与MFSD2a结合参与脑铁代谢的新机制

Deposited-iron in the brain is an important inducement of the iron-related diseases of the central nervous system (CNS). As the brain iron metabolism mechanism are not clear, there are limited treatment methods in reducing the deposited brain iron and the effects of this methods exsit great differences. Thus, exploring the detailed mechanisms of brain iron metabolism may provide a novel therapeutic methods for reducing the deposited iron in these CNS diseases. Hepcidin is the most important iron regulating hormone in the body, and when it.binded to the ferroportin (FPN) located on the membranes of brain microvascular endothelial cells (BMVEs) could regulate the exporting iron function of cells. While the FPN knockout cell also exhibited the function of exporting the intracellular iron. Considering that the key transport function of MFSD2a in the blood-brain barrier (BBB) was specifically expressed on the BMVEs. Additionally, our preliminary experiment also showed that MFSD2a-/- BMVEs have the lowering ability to export the intracellular iron and the MFSD2a has a direct interaction with hepcidin, suggesting that MFSD2a may be as the novel iron export channel of BMVEs and has a critical role in the brain iron metabolism. Therefore, we put forward the following hypothesis: hepcidin interacts with the MFSD2a may be the novel pathway of brain iron metabolism. Therefore based on this, we proposed the further study aims: (1) to clarify the exact role of MFSD2a in the brain iron.metabolism; (2) to explore the mechanisms of the interactions between hepcidin and MFSD2a in the brain iron metabolism. The study can not only find the novel iron export, clarify mechanism of brain iron metabolism, but also provide new method for the treatment of iron-related CNS diseases.

脑铁沉积是铁相关性中枢神经系统疾病的重要致病因素，由于脑铁代谢机制不清楚，目前降铁治疗手段有限且疗效存在较大差异。因此探索脑铁代谢机制可为降铁治疗提供新方法。铁调素是铁代谢的重要调节激素，其通过与脑血管内皮细胞膜上膜铁转运蛋白（FPN）相互作用调控细胞排铁。但FPN敲除细胞仍有排铁功能，提示还存在新的排铁通道。鉴于特异表达于脑血管内皮细胞膜上的MFSD2a在血脑屏障物质转运中具有重要作用，我们前期发现MFSD2a敲除细胞排铁减少且铁调素与MFSD2a有相互作用，提示MFSD2a通过与铁调素相互作用参与调控细胞排铁功能。据此我们提出如下假说：铁调素与MFSD2a结合是脑铁代谢的新途径。为此本项目拟进行：（1）明确MFSD2a在脑铁代谢中的作用；（2）揭示铁调素通过与MFSD2a结合在脑铁代谢中的机制。该研究不仅可发现细胞排铁新通道，阐明脑铁代谢机制，还可为为铁相关中枢疾病的治疗提供新策略。

脑铁沉积是铁相关性中枢神经系统疾病的重要致病因素，由于脑铁代谢机制不清楚，目前降铁治疗手段有限且疗效存在较大差异。因此探索脑铁代谢机制可为降铁治疗提供新方法。然而，目前已知的细胞摄铁途径有多种，但排铁通道却只有膜铁转运蛋白（FPN1）这一种，这显著的细胞摄铁和排铁通道不匹配促使我们进一步探索是否有其他的细胞排铁通道。鉴于铁调素通过与FPN1相互作用发挥重要的细胞排铁调节，且MFSD2a是转运蛋白家族中的重要一员，为此我们想明确Mfsd2a是否可参与细胞排铁且与铁调素相互作用调节铁代谢。为此，我们首先明确了铁调素在脑出血脑铁代谢中的作用及Mfsd2a在脑出血后BBB损伤中的作用，这些研究结果为脑出血治疗提供了新靶点。随后，我们构建了Mfsd2a-IRES-GFP或GFP稳定转染的HEK293细胞。20 μM枸橼酸铁铵(FAC)添加到细胞培养基中，通过Western blot检测细胞内铁蛋白的表达来明确Mfsd2a是否参与了细胞铁的吸收，结果发现在加入FAC后，Mfsd2a过表达组和对照组细胞内的铁蛋白水平均显著升高，并在24 - 48 h达到峰值。然而，两组细胞内的铁蛋白含量在各时间点均无明显差异，提示Mfsd2a可能不参与细胞铁的吸收。进一步地，Mfsd2a过表达和对照组HEK293细胞分别在20 μM FAC培养48 h后，更换无铁的培养基继续培养。分别在更换培养基后的0 h、6 h、12 h、24 h和48 h时收取细胞，并检测细胞内铁蛋白水平。结果表明， Mfsd2a过表达组和对照组之间的铁蛋白水平在每个检测时间点均无显著差异。此外，免疫共沉淀实验检测也发现铁调素和Mfsd2a间无相互作用。这些结果提示Mfsd2a不能参与细胞铁代谢的调节。尽管我们的研究发现Mfsd2a不能参与铁代谢且与铁代谢关键激素铁调素无相互作用，但我们仍需寻找其他的排铁通道，进而为铁代谢相关疾病的研究奠定基础。