电针通过MicroRNA-223调控GluR2/CaM减轻脑缺血损伤的作用和机制研究

Calcium overload is induced by excessive activation of AMPA-type glutamate receptors (AMPAR), and excitotoxicity mediated by this play a critical role in cerebral ischemic insults. However, the clinical application of AMPAR antagonist is limited due to directly blocking receptor physiological function. Our previous study reveals that electric acupuncture (EA) pretreatment enhances GluR2 protain expression, the subunit adjusted the permeability of AMPAR to calcium ion, to protect against global cerebral ischemia. The relevant article was accepted by Scientific Reports. However, the underlying mechanisms of GluR2 regulated by EA pretreatment were incomletely understood. GluR2 has been reported as one target of MicroRNA-223(miR-223), and miR-223 could effectively ameliorate the pathologic hallmarks of spinal cord injury(SCI) via increasing GluR2 expression. Therefore, we will investigate whether inhibition of miR-223 is involved in neuroprotection induced by EA pretreatment via GluR2, which may be related to calmodulin(CaM) signaling pathway after transient global cerebral ischemia in mice. The aim of the study was to clarify the mechanism of neuroprotection induced by EA through regulating GluR2 with comprehensive methods such as morphology and molecular biology and to bring a new perspective of stroke.

AMPAR过度激活引起胞内钙超载诱发的兴奋毒性是脑缺血损伤的重要机制，而利用其拮抗剂减弱兴奋毒性的临床试验，却因阻断AMPAR的生理作用，以失败告终。申请者前期研究发现，电针预处理可上调缺血后神经元上决定AMPAR钙离子通透性的GluR2亚基表达水平，诱导脑缺血耐受，相关结果已被Scientific Reports(IF=5.078)接收。然而电针如何调控GluR2的表达，通过何种机制减轻脑缺血损伤尚未了解。文献报道MicroRNA-223(miR-223)可影响脑内GluR2蛋白表达，改善脊髓缺血损伤。本课题假设：电针通过抑制MicroRNA-223，上调GluR2的表达，减少钙调蛋白(calmodulin，CaM)介导脑缺血耐受。本项目拟应用功能、形态和分子生物学等方法，阐明电针调节GluR2诱导脑缺血耐受的具体机制，为提出脑卒中防治新策略带来新的视角与契机，具有重要的临床意义。

AMPAR过度激活引起胞内钙超载诱发的兴奋毒性是脑缺血损伤的重要机制。我们前期研究发现，电针预处理可上调缺血后神经元上决定AMPAR钙离子通透性的GluR2亚基表达水平，诱导脑缺血耐受。然而电针如何调控GluR2的表达，通过何种机制减轻脑缺血损伤尚未了解。文献报道MicroRNA-223(miR-223)可影响脑内GluR2蛋白表达，改善脊髓缺血损伤。本课题假设：电针通过抑制MicroRNA-223，上调GluR2的表达，减少钙调蛋白(calmodulin，CaM)介导脑缺血耐受。本课题应用功能学、形态学和分子生物学等方法，探索了电针预处理对全脑缺血再灌注损伤小鼠海马神经元miR-223表达的影响、干预miR-223对电针预处理的脑保护作用的改变、miR-223与GluR2之间的调控关系、干预miR-223对电针预处理调控GluR2作用的影响、抑制GluR2对电针预处理改变CaM的影响、拮抗CaM表达对电针预处理脑保护作用的影响。结果明确了电针预处理可下调小鼠全脑缺血再灌注2h后海马神经元miR-223的表达；抑制miR-223表达可模拟电针预处理的脑保护作用，而过表达miR-223可逆转电针的脑保护效应；miR-223与GluR2之间存在上下游的调控关系，抑制miR-223，GluR2表达增强，过表达miR-223，GluR2表达下降。电针预处理可下调CaM的表达，而干涉GluR2后，CaM表达恢复；拮抗CaM可模拟电针预处理的脑保护作用。本课题的研究结果初步验证了我们的假设，提出了电针通过GluR2诱导脑缺血耐受的具体机制，为脑卒中的电针防治新策略带来临床应用的契机，具有重要的临床意义。