基于星形胶质细胞活化介导Shh-Gli-TJ信号通路探索电针促血脑屏障开放的时间窗效应及机制

Many nervous system drugs, such as NGF, cannot permeate through the brain and exert therapeutic effects due to the existence of the blood-brain barrier (BBB). Astrocytes play an important role in maintaining the integrity of BBB. Much attention has focused on the effect of astrocyte-derived Sonic hedgehog (Shh) on the structure and function of BBB. Evidence has suggested that Shh-Gli signaling pathway can affect the permeability of BBB by regulating tight junctional proteins. It is also known that electro-acupuncture (EA) can regulate the activation of astrocytes. Our previous studies have found that 100Hz EA intervention has opening effects on BBB. In addition, the opening effect of EA on BBB exhibited certain time window property. In this project, EA 100 Hz with different time duration is used to intervene at 2 weeks after cerebral ischemia-reperfusion injury in rats (the convalescence period of cerebral ischemia rat model). Fluorescent quantification and ELIAS will be used to study the permeation of Evans blue. Furthermore, immunofluorescent, Western blotting and RT-PCR, etc will be used to investigate the mechanisms of how EA regulates BBB permeability via Shh/Gli/TJ signaling pathway. The aim of this project is to characterize the effects of EA-aided permeation of nervous system drugs into the BBB and elucidate the underlying mechanisms.

诸如神经生长因子（NGF）等药物因血脑屏障的阻挡难以入脑而丧失其治疗作用。星形胶质细胞在维持BBB的完整性方面发挥着重要作用，其分泌的Shh因子对 BBB结构和功能的影响备受关注。已有研究表明，Shh-Gli信号通路可以调控紧密连接转运蛋白，从而影响BBB通透性,同时电针对星形胶质细胞的活化具有调控作用。我们前期的研究发现，100Hz电针干预具有促血脑屏障开发效应，同时发现该开放效应具有一定的时间窗现象。本项目拟以100Hz电针经不同时长对脑缺血再灌注术后2周大鼠（脑缺血再灌注大鼠恢复期）进行干预，采用荧光定量、ELISA法观察伊文思蓝的透过效应；继而采用免疫荧光、Western Blotting、RT-PCR等技术探索电针如何通过Shh-Gli-TJ信号通路调控血脑屏障通透性的机制。本课题旨在研究电针辅助神经系统治疗药物进入血脑屏障并阐明其效应特征及可能机制。

本课题研究了电针开放MCAO/R恢复期大鼠血脑屏障（blood brain barrier, BBB）促NGF入脑的治疗作用及电针刺激模式的优化，进一步从Shh-Gli-TJ信号通路的角度探讨电针开放BBB的机制，其对电针开放BBB的安全性进行了研究。首先观察电针促NGF透过BBB及其被神经元摄取情况，评价电针结合NGF治疗对MCAO/R恢复期大鼠学习记忆能力的影响及缺血侧海马区神经元凋亡情况。结果显示：电针可使MCAO/R模型大鼠脑组织NGF增加，减少缺血侧海马区神经元凋亡，改善大鼠学习记忆能力。继而我们通过比较不同频率、强度、穴位和刺激时长电针开放BBB的效应，对电针的刺激模式进行优化。结果显示：电针“百会”、“水沟”穴，频率2/100Hz、强度3mA、刺激时长40min、刺激6s停6s（6s-6s）的效应较优，且其开放具有即刻、刺激依赖、可逆性的特点。接下来选择优化后的特定刺激模式电针，利用WB和RT-PCR方法检测大鼠脑组织Shh、Gli蛋白与基因及TJs的表达，结果显示：电针下调Shh与Gli蛋白与基因的表达，Shh激动剂Purmorphain能够干扰电针开放BBB的效应、上调TJs，表明电针通过调控Shh-Gli-TJ信号通路影响大鼠BBB的通透性。进一步采用ELISA、蔗糖实验等方法检测电针干预后脑组织TNF-α和IL-1β的含量及大鼠脑水肿、行为情绪的改变，评估电针开放BBB的安全性。结果显示：电针干预不会导致脑组织炎症和脑水肿，不会导致大鼠焦虑抑郁样情绪，初步证实该刺激模式电针促BBB开放是安全的。本课题对电针开放BBB的刺激模式和分子机制进行了研究，提示特定刺激模式开放BBB可能是向中枢神经系统递送治疗药物的一种新策略，并为电针结合NGF治疗脑卒中后遗症的临床应用奠定了一定的工作基础。