基于PKCβ1亚基调控mdr1表达电针诱导血脑屏障开放效应及机制

。 Currently, 90% of nervous system medications are found difficult to play effective treatment limited by blood-brain barrier (BBB).The research on BBB structure and function ,especially on changing the permeation of BBB has become a research hotspot in the field of neuro-rehabilitation. The research shows BBB is a dynamic regulation membrane and the plasma concentration in brain could be affected by P-glycoprotein mdr1 gene expression in drug efflux system, besides the physical barrier. In our research, cerebral middle cerebral artery occlusion (MCAO) rats are intervened by EA in different duration and the permeation effect of Evan's Blue and nerve growth factor(NGF) are observed by fluorescent quantitation and ELISA. Immunohistochemistry、Western Blot and quantitative RT-PCR are applied to observe P-glycoprotein mdr1 gene expression regulated by PKCβ1 subunit to reveal partial mechanism on EA-induced BBB opening. This study focuses on the impact of high-frequence EA on the permeability of BBB and explores the effect and possible mechanism that nervous system medications represented by NGF play effective action in BBB with the help of EA. Additionally, the clinical transformation of this experimental method has great value of research and development in the treatment of nervous system diseases.

目前，90%的神经系统治疗药因血脑屏障的原因难以发挥有价值的治疗作用，对血脑屏障结构和功能研究，特别是如何改变其"屏障"通透性，成为神经康复领域研究热点。研究表明，血脑屏障是一个动态的调节界面，除物理屏障外，脑组织内的血药浓度高低又受到其药物外排系统P-糖蛋白mdr1基因的表达的影响。本项目以电针对脑缺血再灌注大鼠（术后2周）进行不同时长的干预，采用荧光定量、ELISA观察伊文思蓝及神经生长因子透过血脑屏障效应；采用免疫组化、western blot、定量RT-PCR等技术观察PKCβ1亚基如何调控血脑屏障P-糖蛋白mdr1基因表达，以揭示电针促血脑屏障开放部分机制。本项目着眼高频电针对血脑屏障通透性的影响，探索以神经生长因子（NGF）为代表的神经系统治疗药在电针的辅助作用下进入血脑屏障发挥有效治疗作用的效应及可能机制。该实验方法的临床转化对神经系统病治疗具有巨大的研究和应用价值。

本课题通过观察不同时长以及不同频率电针对脑缺血再灌注（MCAO）恢复期大鼠血脑屏障（BBB）开放效应，对PKCβ1亚基参与调控血脑屏障P-糖蛋白（P-glycoprotein，P-gp）mdr1基因表达进而导致BBB通透性增加的分子机制进行了初步研究。首先通过优化筛选电针促MCAO恢复期大鼠BBB开放的最佳干预时长、波形和频率，探索不同电针刺激参数对大鼠BBB的开放效应。结果显示：“百会”、“水沟”穴不同时长和频率电针可不同程度诱导正常大鼠及MCAO大鼠恢复期血脑屏障开放，其中以刺激时长8min以及刺激频率2Hz、100Hz电针对BBB开放效应最佳，并证实该开放效应不致引起大鼠脑水肿。继而选择优化后的电针刺激参数（8min，2Hz，2mA），用ELISA法验证电针能否促NGF透过BBB。结果显示：电针对NGF透过大鼠血脑屏障具有较好的促透效应。进一步利用免疫组化、Western blot以及定量RT-PCR等技术观察PKCβ1亚基对调控血脑屏障P-gpmdr1基因表达，探索电针调节血脑屏障开放是否与PKC β1/P-gp通路的相关性。结果显示：电针(2Hz；8min)可以提高MCAO恢复期大鼠大脑皮层PKCβ1蛋白表达及活性、降低P-gp的表达。提示电针诱导大鼠BBB开放可能是通过促使PKCβ1磷酸化，下调药物外排系统P-gp mdr1a mRNA，抑制P-gp在大脑皮层毛细血管内皮细胞的表达实现。该研究为促BBB开放的电针刺激参数的优选及其在电针结合NGF治疗脑卒中后遗症中枢神经退行性病变的临床应用奠定了一定的工作基础。