活血利窍，开通玄府干预血脑屏障腺苷受体促进益脑方药脑靶向作用机理研究

The blood brain barrier (BBB) is the bottleneck of drugs for the treatment of brain diseases. The brain" Xuanfu" in Traditional Chinese medicine(TCM) is closely correlated with BBB. "Tong Xuan Fu" to treat brain disease has a long history and excellent effect. Based on the TCM theory"HuoXue（Activating Blood circulation）LiQiao(benefit 'qiao')- Kai Tong 'XuanFu' ", We first suggest that Combination of non-aromatic HuoXue Yao(Herbs for invigorating blood circulation ) and Tonic agent might also promote the opening of BBB to realize the brain-targeting distribution of the active components of the TCM herbs. Our previous study demonstrated that the composition of non-aromatic blood circulation- activating herb ginkgo leaf with tonic ginseng and fleece flower root, promoted their active components to penetrate through BBB and increase their concentration in the brain tissue by 10 more times, and then enhanced the therapeutic efficacy. But the material base and the molecular mechanism of the above effect is not yet clear. Further experiments showed that selective inhibition of P-G glycoprotein only partly weaken its brain targeting effect, suggesting that the mechanism is P-glycoprotein independent, and might be related to adenosine receptor pathway. This project intends to use the in vitro BBB model of the co-culture of the brain microvascular endothelial cells and astrocytes, the transendothelial electrical resistance detection, electron microscopy of BBB tight junction （TJ） structure, selective inhibitors and siRNA interference blocking adenosine receptor, and LC-MS methods to study the effects of active components of Ginkgo biloba leaves on the BBB structure,function and related BBB protein ，such as adenosine receptor, ZO-1, Occludin3 and Claudin-3/5 expression, then to obtain reliable evidence that Ginkgo biloba leaves regulate the permeability of BBB by interfering with adenosine receptor. Our project will clarify the mechanism and material basis of brain targeting effect of Ginkgo biloba leaves, scientifically elucidate the biological essence of the hypothesis "HuoXue（Activating Blood circulation）LiQiao(benefit 'qiao')-Kai Tong 'XuanFu'". That might provide new ideas and experimental supports for improving the efficacy of the therapeutic strategy of "BuQi HuoXue Li'Qiao' -KaiTong 'XuanFu' " and the research and development of innovative Chinese drugs for the treatment of brain disease.

血脑屏障（BBB）是治疗脑部疾病药物的瓶颈。中医脑玄府与BBB关系密切。通玄府治脑疾历史久远，效果显著。我们根据活血利窍-开通玄府理论，首次提出非芳香活血药与补益药配伍也有脑靶向作用，发现银杏叶配伍补益药人参首乌，能促进活性成分数十倍透过BBB、提高脑组织内浓度，增强药效，但其物质基础和分子机制还不清楚。进一步实验显示其脑靶向有P-糖蛋白非依赖性，推测其可能与腺苷受体通路有关。本项目拟利用体外BBB模型，跨内皮电阻检测、紧密结构电镜检查、分子抑制剂和RNA干扰阻断腺苷受体，研究银杏叶活性成分对BBB结构功能和相关腺苷受体、ZO-1、Occludin3和 Claudin-5表达的影响，旨在获得银杏叶干预腺苷受体调节BBB通透性的可靠证据，将明确银杏叶脑靶向作用机理及其物质基础，科学阐析活血利窍、开通玄府的生物学本质。为益气活血利窍开通玄府提高疗效、研发治疗脑部疾病创新中药提供新思路和科学依据

血脑屏障（BBB）是治疗脑部疾病药物研究开发的瓶颈。中医脑玄府与BBB关系密切。申请人提出非芳香活血药与补益药配伍也有脑靶向作用，发现银杏叶配伍补益药人参首乌，能促进活性成分3~5倍透过BBB、提高脑组织内浓度，增强药效，推测其可能与腺苷受体通路有关。利用体外BBB模型，跨内皮电阻检测、紧密连接超微结构电镜检查、和小RNA干扰等细胞分子生物学技术，研究银杏叶活性成分对BBB结构功能和腺苷受体及BBB紧密连接相关蛋白表达的影响，首次发现：.1..活血药银杏叶提取物有脑靶向作用.活血药银杏叶提取物（EGb）与人参、何首乌提取物配伍能促进人参皂苷Rb1、Re、Rg1、Rd、Rf、和二苯乙烯苷等中药活性成分在SD大鼠脑组织中含量增加3~5倍，同样，能显著促进伊文斯蓝、FITC-Dextran透过血脑屏障进入脑组织。.2..EGB及其内酯类活性成分能可逆性开放血脑屏障.人脑微血管内皮细胞（HBMEC）和星形神经胶质细胞(HEB)共培养体外血脑屏障模型实验发现：EGb、银杏内酯A/B和白果内酯，能显著降低细胞跨内皮电阻；并能显著增加荧光素钠的透过率。透射电镜观察发现EGb确能使脑微血管内皮细胞间的紧密连接（TJ）间隙扩大、促进血脑屏障可逆性开放。.3..腺苷受体A1（A1R）和A2a（A2aR）是银杏叶提取物开放血脑屏障的作用靶点.EGb、银杏内酯和白果内酯对体外血脑屏障模型的腺苷受体A1和A2aR的蛋白表达有促进作用、对ZO-1、Claudin3、5和Occludin蛋白等紧密联接蛋白表达有一定抑制影响，并且能上调骨架蛋白结合蛋白MLC，ERM磷酸化水平。.4.银杏叶提取物和白果内酯通过腺苷受体信号通路调节血脑屏障开放.白果内酯可逆性增加血脑屏障通透性作用机制及其信号通路涉及腺苷受体A1R及其相关紧密连接蛋白、细胞骨架蛋白结合蛋白MLC、ERM磷酸化以及PKC、ROCK信号靶点。.本研究获得了银杏叶干预腺苷受体调节BBB通透性的可靠证据，明确了活血药EGb有脑靶向作用及其可逆性开放血脑屏障的作用靶点，基本明确了EGb脑靶向作用机理及其物质基础，初步科学阐析了活血利窍、开通玄府的生物学本质。.发表论文8篇，其中SCI收录1篇；申报发明专利8项，授权1项。