开窍药调控“神经血管单元”Wnt信号通路表达"辛-心-开"性效的机制研究

According to the previous research, the orifice-opening medicine play a role in fighting againt the cerebral ischemia and hypoxia, inhibiting the brain edema, regulating the blood-brain barrier and etc. to induce resuscitation and restore consciousness. With an effect-frame "pungent - staying in heart meridian - orifice-opening and resuscitation-inducing", the new academic idea "Functional System in the Nature of Chinese Pharmacy "has been proposed. The ischemic stroke is one of the critical illnesses leading to the high rate of disability. The latest research shows the neurovascular unit, consisting of neuron and blood-brain barrier, has a close connection with Wnt signal path and has an important effect in brain injury repairing. There are little study in the orifice-opening medicine functioning on Wnt path. This puts forward the hypothesis that the orifice-opening medicine has the efficiency in "pungent-staying in heart meridian-orifice-opening and resuscitation-inducing" by regulating and controlling the neurovascular unit and Wnt signal path. The advanced technologies such as CLSM,immunohistochemistry, immunoblotting and GC-MS will be applied and be combined with multi-disciplinary knowledge. Centering on Wnt/β-catenin path and active substances (such as Occludin、VEGF、EPO、HIF-1α、MMPS9), as well as researching the biological mechanisms from the multi-level "entirety-organ-cell" of the storax, borneol and benzoin in regulating and controlling the neurovascular unit and Wnt signal path of the rats with cerebral ischemia, it reveals the scientific connotation in the effect-relationship of "pungent - staying in heart meridian - orifice-opening and resuscitation-inducing" of the orifice-opening medicine and enriches the nature theory of Chinese pharmacy. So, it not only has significant academic value and future application prospect, but also with innovativeness.

前期研究显示，开窍药有抗脑缺血缺氧、抑制脑水肿、调节血脑屏障等作用而醒神回苏，具"辛味-归心经-开窍醒神"的性效构架，提出"中药药性功能系统"新学术观。缺血性脑卒中是一类致残率高的危重症。最新研究认为"神经血管单元"由神经元和血脑屏障构成，Wnt信号通路与之密切相关，在脑损伤修复中起重要作用，开窍药对Wnt通路研究甚少。本次提出开窍药通过调控神经血管单元及Wnt信号通路表达"辛-心-开"性效假说。采用激光共聚焦显微镜、免疫组化、免疫印迹、GC-MS等先进技术，融合多学科知识，围绕Wnt/β-catenin通路及Occludin、VEGF、EPO、HIF-1α、MMPS9等活性物质，从整体-器官-细胞多层次研究苏合香、冰片、安息香调控脑缺血模型大鼠神经血管单元Wnt信号通路的生物学机制，以揭示开窍药"辛-心-开"性效关联科学内涵，丰富中药药性理论。故有重要的学术价值和远期应用前景，亦具创新。

基于开窍药的“辛-心-开（开启闭阻心窍）”性-效特征及研究背景，立足于中医整体观、动态观思路，围绕神经血管单元（NVU），以Wnt信号通路为切入点，借助多学科手段，从宏观-微观，功能-形态，从整体-器官-组织-细胞多层次水平阐明开窍药保护神经血管单元（NVU）-开窍醒神的生物学机制，比较其作用强弱，揭示其“辛-心-开”的性效关联科学内涵，并为临床合理选药提供参考。. 研究显示：①开窍药能促进pMCAO模型大鼠神经功能恢复、降低脑含水量及脑梗死率、减轻神经元和血管的损伤，保护NVU而发挥“辛-开”效应，其作用强弱依次为艾片>冰片>安息香>苏合香>天然冰片。②开窍药通过调节Wnt/β-catenin信号通路，抑制细胞凋亡、促进微血管新生及保护BBB而重塑神经血管单元，可能是综合表达“辛-心-开”的主要生物学机制。冰片、天然冰片抑制炎症因子和抑制细胞凋亡，可能与其寒凉性有关；苏合香与安息香促微血管新生及保护BBB或许与其温性有关。③三种冰片对正常大鼠及LPS发热模型大鼠均呈现降低体温、降血压、减慢心率、抑制活动度效应，表达寒凉性特点；苏合香呈出先兴奋后抑制趋势，其性温表达与时间窗有关；安息香对发热模型大鼠无显著影响，其持续稳定效应呈艾片＞天然冰片＞合成冰片趋势。④艾片与冰片对铜绿假单胞菌生长代谢抑制效应表达出寒凉特性，余药特征不明显。⑤五种开窍药对异丙肾上腺素致心肌缺血心电均有一定改善作用；苏合香能改善手术致心肌缺血模型大鼠心电及心肌梗死面积，降低血清CK-MB、AST、LDH活性，抑制PI3K总蛋白表达，其机制尚待深入研究。. 创新：动态观平行考察了开窍药对正常与LPS发热模型大鼠的生命体征的影响，解析其性效，并从多维度、多层次阐释了开窍药通过调节Wnt/β-catenin信号通路，稳定重塑神经血管单元表达“辛-心-开”的生物学机制，研究思路有特色和新意；结果发现艾片最能有效改善脑缺血损伤及重塑神经血管单元，尤宜于脑缺血损伤伴发热者，可为临床合理组方选药提供参考，提示其社会意义和应用价值。. 成果：孵育新课题4项，其中国家自然基金面上项目1项、青年基金1项，博士后基金面上项目1项，省教育厅1项；培养研究生6人，发表文章15篇，学位论文2篇，待发文章3篇；参加学术会3次，全国优秀论文1篇，三等奖1篇。拟申报省科技进步奖。总体实现了预期目标。