三七丹参有效组分配伍促进脑中风恢复的脑神经血管单元整合机制研究

Except to thrombolytic treatment in acute phase of ischemic stroke, all neuroprotection agents R&D failed by far in abroad. Although Traditional Chinese medicine have been Widely used in clinic and proved effective, mechanism of multi-component synergies and how they promote brain function recovery still need further study. In present study, according to the treatment method of nourashing Qi and promote blood circulation in TCM theory during chronic phase of post-stroke, effective constituents in Xueshuantong Injection and Danshenduofensuan Injection are selected as representative active component constituents of Panax notoginsen and Danshen, the neurovascular remodeling mechanism of the promotive action by combination use of constituents derived from Pannax notoginsen and Danshen during post-stroke recovery are to be elucidated. Based on the previous preliminary experimental results, both in vitro and in vivo study are included. Primary culture of brain microvascular endothelial cells(BMEC), astrocytes(AS), microglia and BMEC/AS/pericyte coculture blood-brain barrier (BBB) model will be established, MCAo rodent model are to be used. BBB permeability, protection for BBB structure and function, impact on the basement membrane, remodeling of extracellular matrix(ECM) are to be studied. As well, promotion effects to neurogenesis, angiogegesis, synaptic repair, neurologic function recovery of the single and combination use of the active component constituents of Panax notoginsen and Danshen in two injections are to be studied. The aim of the study is to explore multi-component synergic action mechanism of the active constituents of active components of Panax notoginsen and Danshen and their compitibility related to neurovascular remodeling, provide more scientific evidence of the combination use of multi-constituents derived from Traditional Chinese medicine for promoting post-stroke recovery, illustrate the advantages of traditional Chinese medicine’s neuroprotection effects and guide the applications of traditional Chinese medicine in clinic.

缺血性脑中风除急性期溶栓治疗外，国外脑保护剂研发迄未成功。中药临床恢复其应用广泛且疗效显著，但如何发挥多成分协同作用促进脑功能恢复及机制研究有待深入。本研究根据中医学理论在中风恢复期益气活血治则为主指导下，以益气活血代表药三七和丹参注射制剂注射用血栓通和丹参多酚酸有效成分为研究对象，在前期工作基础上，重点从促进恢复期神经血管单元整合修复机制方面，研究两种制剂有效成分对中风后血管神经单元各组成及相互作用的影响。体外采用脑微血管内皮细胞、星形胶质细胞、小胶质细胞培养及微血管内皮细胞/星形胶质细胞/周细胞共培养血脑屏障（BBB）模型，以及大脑中动脉栓塞脑缺血大鼠模型，选择注射用血栓通和丹参多酚酸有效成分，从BBB透过性、保护BBB结构与功能、基底膜细胞外基质重建相关因子表达的影响，以及促进神经元再生、血管新生、突触修复、动物神经功能恢复等方面，探讨中药多成分协同作用。

背景和目的：神经血管单元重塑（NVU Remodeling）是中风研究的重要关注内容，缺血性脑中风恢复期中药治疗的作用机制与优势是什么？益气活血中药丹参三七组分配伍对缺血性脑中风恢复期NVU重塑的整合调控机制是本课题研究重点。主要研究内容：研究建立了Wistar♂大鼠大脑中动脉局灶性阻塞再灌注（MCAO/R)模型和NVU相关细胞体外培养及共培养体系，体内体外结合，研究益气活血代表药丹参三七有效组分（以注射用丹参多酚酸中丹参多酚酸类组分和注射用血栓通中三七总皂苷类组分为代表）对脑缺血中风恢复期NVU重塑的整合调节作用和机制。主要结果：（1）发现丹参多酚酸、三七总皂苷组分可增加大鼠脑缺血后脑血流、改善神经功能评分、提高存活率。组分配伍治疗改善情况优于两类组分单用；（2）两类组分尾静脉注射连续7天、14天，可促进MCAO/R大鼠脑缺血半暗带树突重塑，提高神经元突触素SYN、突触后致密蛋白PSD等表达，且配伍应用改善作用更强。说明两类有效组分对于恢复期神经及突触重塑、血管再生等方面发挥了多成分协同作用；（3）丹参多酚酸与三七总皂苷组分可降低MCAO/R大鼠血脑屏障（BBB）病理性开放，降低周细胞与星型胶质细胞、星型胶质细胞与基底膜分离度；改善体外多细胞共培养BBB模型OGD/R损伤程度；两类组分配伍对BBB结构和功能的保护效果更佳；（4）丹参多酚酸与三七总皂苷组分对NVU各组成细胞的整合调控作用：抑制脑微血管内皮细胞炎症，提高内皮细胞、周细胞之间紧密连接蛋白、星型胶质细胞谷氨酸清除和神经营养因子的表达，促进小胶质细胞M1型极化向M2型转化等，促进神经元突起重塑，促进NVU重塑，恢复中风后期脑功能。（5）丹参多酚酸与三七总皂苷组分单体透过MCAO/R大鼠BBB情况：丹参多酚酸组分SalB、原儿茶醛、丹参素，血栓通三七总皂苷组分人参皂苷Rg1、Rb1、Rd1 、Re、三七皂苷R1能透过MCAO/R大鼠BBB。科学意义：研究为丹参三七益气活血组分及配伍对缺血性中风恢复期NVU形态和功能重塑、恢复脑功能提供了实验依据，为益气活血法在缺血性中风恢复期应用提供了理论基础。发表SCI论文3篇、北大中文核心期刊8篇，国内期刊英文版1篇，另中文核心期刊接收1篇。毕业博士生1人，在读3人，毕业硕士生3人，在读5人。