补阳还五汤调控自噬促进缺血性中风神经再生的作用及机制研究

neurogenesis is an important approach to improve the ischemic stroke neural dysfunction, cell autophagy decided to NSCs survival and function, the mechanism is unclear. TCM holds that qi deficiency blood stasis for ischemic stroke main type, yiqi huoxue as the main therapeutic principle. Buyanghuanwu decoction is the representation of yiqi huoxue. clinical practice and experimental research (including our previous studies) confirmed its exact role of restoring nerve function, but the mechanism need to be researchde. In traditional Chinese medicine, "Qi and blood - kidney essence - brains" is closely related to the NSCs, and "gas\" theory closely associated with autophagy. Based on this, this study put forward the hypothesis that endogenous NSCs autophagy activity changes after cerebral ischemia, affect the survival and function of NSCs. Buyanghuanwu decoction regulates the NSCs’ autophagy, influences their proliferation, differentiation and migration, promotes the neurogenesis. And as a whole, cellular and molecular level, we use the MCAO rats, treated by the autophagy inhibitors and inducers; Hypoxia reoxygenation of fetal rat hippocampal NSCs, knocked down or up autophagy key genes by SiRNA; Cultivated NSCs, treated by the blocker to block autophagy respectively control method such as the two signaling pathways, The behavioral science, transmission electron microscopy (sem), RT-PCR Western-blot, immunofluorescence double standard/three standard detection methods, such as, are used to observe how the autophagy activity change after cerebral ischemia and how to influence the proliferation, differentiation and migration of NSCs, finally to discover the function , mechanism and targets of Buyanghuanwu decoction.

神经再生是缺血性中风神经功能恢复的重要途径，细胞自噬决定神经干细胞生存和功能，机制不明。气虚血瘀为缺血性中风主要中医证型，益气活血为主要治则。补阳还五汤为益气活血代表方，临床和实验研究（包括本课题组前期研究）均证实其恢复神经功能的确切作用，机制不明。中医“气血-肾精-脑髓”与 NSCs密切相关，“气的化生”和细胞自噬密切关联。基于此，本研究提出假说：脑缺血后NSCs自噬活性改变，影响NSCs的生存和功能。补阳还五汤调控NSCs自噬，影响其功能，促神经再生。并从整体、细胞、分子水平，以MCAO大鼠，经自噬抑制剂、诱导剂干预；缺氧复氧的NSCs，SiRNA敲减自噬关键基因；培养的NSCs，经阻断剂阻断两条自噬调控通路等手段处理，经行为学、透射电镜、RT-PCR 、western-blot、免疫荧光双标/三标等检测，观察脑缺血后自噬活性改变对NSC功能的影响及补阳还五汤的作用、机制和靶点。

本研究分整体水平、细胞水平两大部分。.整体实验结果揭示：在大鼠 MCAO-R 模型上，给予20g/kg补阳还五汤，可改善损伤第5天的梗死体积，一定程度恢复行为运动功能和大脑神经功能；补阳还五汤组与自噬激活剂Rapa在调节自噬方面呈现相似趋势；SIRT1、自噬相关指标（LC3、P62、beclin-1）、神经生长类相关蛋白（BDNF、DCX、Nestin）和尼氏体染色的结果反映在大鼠脑缺血再灌注损伤损伤及治疗过程中的表达变化。以上结果说明：补阳还五汤通过促进神经再生作用发挥缺血再灌注损伤后的大脑保护，这一过程功能 SIRT1/自噬通路相关。.细胞实验结果揭示：补阳还五汤上调糖氧剥夺/复氧后的神经干细胞的自噬活性，这可能提供了补阳还五汤对神经干细胞的保护作用的新机制；补阳还五汤通过上调自噬，调节缺氧损伤造成的神经干细胞死亡，并有助于分化；Rapa起到了类似的作用，它可以靶向抑制自噬的上游靶点mTOR活性来激活自噬；3-MA抵消了神经干细胞的保护作用，并且分化能力也受到的影响，这也证明了自噬对于神经干细胞的存活和分化息息相关。. 本研究从整体、细胞水平证实补阳还五汤对 MCAO-R大鼠以及缺氧缺糖损伤NSCs有一定的神经保护作用，这可能与补阳还五汤调控自噬相关通路（SIRT1/自噬，PI3K/AKT，Wnt通路）有关，为缺血性中风的机制研究和临床防治提供重要的实验依据。