基于针刺对脑出血神经重塑作用的信号转导调控机制研究

Previous research in the lab has shown that acupuncture treatment had affirmative effect on cerebral hemorrhage. It has been demonstrated that the repair and remodeling of neural functions were associated with the activation of autologous neural stem cells (NSCs) through the expression of the endogenous neurotrophic factors. This study further demonstrated the mechanism of neural function remodeling after acupuncture treatment and found that the Wnt,Notch and ERK signal pathways were involved in the process. By using rat cerebral hemorrhage model,we monitored dynamically the proliferation and differentiation of NSCs as well as the mRNA/protein levels of the key factors in the Wnt, Notch and ERK pathways. Methods used in this study include immunohistochemistry,double-label immunofluorescence assays,immune imprinting and real-time fluorescence quantitative PCR. This study is a comprehensive analysis of the effect of acupuncture treatment on NSCs proliferation and differentiation though endogenous signal pathways. It helps to understand the mechanisms underlying neuronal repair and remodeling following acupuncture treatment after cerebral hemorrhage. It supports the hypothesis that acupuncture majorly triggers endogenous repair and promotes neuronal reshape through endogenous signal pathways. This study provides new scientific evidences and broadens the horizon for the treatment of cerebral hemorrhage.

基于前期国家自然基金课题研究发现：针刺治疗脑出血疗效肯定，其神经功能的修复、重塑与调控内源性神经营养因子表达、激活自体神经干细胞（NSCs）有关。本项目进一步从Wnt、Notch和ERK信号通路，探讨针刺对脑出血后神经功能重塑的调控机制。制备脑出血大鼠模型，应用免疫组化、免疫荧光双标、免疫印迹、实时荧光定量PCR等研究方法，动态观察各组大鼠内源性NSCs增殖、分化状况以及上述信号通路中关键因子的mRNA/蛋白表达变化；综合分析针刺对脑出血后NSCs增殖分化的影响规律，以及胞内信号通路对内源性NSCs增殖和分化的调控作用，以期从胞内信号通路的整体调控作用，阐明针刺促进脑出血后神经功能修复与重塑的作用机理，进而验证"针刺刺激-信号调控-内源性修复启动是针刺促进脑出血神经功能修复与重塑的关键作用机制之一"的科学假说，为针刺治疗脑出血提供新的科学依据，为脑出血的治疗研究拓宽思路。

课题组前期研究发现，针刺促进脑出血大鼠神经功能修复、重塑与调控内源性神经营养因子表达、激活自体神经干细胞（NSCs）有关。本项目旨在进一步从Wnt、Notch和ERK信号通路，探讨针刺对脑出血后神经功能重塑的调控机制。制备脑出血大鼠模型，应用免疫荧光双标、Western blot、实时荧光定量PCR等方法，动态观察各组大鼠内源性NSCs增殖、分化状况以及Wnt、Notch和ERK信号通路中关键因子的蛋白/mRNA表达变化；综合分析针刺对脑出血后NSCs增殖分化的影响规律，以及胞内信号通路对内源性NSCs增殖和分化的调控作用，从而阐明针刺促进脑出血后神经功能修复与重塑的作用机理。结果表明：（1）脑出血急性期脑损伤能够激活大鼠自体内源性NSCs的原位增殖；脑出血恢复期，大鼠自体内源性NSCs由海马齿状回的颗粒细胞下层逐渐迁移至颗粒细胞层，并逐渐由未成熟神经元向成熟神经元分化。（2）Wnt通路的正反馈因子wnt1、β-catenin、Neurogeninl在脑出血后内源性NSCs增殖期处于高表达，而其负反馈因子GSK-3β在内源性NSCs迁移分化期表达最高，Wnt信号通路对大鼠脑出血后内源性NSCs的增殖和分化有一定的调控作用。（3）Notch通路的正反馈因子notch1、Hes1在脑出血后内源性NSCs增殖期处于高表达，而其负反馈因子Mashl在内源性NSCs迁移分化期表达最多，Notch信号通路对大鼠脑出血后内源性NSCs的增殖和分化有一定的调控作用。（4）ERK通路的MEK、ERK、Cyclin D1在脑出血后内源性NSCs增殖期处于高表达，在内源性NSCs迁移分化期表达有所减低，ERK信号通路对大鼠脑出血后内源性NSCs的增殖和分化有一定的调控作用。（5）针刺头部腧穴可促进大鼠脑出血后脑组织中Wnt、Notch、ERK通路关键因子的蛋白/mRNA表达，激活细胞内Wnt、Notch、ERK通路，调节下游靶基因转录，进而影响机体NSCs的增殖、分化，从而启动机体自身的内源性修复机制---验证了“针刺刺激-信号调控-内源性修复启动”是针刺促进脑出血神经功能修复与重塑的关键作用机制之一”的科学假说，为针刺治疗脑出血提供了新的科学依据，为脑出血的治疗研究拓宽了思路。