栝楼桂枝汤对缺血性脑卒中后神经元-小胶质细胞crosstalk的调控机制研究

TThe activation of microglia (MG) is the key point to initiate the inflammation reaction after ischemic injury. In our previous study, something can be found that GLGZD based on Yi Jing and blood essence method can exert the neuroprotective effects by inhibiting the activation of MG. What’s more, the neuron is, to large extent, not only the target for MG, but also participating in administrating the activity of MG. that is to say there is a crosstalk between MG and neuron. So we draw a hypothesis that GLGZD can Alleviate the cascade of inflammation and repair the ischemic injury by regulating the MG-neuron crosstalk, which is the potential mechanism of GLGZD on treating limb spasticity after stroke. Our study is guided by the holistic opinion of traditional Chinese medicine, which takes neuron-MG crosstalk as the research point and applies pharmacodynamics and cell biology as well as molecular biology methods to to explore the mechanism of GLGZD in alleviating limb spasticity after stroke. In this section, the therapeutic effect of GLGZD and its effects on neuronal apoptosis and neonatal, microglial activation and phenotype, the expression of inflammatory factor and neuroprotective factors as well as main factors in MG-neuron crosstalk can be observed via the establishment of rat MCAO model in vivo and OGD model of MG-neuron co-cultivate system in vitro. The mention above provides a scientific basis for the clinical application of GLGZD in rehabilitation of neurological function after stroke, and provides a modern scientific explanation for the treatment of spasms with the method of Yi Jin and blood essence.

小胶质细胞(MG)活化是启动脑缺血炎性反应的关键。前期研究发现，以益津和血养筋法立方的栝楼桂枝汤(GLGZD)可通过抑制MG活化发挥神经保护作用。新近研究发现神经元不仅是MG的靶标，还是调节MG活性的重要参与者，即神经元和MG间存在crosstalk，故推测：通过影响神经元-MG间crosstalk调控MG活化，减轻脑卒中后炎症级联反应，修复损伤，是GLGZD治疗脑卒中后肢体痉挛整体效应的重要机理。本项目在中医整体观理论指导下，以神经元-MG间crosstalk为切入点，运用药效学、细胞生物学、分子生物学等方法，制备MCAO大鼠模型，体外建立氧糖剥夺的神经元-MG共培养体系，观察GLGZD对神经元凋亡和新生、MG活化和表型变化、炎症因子、神经保护因子、神经元-MG间crosstalk关键分子表达的影响，探索GLGZD缓解脑卒中后肢体痉挛的作用机制，并为益津和血养筋法治疗痉证进行科学阐释。

目前研究认为，脑缺血后的急性炎性反应是导致脑组织缺血损伤加剧的重要因素，小胶质细胞的迅速激活，是启动脑缺血炎性反应的关键，对脑缺血后神经功能重构具有重要影响。最新研究发现神经元不仅是小胶质细胞的靶目标，而且是调节小胶质细胞活性的重要参与者。神经元和小胶质细胞之间存在crosstalk，即小胶质细胞的活化程度受到神经元的严密调控。.栝楼桂枝汤（GLGZD）出自《金匮要略》，现代临床研究发现，其对改善脑卒中后偏瘫患者肢体痉挛状态、运动功能和生存质量有较好的疗效，但其作用机制尚未阐明。故本研究采用HPLC分析了GLGZD中主要成分和含量，获得GLGZD指纹图谱，为GLGZD的质量控制和药效物质基础的进一步研究奠定基础。通过线栓法制备大鼠MCAO模型，并构建氧糖剥夺（OGD）神经元-小胶质细胞共培养体系，研究GLGZD对脑缺血/再灌注损伤的保护作用及调控神经元-小胶质细胞crosstalk的可能机制。体内外实验结果显示：GLGZD对脑缺血/再灌注损伤具有较好的保护作用，可促进脑缺血损伤后神经和运动功能恢复，缓解肢体痉挛，改善缺血侧皮层神经细胞的病理状态，并可减少OGD后对神经细胞的损伤，而这一作用与其通过影响TWEAK/Fn14/CCL21/CXCR3信号通路与CD200-CD200R信号传导介导的神经元调控小胶质细胞活化及表型转化，抑制过度炎症反应，促进组织修复有关。.本研究采用大鼠原代皮层神经元及大鼠原代皮层小胶质细胞进行混合共培养，并以OGD模拟脑缺血，较好的模拟脑缺血再灌注损伤后神经元受损、小胶质细胞增殖活化的体内过程。为神经血管单元涉及多种结构、多种作用环节和众多信号通路在缺血性脑卒中的研究提供了更符合体内过程且可靠的实验方法！本研究为GLGZD的临床应用和深入开发奠定基础。亦对益津和血养筋法治疗痉证的中医传统治法进行现代科学阐释，并为古方今用提供研究思路和方法。这对充分挖掘中药科学内涵、推动和发展中药产业，具有十分重要的现实意义和广阔的市场前景。