基于MRI多模态成像的栝楼桂枝颗粒治疗缺血性脑卒中后肢体痉挛及其调控钙稳态的作用机制研究

Traditional Chinese medicines are always employed to treat stroke and post-stroke sequelae. However, the unclear mechanisms and targets still restrict the development of traditional Chinese medicine. The famous preparation Gualou-guizhi decotion is good for the recovery of illness after stroke happening in clinical practice. In the previous study, it is found that Gualou-guizhi decotion has anti-post-ischemia reperfusion injury (MCAO) spasm effect, can inhibit neuronal apoptosis and decrease intracellular Ca2+ concentration, up-regulate the expression of some protein related to calcium signal transduction pathway, suggesting that Gualouguizhi Granule inhibit neuronal apoptosis through regulating of intracellular calcium homeostasis and neurotransmitter balance, thereby activating calcium signal transduction pathway. To carify this mechanism of Gualouguizhi Granule, we intend to develop ischemia reperfusion injury (MCAO) model and NMDA induced neuronal excitability toxicity model, observe the effect of Gualouguizhi Granule combining with MRI multimodal imaging technology. Meanwhile, intend to illustrate the mechanism of Gualouguizhi Granule from calcium signal transduction pathway, and analyse the relation between protein related to calcium signal transduction pathway and neuronal apoptosis. Then use siRNA to validate the target influenced by Gualouguizhi Granule. This study is based on the “holistic concept” of TCM and previous proteomic research with whole characteristic. It will make a new important theory complement to “whole regulation” of Chinese medicine anti- ischemic stroke mechanism.

中医药治疗脑卒中及其后遗症是常用治疗手段，然而其作用机制及靶点不明制约着中医药的发展。经方栝楼桂枝汤临床上用于脑卒中后康复有独特疗效。前期研究表明该方具有显著抗大鼠脑缺血再灌注损伤（MCAO）后肢体痉挛的作用，明显抑制神经元凋亡，显著降低细胞内Ca2+浓度，上调钙信号转导通路中多个关键分子的表达，提示该方可能通过调控细胞内钙稳态和神经递质平衡，从而激活钙信号转导通路、抑制神经元凋亡治疗缺血性卒中后肢体痉挛。本研究拟建立MCAO模型及NMDA诱导神经元兴奋性毒性损伤模型，结合MRI多模态成像技术观察药物抗大鼠MCAO后肢体痉挛及兴奋性毒性作用，并从钙信号转导通路阐释栝楼桂枝颗粒的作用机制，分析关键信号分子改变与神经元细胞凋亡之间的相关性，初步筛选出栝楼桂枝颗粒可能作用的分子靶点并进行反向验证。本研究符合中医“整体观念”和中药“整体调节”的特点，将为中药复方治疗缺血性卒中提供新的理论依据。

栝楼桂枝颗粒组方来源于张仲景《金匮要略》卷上，临床研究表明栝楼桂枝颗粒对缺血性脑卒中疗效确切，明显改善患者运动功能障碍及活动能力。前期研究表明该方具有显著抗大鼠脑缺血再灌注损伤（MCAO）后肢体痉挛的作用，明显抑制神经元凋亡，显著降低细胞内Ca2+浓度，上调钙信号转导通路中多个关键分子的表达。本项目在此基础上，以体内与体外实验研究相结合发现栝楼桂枝颗粒改善MCAO大鼠神经缺损、大鼠前爪抓握能力及肌力情况，减小脑梗死面积，改善脑组织病理损伤、减少尼氏小体的丢失，保护细胞器的形态，减少细胞的损伤，抑制神经元凋亡等作用。并且可降低细胞内Ca2+、减少兴奋性神经递质的含量，提示其抗缺血性脑卒中的作用机制与调控Ca2+相关，进一步从mRNA、蛋白质等层次揭示栝楼桂枝颗粒对细胞凋亡和钙信号转导相关通路，包括Ca2+/CaMKⅡ/CREB 信号通路、PI3K/Akt通路的调控机制。结果显示栝楼桂枝颗粒具有调控Akt、pAkt(Ser473)、PI3K(p85)、PI3K(P110α)、PDK1、pPDK1、PTEN、pPTEN、CREB、CaMKⅡ、CaM 等相关信号通路上的关键分子靶点的作用，初步阐明了其抗缺血性脑卒中的作用机制。本项目在中医“整体观念”理论指导下采用现代生命科学的技术手段揭示了复方中药多成分、多途径、多靶点的特点。尽管研究仍有待进一步深入，但已获得的研究结果将为栝楼桂枝颗粒在临床上更广泛和安全地应用提供科学依据。