基于NMMHCⅡA-TF信号通路探讨麦冬皂苷防治血栓性疾病的新机制研究

In our previous study, we found ophiopogonins inhibited tissue factor (TF)activity and expression,decreased thrombosis,and firstly indentified that non-muscle myosin heavy chain 9 (NMMHCⅡA) was potential protein target of ophiopogonins. Basing on these findings, we propose a novel study hypothesis that ophiopogonins exert anti-thrombotic activities via binding non-muscle myosin heavy chain 9 (NMMHCⅡA) so as to modulate TF expression and related signal pathways in the present project. We try to explore the function of NMMHCⅡA in TF activity, expression and related signal pathway in endothelial cells and monocytes induced by inflammatory cytokines, hypoxia etc. We would observe the expression, phosphoralytion or possible binding protein and TF changes when its ATPase was inhibited by specific inhibitors or its expression was knocked down by RNAi, by various methods including western blot, immunofluoresence and coimmunoprecipation. Meanwhile, the modulation of representative ophiopogonins on NMMHCⅡA-TF pathway will be determined in vitro and verified in three thrombosis models, which was induced by inferior vena cava ligation, pulmonary embolism and middle cerebral artery occlusion. We will also analysize the interaction mode, and common character between ophipogonin saponins or other active components from Chinese medicines with similar effects and NMMHCⅡA by molecular docking,affinity capillary electrophoresis and ATPase activity assay. We expect to elucidate the molecular mechanism of ophiopogonins, for the treatment of thrombosis, so as to provide some evidence for their further clinical application. Our findings would also provide some new insights and multidisciplinary methodology for exploring pharmacological mechanism of traditional Chinese medicine. New data would be accumulated for enriching the scientific connotation of "ziyinhuoxue (nourishing yin and invigorating the blood)" and some new clues for drug development for the thrombotic disease.

本课题组在前期发现麦冬皂苷类成分下调组织因子（TF）活性及表达、抑制血栓形成，并识别非肌肉肌球蛋白（NMMHCⅡA）为其特异性结合的功能靶标基础上，提出"麦冬皂苷通过调节NMMHCⅡA影响下游TF表达及相关信号通路防治血栓形成"的研究假说。拟采用缺氧、炎症因子等病理因素活化的内皮细胞/单核细胞模型，并利用蛋白抑制剂和RNA干扰、蛋白印迹、免疫荧光、共沉淀等方法，考察NMMHCⅡA的表达、磷酸化及可能的相互作用蛋白，探讨其对TF表达及信号通路的影响及代表性麦冬皂苷成分的调节作用；并在静脉血栓、肺栓塞、脑血栓模型上加以验证；采用计算机虚拟分析、亲和毛细管电泳结合酶活性测定等方法，分析麦冬皂苷及类似功效中药有效成分与NMMHCⅡA相互作用模式位点的共性和特性，以期深入阐明麦冬防治血栓性疾病的分子机制；为体现中医药特色的中药药理研究提供思路和方法，为丰富滋阴活血治则防治血栓性疾病提供参考

本课题在大量前期工作基础上，结合国际前沿进展，提出“麦冬皂苷类成分通过调节NMMHCⅡA 影响下游组织因子（TF）表达及相关信号通路防治血栓形成”的研究假说，综合利用细胞分子生物学、免疫学、药理学、病理学等多学科交叉技术，在分子、细胞、组织与整体水平，进一步探讨NMMHCⅡA 在缺氧、炎症因子等病理因素诱导内皮细胞/单核细胞TF 表达途径中的功能，阐释麦冬皂苷通过调节NMMHCⅡA及下游信号通路抑制TF 途径防治血栓形成的分子机制；并探讨功效类似中药有效成分与NMMHCⅡA 相互作用特性。研究证实NMMHC IIA主要通过与TNFR2/HIF-1a相互作用，上调PI3K/Akt通路，下调NF-κB通路，抑制p65的核转录，从而抑制TF表达，并在DVT动物体内模型加以证实。并利用分子对接、亲和色谱、干扰、过表达和PLA等技术，通过计算机虚拟分析与体内外实验验证相结合的方法，识别确证麦冬皂苷类成分直接结合NMMHC IIA，调节NMMHC IIA与TNFR2/HIF-1a相互作用，影响下游信号通路，调节内皮细胞TF表达并抑制静脉血栓形成。同时证实NMMHC IIA 参与调节脂多糖诱导的单核细胞和肺损伤小鼠肺组织TF 表达，并发现NMMⅡA在局灶性脑缺血再灌注损伤模型小鼠脑组织表达增加，而NMMHC IIA抑制剂可明显降低肺部TF表达和纤维蛋白沉积，为进一步探讨NMMⅡ蛋白的病理功能提供实验依据。. 上述研究结果，为基于NMMHC IIA靶标发现防治血栓性疾病提供可能的先导化合物或重要候选药物提供参考线索，为深入阐明麦冬皂苷防治血栓性疾病的分子机制提供科学数据，为开展原创性强、体现中医药特色的中药药理作用机制研究提供一定思路和方法借鉴， 也为丰富滋阴活血的中医治则阐释其科学内涵提供一定参考依据。总体完成课题的预定目标，共发表论文14篇，其中10篇为SCI收载，国内外会议交流7人次，培养博士后1名，博士生2名，硕士生6名，并为进一步构建新的疾病动物模型奠定基础。