基于NMMHCⅡA-TLR4相互作用探讨麦冬皂苷防治急性肺损伤的机制研究

This study is based on the pathogenesis of acute lung injury (ALI) in Chinese and western medicine. According to the previous experimental results that non-muscle myosin heavy chain 9 (NMMHCⅡA), which was identified as the potential protein target of ruscogenin, interacted with toll-like receptor 4 (TLR4) in ALI models, we proposed a novel study hypothesis that ophiopogonin might exert anti-hyperpermeability activity via modulating NMMHCⅡA-TLR4 interaction so as to modulate related signaling pathways downstream in LPS-induced ALI, based on the critical pathological feature of ALI -- endothelial barrier dysfunction. We would comprehensively use molecular biological techniques (inhibitors, RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR）technology and gene overexpression etc), and various models including endothelial-targeted NMMHC IIA conditional knockout mice and endothelial cells. To find out the key point in which functional domain of NMMHC IIA-TLR4 interaction mediates the pathomechanism. We expect to elucidate the exact molecular mechanism of ophiopogonin, for the mediation of NMMHC IIA and TLR4 interaction, so as to provide some evidence for their further clinical application. This research would not only enrich the pathological mechanism of ALI, but also provide candidate molecules or leading compounds for the protection of endothelial barrier function. On the other hand, new data would be accumulated for enriching the scientific connotation of “Tongtiaoshuidao (dredging and regulating water passage)” and some new clues for drug development for acute lung injury.

本项目基于急性肺损伤（ALI）发生的中西医病机，结合前期发现滋阴润肺中药麦冬的皂苷类成分特异性结合蛋白非肌肉肌球蛋白（NMMHCⅡA）与Toll样受体4（TLR4）相互作用可能参与ALI的实验证据，提出“麦冬皂苷可能通过干预NMMHC IIA与TLR4 相互作用，调节下游信号通路，保护肺内皮屏障，改善脂多糖（LPS）诱导的急性肺损伤”的科学假说，拟综合利用抑制剂阻断、RNA干扰、CRISPR敲除及过表达技术，及内皮条件性敲除小鼠模型，进一步确证NMMHCIIA与TLR4相互作用在LPS损伤的肺血管内皮屏障中的作用，解析二者相互作用介导下游信号通路的关键位点或功能结构域，继而深入阐释麦冬皂苷干预NMMHC IIA与TLR4相互作用，改善ALI的分子机制，为临床防治ALI提供可能的新靶点和先导化合物，为丰富ALI的病理机制和滋阴润肺通调水道的科学内涵，扩展麦冬皂苷的临床应用提供参考依据。

本项目结合急性肺损伤的中西医病机和国际前沿进展，综合应用基因敲除技术、RNA干扰、微量热涌动、邻位链接技术、计算机虚拟分析、CRISPR、基因突变技术等多学科技术方法，成功构建了NMMHC IIA内皮敲除/敲低小鼠模型，并从分子靶点、细胞水平、蛋白相互作用、信号转导通路、整体效应等多种层次角度，确证了MMHC IIA与TLR4相互作用参与调节LPS诱导的肺内皮屏障功能障碍。并且本项目识别出NMMHC IIA与TLR4结合的关键功能域，揭示LPS可诱导NMMHC IIA与TLR4解离，激活TLR4下游信号通路，Src激酶磷酸化增加，VE-cadherin含量减少，阐明了内皮屏障破坏的新病理机制，提示NMMHC IIA是调节血管稳态的重要分子，参与脓毒血症和细颗粒物诱导的肺损伤。此外，本研究证实麦冬皂苷类成分RUS通过直接作用于NMMHC IIA氨基端，干预NMMHC IIA与TLR4 相互作用，调节下游信号通路，保护肺内皮屏障，从而改善脂多糖诱导的急性肺损伤。上述研究成果进一步丰富了急性肺损伤形成的病理机制和中医滋阴润肺的科学内涵，阐释了麦冬皂苷防治肺损伤的作用靶点和特色，为麦冬的临床应用提供参考依据。本研究为探讨中药活性成分的原创机制和中药药理研究提供一定思路和方法学借鉴和研究范例；为细菌感染、细颗粒物、脓毒症等引起的肺损伤防治提供潜在的新靶点及先导化合物，为内皮功能障碍相关疾病的防治提供可能的新线索或新策略，为后续研制具有自主知识产权的中药新药奠定基础。通过本项目的实施，共发表基金标注论文8篇，其中SCI论文5篇，包括1区期刊APSB(IF=11.413) 1篇，APS (IF=6.150) 1篇，Biomed Pharmacother (IF=6.529) 2篇及JPP (IF=3.765) 1篇；申请并授权专利1项；培养青年骨干2名，博士生1名，硕士研究生7名。