生脉散有效成分群调控AMPK/NMMHCIIA/14-3-3防治心肌缺血再灌注损伤多靶点整合机制

In order to elucidate the complicated pathogenesis of myocardial ischemia-reperfusion injury and solve the major problem that characteristic of multi-components, multi-targets and multi-pathways mechanism of traditional Chinese medicine is difficult to be explored in the modern studies, we hypothesized that "Effective components derived from Shengmai-san have a synergistic integrated effect on improving myocardial ischemia-reperfusion injury via regulating multi-targets including AMP-activated protein kinase-alpha (AMPK-alpha), non-muscle myosin heavy chain IIA (NMMHC IIA) and 14-3-3-theta, respectively, then modulating the related signal transduction pathways, ameliorating oxidative stress, inflammation and energy metabolism, and ultimately inhibit myocardial apoptosis and myocardial ischemia-reperfusion injury". In the present study, we will comprehensively use molecular biology techniques (RNA interference, clustered regularly interspaced short palindromic repeats (CRISPR） technology and gene overexpression etc), imaging technology (confocal laser, PET etc) and modern analytical techniques (computer simulation analysis and microcalorimetry swimming technology etc) combining using whole animal and cell models to explore the integrated mechanism of effective components from Shengmai-san in preventing and treating myocardial ischemia injury from the view of molecular targets, signaling networks, biochemical indexes and pharmacological effects. Our findings will provide a better understanding of the characteristics and signaling pathways of Shengmai-san active components in prevention and treatment of myocardial ischemic disease. Our results will also provide some new insights and methodology reference for exploring pharmacological mechanism of traditional Chinese medicine.

课题组针对心肌缺血再灌注损伤病理机制的复杂性及中药复方多成分多靶点多效应通路的作用特点，结合国际前沿进展和前期基础，提出本项目的研究假说：“生脉散有效成分群通过各自调控AMPK/NMMHC IIA/14-3-3多靶点，调节相关信号转导通路，改善能量代谢，炎症反应及氧化应激等多途径，抑制心肌细胞凋亡，协同整合发挥改善心肌缺血再灌注损伤的效应”，将综合应用RNA干扰，CRISPR技术，基因过表达等分子生物学技术，激光共聚焦、PET等影像学技术，计算机虚拟分析、微量热泳动技术等分析技术，采用整体动物和体外细胞模型，宏观与微观相结合，从分子靶点、信号网络、生化指标、药理效应等多层次多途径，探索发现生脉散有效成分群协同整合防治心肌缺血的分子机制及作用特性，为进一步揭示该方防治心肌缺血性疾病的作用特色与信号途径，指导其临床合理应用提供参考依据，为探讨中药复方活性成分群的多靶点多途径研究提供方法借鉴

课题组针对心肌缺血再灌注损伤病理机制的复杂性及中药复方多成分多靶点多效应通路的作用特点，结合国际前沿进展和前期基础，综合应用RNA干扰，CRISPR技术，基因过表达等分子生物学技术，激光共聚焦、PET等影像学技术，计算机虚拟分析、微量热泳动技术等分析技术，证实了本项目的研究假说：“生脉散有效成分群通过各自调控AMPK/NMMHC IIA/14-3-3多靶点，调节相关信号转导通路，改善能量代谢，炎症反应及氧化应激等多途径，抑制心肌细胞凋亡，协同整合发挥改善心肌缺血再灌注损伤的效应”，本研究采用整体动物和体外细胞模型，宏观与微观相结合，从分子靶点、信号网络、生化指标、药理效应等多层次多途径，阐释了AMPK/NMMHC IIA/14-3-3介导心肌细胞凋亡的关键功能，同时，证实了关键功能蛋白myosin IIA-actin相互作用途径在MI/R损伤中线粒体分裂诱导心肌凋亡中的病理功能；进一步探索发现了生脉散有效成分群协同整合防治心肌缺血的分子机制及作用特性，确证了介导生脉散活性成分群抑制MI/R诱导心肌细胞凋亡从而改善心肌损伤的关键功能蛋白，阐明了生脉散活性成分群通过调控各自关键功能蛋白及相关信号转导通路，人参皂苷Rb1能显著激活AMPKα，鲁斯可皂苷元对NMMHC II表达有显著的抑制作用，而五味子醇甲能显著增加14-3-3θ蛋白的表达，改善能量代谢、炎症反应及氧化应激等多个途径，抑制心肌细胞凋亡，发挥心肌保护效应的作用机制，为进一步揭示该方防治心肌缺血性疾病的作用特点，指导其临床更合理应用提供参考依据，为研究中药活性成分群的多靶点多途径作用机制提供方法学借鉴及相关疾病的防治提供新的线索，为进一步创制成分明确、机理清楚、质量可控、安全有效的新复方制剂提供了参考依据。.综上，经过四年的努力，已全面完成课题的预定目标，共发表SCI论文9篇，国内外会议交流多次，培养博士后1名，硕士研究生共5名。.