Notch1介导的心肌细胞重塑在乌头碱慢性心肌毒性中的作用

Aconitine, the main effective and toxic component of Aconitum L. herbs, has always raised toxicological concerns due to its cardiotoxicity. Our previous study has demonstrated that aconitine could produce a dose-response effect on Notch1 signaling pathway characterized by a low-dose stimulation and a high-dose inhibition in myocardial cell in a short time. However, few systematic research focuses on aconitine-induced chronic myocardial toxicity and related molecular toxicological mechanisms. In this project, we will employ the research approach integrating the disease and syndrome, in vitro and in vivo, effects and mechanisms, and build an animal model of disease, firstly to characterize the chronic toxic effects of aconitine on cardiomyocytes, and to analyze the effects on Notch1 signaling pathway and cardiomyocyte remodeling under the two models; Then, combining with in vitro study, to identify the role of Notch1-mediated cardiomyocyte remodeling in aconitine-induced chronic myocardial toxicity; Lastly to preform comparative analysis of molecular toxicological mechanisms induced by aconitine under the physiological and pathological condition and to analyze the role of Notch1-mediated cardiomyocyte remodeling in the transformation between aconitine-induced toxicity and efficacy. Overall, our study will the investigate the myocardial toxicity of Aconitine and the molecular mechanisms involved based on toxicological effect and molecular toxicological mechanism levels. The anticipated outcomes will be benefit for identification of the chronic myocardial toxicity of Aconitum L. herbs, and provide scientific advice for clinical rational administration of Aconitine.

乌头碱作为含乌头碱类中药的主要效-毒物质，其心脏毒性备受关注。前期研究发现，乌头碱短时间内可对心肌细胞内Notch1信号通路产生低剂量激活高剂量抑制效应。但目前，针对乌头碱慢性心肌毒性及相关分子毒理学机制研究较少。因此，本研究拟采用病症结合、体内与体外结合、效应与机制结合的研究方式，通过建立疾病动物模型，首先系统表征生理和病理状态下乌头碱的慢性心肌毒性效应，分析乌头碱暴露过程中Notch1信号通路、心肌细胞重塑相关指标的变化；继而结合体外试验，明确Notch1介导的心肌细胞重塑在乌头碱慢性心肌毒性中的作用；最后，对比分析生理与病理状态下相关分子机制的变化差异，解析Notch1介导的心肌细胞重塑在乌头碱“毒-效”关系转换中的作用。本项目从毒理学效应和分子毒理学机制两个层面探讨乌头碱的慢性心肌毒性作用及相关毒性机制，为含乌头碱类中药慢性心肌毒性的认识，以及指导其临床合理用药提供科学依据。

乌头碱作为含乌头碱类中药的主要效-毒物质，其心脏毒性备受关注。但目前，针对乌头碱慢性心肌毒性及相关分子毒理学机制研究较少。本研究利用多种心肌细胞，首次发现，低小剂量乌头碱诱发的Notch1信号及其介导的NICD-KDM5A-HCN4毒性信号通路，促进成熟心肌细胞起博电流（If）恢复；同时，低剂量乌头碱呈剂量依赖性减少心肌纤维蛋白（α-Actinin），上调多能干细胞标志分子（Oct4A、Sox2和c-Myc），心肌收缩能力逐渐降低，揭示了Notch1介导心肌重塑在“安全剂量”乌头碱慢性心肌毒性发生中分子毒理学机制。其次，本研究还发现，低剂量乌头碱重复给药可通过激活AMPK信号，促使心肌细胞代谢模式线粒体氧化磷酸化向糖酵解方向转移，底物选择从脂肪酸向葡萄糖转变，为快速搏动的心肌提供能量。从分子机制层面，本研究证实低剂量乌头碱激活的AMPK信号，可上调OPA1，促进线粒体融合，诱发线粒体蛋白酶复合物V（ATP合酶）变化（ATP5a1表达增加），参与心肌快速收缩供能，构建出乌头碱正向肌力作用关键药效信号——“AMPK-OPA1-ATP5A1”。通过动物实验，证实低小剂量乌头碱持续给药，可上调小鼠心肌NICD和OPA1蛋白表达。最后，基于上述毒性或药效关键信号通路，利用高内涵分析方法，模拟乌头碱诱导心肌细胞离子通道异常变化以及能量代谢调控的相关毒性通路-剂量反应曲线及其扰动情况，初步预测乌头碱人体安全剂量范围（低于5.6nM）。本研究不仅系统解析低小剂量乌头碱诱发Notch1介导心肌重塑相关分子机制，及其在乌头碱慢性心肌毒性中作用，还进一步明确乌头碱重复用药安全阈值，为乌头类中药临床合理用药和长期服用安全限量标准的制订提供科学依据。