基于能量代谢通路MCU蛋白的“附子无干姜不热”的生物学机制研究

Radix Aconite Lateralis Preparata and Rhizome Zingiberis are both pungent in taste and hot in nature. As a common clinical medicine pair from ancient time to nowadays, they are one of the most typical representatives reflecting the very essence of the theory of Chinese material medica compatibility. In the previous researches, the objective truth that “Radix Aconite Lateralis Preparata not exhibiting its hot property without Rhizome Zingiberis” has been proved in animalsby energy metabolism of mitochondria. However, the key mechanism regulating the energy metabolism remains poorly understood. It has been demonstrated in the latest researches that mitochndrial calcium uniporter (MCU) is the key protein mediating the energy metabolism and dominantly regulating the dynamic equilibrium of energy information in mitochondria. And based on this phenomenon, this project proposes a hypothesis that the combination of Radix Aconite Lateralis Preparata and Rhizome Zingiberis exhibits the “hot property” by modulating the MCU signaling pathway. Beginning with molecular pathway of energy metabolism mediated by MCU, we employ the technique of bio-thermodynamics and metabolomics. And with molecular biology technique such as high-content screening mitochondria tomography, the target of reaction from the combination of Radix Aconite Lateralis Preparata and Rhizome Zingiberis to energy metabolism in mitochondria and biological information characteristics of substance and energy in the relevant pathways will be studied systematically. Besides, we will illuminate the inner relation and biological mechanism of both chemical substance and energy in the Radix Aconite Lateralis Preparata and Rhizome Zingiberis to provide reference for elucidating the scientific essence of “Radix Aconite Lateralis Preparata not exhibiting its hot property without Rhizome Zingiberis”. Finally, this project will provide theoretical fundament for the further acquaintance of the compatibility theory of Chinese material medica and have a critical significance for the guidance of clinic practice of traditional Chinese medicine.

附子与干姜味辛性热，是古今临床常用药对，也是最能反映中药配伍理论精髓的代表之一。在前期研究中，课题组从线粒体能量代谢角度证实了“附子无干姜不热”的客观真实性，但其调节能量代谢的关键机制尚不清楚。最新研究认为：线粒体钙离子单向转运体（MCU）是介导能量代谢的关键蛋白，主导并调节了线粒体能量信息的动态平衡。为此，本项目提出附子配干姜调控MCU蛋白信号通路是表达其“热性”关键机制的工作假说。拟以MCU介导的能量代谢分子通路为切入点，采用生物热动力学与代谢组学技术，结合以高内涵线粒体断层成像为主要手段的分子生物学技术，系统探讨附子配干姜对线粒体能量代谢的作用靶标及相关分子通路的物质、能量的生物学变化信息，阐明附子配干姜的化学物质-能量相关的内在联系及其生物学机制，为阐释“附子无干姜不热”的科学内涵提供参考。通过本项目研究，可为深入认识传统中药配伍理论提供理论依据，对指导中医药临床实践具有重要意义。

附子配伍干姜是古今临床常用药对，也是最能反映中药配伍理论精髓的代表之一。课题组前期研究已证实了“附子无干姜不热”的客观真实性。但是，附子配伍干姜化学物质与能量代谢的内在联系及生物学机制尚不明确。为此，本项目以附子、干姜及其配伍水煎液/效应组分为研究对象，以心力衰竭/心阳虚证大鼠为研究载体，从线粒体能量代谢角度，结合分析化学、药理药效学、代谢组学、网络药理学和分子生物学等多学科技术，围绕线粒体钙离子单向转运体（MCU）介导的线粒体能量信息动态平衡，系统考察了附子、干姜及其配伍水煎液/效应组分对线粒体能量代谢的作用靶标及相关分子通路物质和能量的生物学变化信息。从物质基础层面，通过主要成分含量测定、化学指纹图谱分析和化学组分鉴定，考察了附子配伍干姜主要成分的变化规律和化学成分信息；从药理药效学层面，明确了附子配伍干姜治疗心力衰竭/心阳虚证的量效关系，且证实了附子发挥量效关系的主要效应成分为去甲猪毛菜碱和去甲乌药碱等水溶性生物碱，而干姜的主要效应成分为6-姜酚；从作用机制层面，采用分子生物学方法，考察了不同剂量附子、干姜及其配伍水煎液/效应组分通过调控MCU能量代谢通路、AMPK-UCP2线粒体能量代谢通路、Prx3/SOD2线粒体氧化应激通路、Sirt1/PGC-1α线粒体合成通路、PPARα/PGC-1α/Sirt3线粒体能量代谢通路、LKB1/AMPK/Sirt1线粒体能量代谢通路以及PDH、MDH、NNT等三羧酸循环关键酶发挥治疗心力衰竭/心阳虚证的作用，阐明了附子配伍干姜治疗心力衰竭/心阳虚证的生物学机制；采用代谢组学与网络药理学方法，探究了附子配伍干姜治疗心力衰竭/心阳虚证的特征代谢标志物、特征作用靶点及信号网络，进一步验证了附子、干姜及其配伍水煎液/效应组分治疗心力衰竭/心阳虚证的物质基础及生物学机制。本项目以附子配伍干姜改善线粒体能量代谢为切入点，将“附子无干姜不热”客观真实存在的宏观效应深入到物质基础及作用机制层次，多方位阐释了附子、干姜及其配伍水煎液/效应组分治疗心力衰竭/心阳虚证的科学内涵，明确了附子配伍干姜化学物质与能量相关的内在联系，诠释附子配伍干姜调节MCU介导的线粒体钙通路的关键生物学机制。通过揭示“附子无干姜不热”的“热性”本质特征，有助于诠释中药药性理论的现代科学内涵，为深入认识传统中药配伍理论提供理论依据与直接指导。