基于“肝肾同源”理论和代谢组学技术的二至丸实现肝肾同治的作用机制及效应物质基础研究

Erzhiwan, the best formula of “Qingshangbuxia”, consisted of Fructus Ligustri Lucidi and Herba Ecliptae for liver and kidney, which is the commonly used formula for the remedy of liver and kidney at the same time in traditional Chinese medicine according to the theory of “liver and kidney from the same source”. Early completion of the National Youth Fund has also indicated that it can regulate liver metabolism and help the recovery of liver and kidney function. Therefore, we believe there is a certain link of metabolic network between liver and kidney, and Erzhiwan could improve liver and kidney function by modulating the metabolic network for the remedy of liver and kidney at the same time. For this point of view, these work will be carried out as followings: Based on the injury model of liver and kidney of rats with deficiency of yin and the injury model of liver and kidney cell of rats, metabolomics technology will be applied to systematically study the metabolite spectrum of effects, significant metabolites and metabolic pathway by the intervention of Erzhiwan, and establish the metabolic regulatory networks between the liver and kidney. Based on the modern chromatographic separation techniques guided by adopting targeted quantitative significant metabolites and quality assessment techniques, the ingredient for the remedy of liver and kidney will be screened and separated from Erzhiwan; Network pharmacology method will be used to build "effective ingredients-targets-pathways" model to explore targets and ways to achieve the remedy of liver and kidney at the same time. The aim of this project is to clarify the mechanism of action and the material basis of Erzhiwan for the role of Erzhiwan to the liver and kidney, the regulation to liver and kidney metabolism, and the achievement of remedy of liver and kidney at the same time, and to provide the exploration of ideas and methods for the confirmation of the scientificity of clinical medicine in traditional Chinese medicine.

清上补下第一方二至丸由归肝肾经的女贞子和墨旱莲组成，是中医基于“肝肾同源”理论的常用肝肾同治方剂。前期完成国家青年基金亦表明其能够调控肝肾代谢、恢复肝肾功能。因此，我们认为肝肾之间存在一定的代谢网络联系，二至丸可通过调控该代谢网络改善肝肾功能、实现肝肾同治。针对此，拟开展下列研究工作：应用代谢组学技术对二至丸干预阴虚型肝肾损伤动物模型及非接触共培养的肝肾细胞损伤模型代谢物谱的效应、显著性代谢物及代谢通路进行系统研究，建立二至丸干预下肝肾之间的代谢调控网络；采用以靶向定量的显著性代谢物为导向的分离技术和现代色谱分析质量评价技术，筛选二至丸实现肝肾同治的效应成分（群）；基于网络药理学构建“效应成分-靶标-通路”模型，探讨二至丸实现肝肾同治效应成分的作用靶点及途径。以期科学阐明二至丸作用于肝肾、调控肝肾代谢、实现肝肾同治的作用机制及物质基础，为证实中医临床用药的科学性提供思路与方法等的探索。

由归肝肾经的女贞子和墨旱莲组成的二至丸，是中医基于“肝肾同源”理论的肝肾同治经典方剂。本项目采用色谱质谱联用技术已较清晰地完成了二至丸及其组方药材女贞子的体内外成分分析和质控研究；基于RPLC-Q/TOF-MS和 HILIC-Q/TOF-MS模式下的宽范围代谢组学并结合网络分析系统讨论二至丸治疗肝肾阴虚的可能作用机制及肝肾之间的代谢联系，通过整合差异性代谢物和代谢通路，发现五种核心代谢物，即花生四烯酸，L-精氨酸，睾丸激素，牛磺酸和氧戊二酸，可能与二至丸治疗肝肾阴虚证相关，建立以衰老基因为起点的最短通路图，发现衰老基因如CAV1和ACO1可能是治疗肝肾阴虚证并解释其病理机制的潜在靶标。借助网络药理学的“分子（药物）-靶点-通路”多层次网络研究分析二至丸保护肝肾的药效活性成分和可能的分子网络机制，共有89个不同的活性成分通过调控不同的靶点，作用于不同的通路，协同配伍从而对肝肾起到保护作用、体现了肝肾同治的作用机制。为探索二至丸抗衰老作用机制及肝肾细胞之间的可能联系，借助非靶向代谢组学对非接触共培养体系中衰老的肾细胞进行代谢组学研究，结果表明主要与TCA循环和糖酵解有关，进一步采用靶向代谢组学准确定量两个代谢通路，所测定的代谢物如丙酮酸、琥珀酸、苹果酸、α-酮戊二酸、柠檬酸和葡萄糖的含量都有显著回调，明确二至丸可通过调控TCA循环和糖酵解通路而实现抗衰老的作用。采用靶向细胞代谢组学结合多元统计分析和AHP分析对二至丸以及不同极性部位含药血清的抗衰老作用进行评价，结果显示石油醚部位其次是环烯醚萜苷类部位具有着更好的抗衰老作用，此前的二至丸及不同极性部位化学成分及入血成分定性分析研究结果也表明石油醚部位中种类最多的是三萜类化合物（39.3%）和环烯醚萜苷类（16.4%），响应强度较高的成分为三萜类，提示发挥药效的成分群可能为三萜类和环烯醚萜苷类。上述研究揭示了二至丸在“肝肾同源”理论指导下实现肝肾同治的作用机制及物质基础，为证实中医临床用药的科学性提供了思路与方法等的探索。