Table_1_Dose-Dependent Variation of Synchronous Metabolites and Modules in a Yin/Yang Transformation Model of Appointed Ischemia Metabolic Networks.docx

AimChinese medicine Danhong injection (DHI) is an effective pharmaceutical preparation for treating cerebral infarction. Our previous study shows that DHI can remarkably reduce the ischemic stroke-induced infarct volume in a dose-dependent manner, but the pharmacological mechanism of the DHI dose-dependent relationship is not clear. Therefore, the dose-dependent efficacy of DHI on cerebral ischemia and the underlying mechanisms were further investigated in this study.
MethodsA middle cerebral artery occlusion (MCAO) model was established and the rats were randomly divided into six groups: sham, vehicle, DHI dose-1, DHI dose-2, DHI dose-3, and DHI dose-4. Forty-one metabolites in serum were selected as candidate biomarkers of efficacy phenotypes by the Agilent 1290 rapid-resolution liquid chromatography system coupled with the Agilent 6550 Q-TOF MS system. Then, the metabolic networks in each group were constructed using the Weighted Correlation Network analysis (WGCNA). Moreover, the Yang and Yin transformation of six patterns (which are defined by up- and downregulation of metabolites) and synchronous modules divided from a synchronous network were used to dynamically analyze the mechanism of the drug’s effectiveness.
ResultsThe neuroprotective effect of DHI has shown a dose-dependent manner, and the high-dose group (DH3 and DH4) effect is better. The entropy of the metabolic network and the Yin/Yang index both showed a consistent dose–response relationship. Seven dose-sensitive metabolites maintained constant inverse upregulation or downregulation in the four dose groups. Three synchronous modules for the DH1–DH4 full-course network were identified. Glycine, N-acetyl-L-glutamate, and tetrahydrofolate as a new emerging module appeared in DH2/DH3 and enriched in glutamine and glutamate metabolism-related pathways.
ConclusionThis study takes the DHI metabolic network as an example to provide a new method for the discovery of multiple targets related to pharmacological effects. Our results show that the three conservative allosteric module nodes, taurine, L-tyrosine, and L-leucine, may be one of the basic mechanisms of DHI in the treatment of cerebral infarction, and the other three new emerging module nodes glyoxylate, L-glutamate, and L-valine may participate in the glutamine and glutamate metabolism pathway to improve the efficacy of DHI.

中药丹红注射液（DHI）是一款用于治疗脑梗死的有效药物制剂。既往研究表明，DHI可通过剂量依赖性方式显著降低缺血性脑卒中诱导的脑梗死体积，但目前其剂量依赖性关系的药理学机制尚不明确。因此，本研究进一步探讨了DHI对脑缺血的剂量依赖性疗效及其潜在作用机制。
方法：构建大脑中动脉闭塞（Middle Cerebral Artery Occlusion, MCAO）模型，将大鼠随机分为6组：假手术组、溶剂对照组、DHI剂量1组、DHI剂量2组、DHI剂量3组及DHI剂量4组。采用安捷伦1290超高效液相色谱系统联用安捷伦6550 Q-TOF质谱系统，筛选得到41种血清代谢物作为疗效表型的候选生物标志物。随后，采用加权基因共表达网络分析（Weighted Correlation Network Analysis, WGCNA）构建各组的代谢网络。此外，基于代谢物上调与下调定义的6种模式的阴阳转化，以及从同步网络中划分得到的同步模块，用于动态解析药物的疗效机制。
结果：DHI的神经保护作用呈剂量依赖性，且高剂量组（DHI剂量3组、DHI剂量4组）疗效更优。代谢网络的熵值与阴阳指数均呈现出一致的剂量响应关系。7种剂量敏感性代谢物在4个剂量组中始终呈现反向的上调或下调表达模式。针对DHI剂量1组至剂量4组的全疗程网络，共鉴定得到3个同步模块。甘氨酸、N-乙酰-L-谷氨酸与四氢叶酸构成的新兴模块出现在DHI剂量2组/剂量3组中，并显著富集于谷氨酰胺与谷氨酸代谢相关通路。
结论：本研究以DHI代谢网络为范例，为发掘与药理学效应相关的多靶点提供了全新方法。研究结果表明，牛磺酸、L-酪氨酸与L-亮氨酸这3个保守变构模块节点，可能是DHI治疗脑梗死的核心机制之一；而乙醛酸、L-谷氨酸与L-缬氨酸这3个新兴模块节点，则可能通过参与谷氨酰胺与谷氨酸代谢通路，增强DHI的治疗疗效。