Antipyretic effects of Xiangqin Jiere granules on febrile young rats revealed by combining pharmacodynamics, metabolomics, network pharmacology, molecular biology experiments and molecular docking strategies

Xiangqin Jiere granules (XQJRG) is a proprietary Chinese medicine treating children’s colds and fevers, but its mechanism of action is unclear. The aim of this study was to explore the antipyretic mechanisms of XQJRG based on pharmacodynamics, non-targeted metabolomics, network pharmacology, molecular biology experiments, molecular docking, and molecular dynamics (MD) simulation. Firstly, the yeast-induced fever model was constructed in young rats to study antipyretic effect of XQJRG. Metabolomics and network pharmacology studies were performed to identify the key compounds, targets and pathways involved in the antipyretic of XQJRG. Subsequently, MetScape was used to jointly analyze targets from network pharmacology and metabolites from metabolomics. Finally, the key targets were validated by enzyme-linked immunosorbent assay (ELISA), and the affinity and stability of key ingredient and targets were evaluated by molecular docking and MD simulation. The animal experimental results showed that after XQJRG treatment, body temperature of febrile rats was significantly reduced, 13 metabolites were significantly modulated, and pathways of differential metabolite enrichment were mainly related to amino acid and lipid metabolism. Network pharmacology results indicated that quercetin and kaempferol were the key active components of XQJRG, <i>TNF</i>, <i>AKT1</i>, <i>IL6</i>, <i>IL1B</i> and <i>PTGS2</i> were core targets. ELISA confirmed that XQJRG significantly reduced the plasma concentrations of IL-1β, IL-6, and TNF-α, and the hypothalamic concentrations of COX-2 and PGE2. Molecular docking demonstrated that the binding energies of kaempferol to the core targets were all below −5.0 kcal/mol. MD simulation results showed that the binding free energies of TNF-kaempferol, IL6-kaempferol, IL1B-kaempferol and PTGS2-kaempferol were −87.86 kcal/mol, −70.41 kcal/mol, −69.95 kcal/mol and −106.67 kcal/mol, respectively. In conclusion, XQJRG has antipyretic effects on yeast-induced fever in young rats, and its antipyretic mechanisms may be related to the inhibition of peripheral pyrogenic cytokines release by constituents such as kaempferol, the reduction of hypothalamic fever mediator production, and the amelioration of disturbances in amino acid and lipid metabolism.

香芩解热颗粒（Xiangqin Jiere granules，XQJRG）是一款用于治疗儿童感冒发热的专利中药，但其具体作用机制尚未阐明。本研究旨在结合药效学、非靶向代谢组学、网络药理学、分子生物学实验、分子对接及分子动力学（Molecular Dynamics，MD）模拟技术，探究XQJRG的解热作用机制。
首先，构建酵母诱导的幼龄大鼠发热模型，以评估XQJRG的解热药效。通过代谢组学与网络药理学分析，筛选XQJRG发挥解热作用过程中的关键活性成分、作用靶点及信号通路。随后，采用MetScape软件对网络药理学所得靶点与代谢组学鉴定得到的差异代谢物进行联合分析。最后，通过酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）验证关键靶点，并通过分子对接与MD模拟评估关键活性成分与靶点的结合亲和力及稳定性。
动物实验结果表明，经XQJRG干预后，发热大鼠的体温显著降低；共筛选得到13种差异代谢物，差异代谢物的富集通路主要与氨基酸代谢及脂质代谢相关。网络药理学分析结果显示，槲皮素（quercetin）与山柰酚（kaempferol）为XQJRG的核心活性成分，肿瘤坏死因子（Tumor Necrosis Factor，TNF）、AKT1、白细胞介素6（Interleukin 6，IL6）、白细胞介素1β（Interleukin 1β，IL1B）及前列腺素内过氧化物合酶2（Prostaglandin-Endoperoxide Synthase 2，PTGS2）为核心靶点。ELISA实验证实，XQJRG可显著降低发热大鼠血浆中IL-1β、IL-6及TNF-α的浓度，同时下调下丘脑组织中环氧化酶2（Cyclooxygenase 2，COX-2）与前列腺素E2（Prostaglandin E2，PGE2）的表达水平。分子对接结果显示，山柰酚与各核心靶点的结合能均低于−5.0 kcal/mol。MD模拟结果显示，山柰酚与TNF、IL6、IL1B及PTGS2的结合自由能分别为−87.86 kcal/mol、−70.41 kcal/mol、−69.95 kcal/mol及−106.67 kcal/mol。
综上，XQJRG对酵母诱导的幼龄大鼠发热模型具有显著解热作用，其解热机制可能与山柰酚等活性成分抑制外周致热细胞因子释放、减少下丘脑发热介质生成，以及改善氨基酸代谢与脂质代谢紊乱密切相关。