Unraveling the mechanisms of Tou-Tong-Xin-1-Hao granules in treating migraine: A multi-modal study integrating microbiome, metabolomics, and network pharmacology, and experimental validation

Migraine is a highly prevalent and disabling primary headache disorder that imposes a severe socioeconomic burden. Tou-Tong-Xin-1-Hao (XYH), an “acrid-warm” traditional Chinese medicine (TCM) formula characterized by its ability to dispel cold, relieve pain, and resolve blood stasis, has demonstrated remarkable clinical efficacy and gained widespread recognition among migraine patients. In this study, the chemical constituents of XYH were characterized by ultra-high-performance liquid chromatography–quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) using an ethanol extract prepared from the 21 constituent medicinal herbs. Through this analysis, representative compounds including adenosine, quercetin, ononin, and others were identified. For the in vivo pharmacological experiments, XYH was administered as a granular formulation prepared from commercially standardized single-herb extract granules. A multi-modal approach integrating 16S rRNA microbiome sequencing, behavioral tests, Nissl staining, immunofluorescence, untargeted metabolomics, network pharmacology, and molecular docking was employed to explore its anti-migraine effects and potential underlying mechanisms.

In vivo experiments demonstrated that XYH prolonged pain response latencies and preserved neuronal integrity in the cortex and hippocampus. Furthermore, immunofluorescence assays demonstrated its anti-neuroinflammatory effects. Mechanistically, metabolomic analysis indicated that XYH modulated TCA cycle-related metabolic alterations and was associated with changes in cerebral energy metabolism. In parallel, 16S rRNA sequencing identified changes in gut microbiota composition, including increased abundance of bacteria associated with SCFA production. Correlative analyses suggested a potential gut–brain interaction linking microbiota alterations with central metabolic-inflammatory phenotypes. Network pharmacology identified representative candidate compounds (quercetin, rutin, kaempferol, and luteolin), predicting their potential migraine-related targets and potential signaling pathways. Molecular docking further revealed favorable theoretical binding affinities between these compounds and predicted target proteins.

Collectively, XYH exhibits anti-migraine effects through a “multi-component, multi-target, and multi-pathway” approach, which may involve the modulation of neuroinflammation and energy metabolism. The present findings provide preliminary experimental evidence supporting its therapeutic potential, as well as new perspectives for the modern research of traditional Chinese medicine targeting the “gut-brain-energy metabolism” axis.