Metabolomics and bio-layer interferometry combined with molecular biology validation uncover the potential active components and mechanism of Guangtongxiao in alleviating anal fissure pain

Guangtongxiao (GTX) is a traditional Chinese medicine formulation used in the clinical management of anal fissure (AF). However, its active components and underlying mechanisms of action remain unclear. Therefore, this study aimed to investigate the potential active components and mechanisms underlying the pain-relieving effects of GTX in AF using serum pharmacochemistry, integrated metabolomics, network pharmacology, biolayer interferometry (BLI), and molecular biological validation. The analgesic and anti-inflammatory effects of GTX were evaluated in a rat model of AF. Our findings revealed that GTX significantly reduced pain and tissue inflammation in the animal model. Concurrently, chemical analysis of GTX identified 82 compounds, including five blood-absorbed components. Integrated analysis of network pharmacology and metabolomics were used to elucidate the key targets and signalling pathways. Integrated analysis revealed 15 core target proteins, including NF-κB, NLRP3, and TLR4. Metabolomics identified the glycerophospholipid pathway and dynorphin B as key regulatory elements. Then interactions between potential active components and target proteins were further validated using molecular docking and BLI, followed by further confirmation in microglial cells. Five blood-absorbed components (corydaline, albiflorin, astragaloside IV, cianidanol, and daucosterin) exhibited effective binding toward the inflammatory targets NLRP3 and TLR4. Molecular biological validation demonstrated that 5 Mix inhibit the TLR4/NF-κB/NLRP3 signalling pathway. In summary, GTX alleviated AF pain primarily by inhibiting the TLR4/NF-κB/NLRP3 inflammatory signalling axis via these five components. These findings offer a scientific foundation for the development of GTX-derived therapeutics and support the modernisation of complex traditional herbal formulae.