Multi-Pathway and Multi-Gene Molecular Mechanisms of Huoxue San in Fracture Healing and Blood Stasis

INTRODUCTION: Huoxue San (HXS) is a traditional Chinese medicinal formulation widely used to treat bone fractures and blood stasis. Comprising seven herbs-Siphonostegia chinensis Benth, Kochia scoparia (L.) Schrad, Scutellaria barbata D.Don, Polygonum cuspidatum Sieb. et Zucc, Arisaema erubescens (Wall.) Schott, Phellodendron chinense Schneid, and Eupolyphaga sinensis Walker-HXS has been administered at Nanjing Chinese Medicine Hospital for over 50 years. It is effective in promoting fracture healing, supporting soft tissue repair, and rarely causing adverse reactions such as skin allergies. The present study aimed to elucidate the molecular mechanisms underlying HXS's therapeutic effects. METHODS: Ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF MS) was used to identify HXS components absorbed into the bloodstream. Network pharmacology, molecular docking, and molecular dynamics simulations were then conducted to explore the active ingredients and their regulatory mechanisms in fracture healing and blood stasis. RESULTS: Transdermal absorption tests identified 20 active compounds from HXS. Network pharmacology analyses using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform highlighted vanillic acid, demethyleneberberine, palmatine hydrochloride, luteolin, apigenin, and wogonin as key active ingredients. Molecular dynamics simulations further validated the stability, conformational changes, and interactions of these compounds with their target proteins. DISCUSSION: Analysis of the transdermal absorption samples revealed 291 potential active targets for HXS in treating fractures and blood stasis, of which 159 were common to both conditions. Protein-protein interaction (PPI) network analysis identified core targets including AKT1, ALB, EGFR, STAT3, and CTNNB1. Molecular docking confirmed strong binding interactions between HXS compounds and these core targets, while molecular dynamics simulations validated the stability and mechanistic plausibility of these interactions. CONCLUSION: This study provides a systematic elucidation of HXS's molecular mechanisms in fracture healing and blood stasis. Identification of active compounds, core targets, and their interactions offers a scientific basis for the therapeutic effects of HXS and supports the rational development of herbal-medicine-based interventions for fracture management and blood stasis treatment.