Effects and Action Mechanism of Huoxue Tongluo Formula on the Formation of Neutrophil Extracellular Traps

Excessive infiltration and uncontrolled activation of neutrophil extracellular traps (NETs) are likely to destroy normal tissue architecture and cause uncontrolled inflammation. The present research attempted to screen potential signaling pathways of Huoxue Tongluo Formula (HXTLF) affecting the formation of NETs using network pharmacology technique. Active chemical components of HXTLF and therapeutic targets related to vasculitis were screened, and a chemical components-targets network diagram of HXTLF was constructed by Cytoscape. Finally, the inhibitory effect and mechanism of HXTLF on the formation of NETs were explored in vitro using LPS-induced NETs. Immunofluorescence and Western blot were conducted to determine the protein fluorescence intensity and relative expression. The experimental results illustrated that HXTLF mediated the expression levels of H3Cit and myeloperoxidase (MPO) protein in neutrophils activated by LPS, inhibited NETs formation, and reduced the concentration of interleukin- (IL-) 1β, a proinflammatory factor in cells. Additionally, we activated and inhibited the AKT1 signaling pathway using the corresponding activator and inhibitor to explore the regulatory mechanism of HXTLF on AKT1 and other molecules in the treatment of vasculitis. The results demonstrated that HXTLF could inhibit the phosphorylation of AKT1, IKK, and NF-κB proteins, inhibit NETs formation, and reduce IL-1β concentration, indicating that AKT1 exerts a vital role in the treatment of vasculitis after HXTLF administration. The current study initially revealed the pharmacological mechanism of HXTLF for vasculitis management using network pharmacology techniques and tests in vitro, which is expected to provide important theoretical basis for elucidating the molecular mechanism of HXTLF and promoting its clinical application.