Danshen, the dried root of Salvia miltiorrhiza, one of the most investigated medicinal plants with well-defined phytochemical constituents, has shown prominent clinical outcomes for antioxidant, anti-inflammatory, and anticoagulant activities to attain vascular protection and additional benefits for cancer therapy. More recently, activation of neutrophil and excessive formation of neutrophil extracellular traps (NETs) have been observed in pathological conditions of metastatic cancers; thus, we hypothesized that suppression of NETs could account for an essential cellular event underlying Danshen-mediated reduction of the incidence of metastasis. Using an experimental pulmonary metastases model of red fluorescent protein- (RFP-) labeled gastric cancer cells in combination with macroscopic ex vivo live-imaging system, our data indicated that Danshen impaired the fluorescent intensity and quantity of metastatic nodules. Moreover, Danshen could prevent neutrophil trafficking to the metastatic sites with decreased plasma levels of neutrophil elastase (NE) and procoagulant potential featured by fibrinogen. We further established phorbol 12-myristate 13-acetate- (PMA-) induced NET formation of human neutrophils and screened representative active compounds derived from the hydrophilic and hydrophobic fractions of Danshen using qualitative and quantitative methods. As a result, we found that salvianolic acid B (Sal B) and 15,16-dihydrotanshinone I (DHT I) exhibited superior inhibitory activities on NET formation and significantly attenuated the levels of citrullinated histone H3 (citH3), a biomarker for NET formation. Multitarget biochemical assays demonstrated that Sal B and DHT I distinctly modulated the enzymatic cascade involved in NET formation. Sal B and DHT I could disrupt NET formation at the earlier stage by blocking the activities of myeloperoxidase (MPO) and NADPH oxidase (NOX), respectively. Lastly, combining treatment of Sal B and DHT I under subED50 doses displayed remarkable synergism effect on NET inhibition. Altogether, these data provide insight into how promiscuous compounds from herbal medicine can be effectively targeted NETs towards hematogenous metastasis of certain tumors.

Upon interaction, neutrophils can potentially release neutrophil extracellular traps (NETs) on the surface of an implanted electrospun template, which may be a significant preconditioning event for implantable biomaterials of yet unknown consequences. In this study, we investigated the potential of polydioxanone templates as a delivery vehicle for Cl-amidine, an inhibitor of peptidyl arginase deiminase 4 (PAD4), and if drug elution could attenuate PAD4-mediated NETosis in the vicinity of implanted templates. Electrospun polydioxanone templates were fabricated with distinct architectures, small diameter (0.4 μm) or large diameter (1.8 μm) fibers, and incorporated with 0-5 mg/mL Cl-amidine to examine dose-dependent effects. Acute neutrophil-template interactions were evaluated in vitro with freshly isolated human neutrophils and in vivo with a rat subcutaneous implant model. The in vitro results suggest large diameter templates with 0 mg/mL Cl-amidine significantly attenuate NETosis compared to small diameter templates. As the drug concentration increased, NETosis was significantly decreased on small diameter templates in a dose-dependent manner. The opposite was observed for large diameter templates, indicating multiple mechanisms of NETosis may be regulating neutrophil template preconditioning. Similar results were observed in vivo, verifying local NETosis inhibition by Cl-amidine eluting templates in a physiological environment. Importantly, large diameter templates with Cl-amidine enhanced neutrophil invasion and survival, supporting the potential for long-term modulation of tissue integration and regeneration. This preliminary study demonstrates a novel delivery vehicle for Cl-amidine that can be used to regulate acute NETosis as the potential critical link between the innate immune response, inflammation, and template-guided tissue regeneration.