Ginsenoside Rg5 allosterically interacts with P2RY12 and ameliorates deep venous thrombosis by counteracting neutrophil NETosis and inflammatory response

BackgroundDeep venous thrombosis (DVT) highly occurs in patients with severe COVID-19 and probably accounted for their high mortality. DVT formation is a time-dependent inflammatory process in which NETosis plays an important role. However, whether ginsenoside Rg5 from species of Panax genus could alleviate DVT and its underlying mechanism has not been elucidated. MethodsThe interaction between Rg5 and P2RY(12) was studied by molecular docking, molecular dynamics, surface plasmon resonance (SPR), and molecular biology assays. The preventive effect of Rg5 on DVT was evaluated in inferior vena cava stasis-induced mice, and immunocytochemistry, Western blot, and calcium flux assay were performed in neutrophils from bone marrow to explore the mechanism of Rg5 in NETosis via P2RY(12). ResultsRg5 allosterically interacted with P2RY(12), formed stable complex, and antagonized its activity via residue E188 and R265. Rg5 ameliorated the formation of thrombus in DVT mice; accompanied by decreased release of Interleukin (IL)-6, IL-1 beta, and tumor necrosis factor-alpha in plasma; and suppressed neutrophil infiltration and neutrophil extracellular trap (NET) release. In lipopolysaccharide- and platelet-activating factor-induced neutrophils, Rg5 reduced inflammatory responses via inhibiting the activation of ERK/NF-kappa B signaling pathway while decreasing cellular Ca2+ concentration, thus reducing the activity and expression of peptidyl arginine deiminase 4 to prevent NETosis. The inhibitory effect on neutrophil activity was dependent on P2RY(12). ConclusionsRg5 could attenuate experimental DVT by counteracting NETosis and inflammatory response in neutrophils via P2RY(12), which may pave the road for its clinical application in the prevention of DVT-related disorders.

Automated summary

The study found that ginsenoside Rg5 can alleviate the occurrence of experimental DVT by inhibiting NETosis and inflammatory response in neutrophils. This provides a new perspective for the clinical application of Rg5 in the prevention of DVT-related disorders.