ABSTRACT
Background: Deep 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. Methods: The interaction between Rg5 and P2RY12 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 P2RY12. Results: Rg5 allosterically interacted with P2RY12, 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-1ß, and tumor necrosis factor-α 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-κB 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 P2RY12. Conclusions: Rg5 could attenuate experimental DVT by counteracting NETosis and inflammatory response in neutrophils via P2RY12, which may pave the road for its clinical application in the prevention of DVT-related disorders.
Subject(s)
COVID-19 , Venous Thrombosis , Animals , Ginsenosides , Mice , Molecular Docking Simulation , NeutrophilsABSTRACT
Lonicerae japonicae flos (L. japonicae flos, Lonicera japonica Thunb.) is one of the most commonly prescribed botanical drugs in the treatment or prevention of corona virus disease 2019. However, L. japonicae flos is often confused or adulterated with Lonicerae flos (L. flos, Lonicera macrantha (D.Don) Spreng., Shanyinhua in Chinese). The anti-SARS-CoV2 activity and related differentiation method of L. japonicae flos and L. flos have not been documented. In this study, we established a chemical pattern recognition model for quality analysis of L. japonicae flos and L. flos based on ultra-high performance liquid chromatography (UHPLC) and anti-SARS-CoV2 activity. Firstly, chemical data of 59 batches of L. japonicae flos and L. flos were obtained by UHPLC, and partial least squares-discriminant analysis was applied to extract the components that lead to classification. Next, anti-SARS-CoV2 activity was measured and bioactive components were acquired by spectrum-effect relationship analysis. Finally, characteristic components were explored by overlapping feature extracted components and bioactive components. Accordingly, eleven characteristic components were successfully selected, identified, quantified and could be recommended as quality control marker. In addition, chemical pattern recognition model based on these eleven components was established to effectively discriminate L. japonicae flos and L. flos. In sum, the demonstrated strategy provided effective and highly feasible tool for quality assessment of natural products, and offer reference for the quality standard setting.