Diosmetin Inhibits NETs Formation in Neutrophils Through Regulating Nrf2 Signaling

Background: Neutrophil extracellular traps (NETs) are important pieces of equipment for neutrophils. Excess NETs play promoting roles in cancer-associated thrombosis (CAT). Therefore, directing NETs formation is a promising therapeutic strategy in thrombosis and related diseases. Diosmetin, an antioxidant flavonoid derived from dietary sources, might be involved in NETs formation and CAT.

Methods: Firstly, the tests of cell-free DNA and Immunofluorescence were applied to evaluate the NETs levels of neutrophils. Luminol-based chemiluminescence and the DCFH-DA probe were used to detect the levels of Reactive Oxygen Species (ROS) in neutrophils. Then, network pharmacological analysis and molecular docking were used to predict potential target molecules of diosmetin. The RT-qPCR was performed to measure the levels of Nrf2 and HO-1. A series of functional assays of neutrophils were used to examine the effect of diosmetin on Other neutrophil functions. Finally, an animal model of deep vein thrombosis was constructed to assess the effect of diosmetin on thrombosis.

Results: Diosmetin reduced NETs and ROS levels in neutrophils. Then, molecular mechanisms analysis suggested that Nrf2 might be the primary target of diosmetin. Diosmetin treatment increased the levels of Nrf2 and HO-1 in NETs-generating neutrophils. An inhibitor of Nrf2 diminished the negative effect of diosmetin on NETs generation. Lastly, the murine thrombosis model results indicated that diosmetin treatment reduced thrombosis via NETs formation.

Conclusion: Diosmetin exerts as anti-NETs effect through Nrf2 signaling in neutrophils, showing the therapeutic potential in thromboembolism and related pathological processes, such as CAT.

NETs; Nrf2; diosmetin; thrombosis.