HSYA ameliorates venous thromboembolism by depleting the formation of TLR4/NF-κB pathway-dependent neutrophil extracellular traps

Abstract

Neutrophil extracellular traps (NETs), released by activated neutrophils, are implicated in various medical conditions, including venous thromboembolism (VTE). To develop effective therapeutic strategies for VTE, it is crucial to elucidate the mechanisms involved. In this study, we explored the role of NETs in VTE pathogenesis and assessed the impact of hydroxyl safflower yellow pigment A (HSYA) treatment on VTE pathogenesis. Various biochemical, pharmacological, and functional assessments were performed in human samples and VTE mouse models. Our findings revealed that NETs formation was enhanced in VTE patients and mouse model. NETs were shown to reduce the viability and integrity of endothelial cells and facilitated ferroptosis in human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. Depletion of NETs using the NE inhibitor Alvelestat significantly alleviated ferroptosis in VTE mice. Similarly, NETs depletion markedly attenuated thrombus formation and vein wall thickness in VTE mice. Notably, NETs treatment induced a significant elevation in total N6-Methyladenosine (m6A) RNA methylation level in HUVECs, with the most significant increase observed in methyltransferase-like 3 (METTL3). Mechanistically, the TLR4/NF-κB pathway was activated, and silencing METTL3 reversed the NETs-induced activation of this pathway in HUVECs. Rescue assays illustrated that METTL3 regulated the viability and ferroptosis of NETs-stimulated HUVECs by mediating TLR4 mRNA stability. Additionally, we found that HSYA exerted protective effects against ferroptosis in NETs-induced HUVECs and VTE mice. In summary, HSYA ameliorates VTE by depleting neutrophil extracellular traps through the inhibition of the TLR4/NF-κB pathway, thus providing a novel therapeutic strategy for treating VTE.