Leonurine ameliorates hypoxic pulmonary hypertension by inhibiting cross-talk between neutropHils and endothelial cells via SERPINB3 targeting.

Abstract

The pharmacological mechanism of leonurine (LEO) in treating hypoxic pulmonary hypertension (HPH) remained unclear. We established a rat model of HPH to assess the preliminary therapeutic effects of LEO. We examined how LEO alters cell populations and affects S100A9 expression in lung tissue using single cell RNA sequencing. We developed an in vitro model of neutrophil activation and migration and a co-culture system of neutrophils and pulmonary artery endothelial cells (PAECs) to investigate the mechanistic basis of this process. We also employed molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) to identify the molecular target and used gene silencing approach to validate the mechanism. LEO treatment significantly alleviated associated pathological changes in lung tissue. LEO suppressed hypoxia-induced S100A9 production in neutrophils. Interestingly, LEO did not directly mitigate PAEC dysfunction. However, supernatants from LEO-treated neutrophils significantly reduced the expression of inflammatory cytokines, as well as the mesenchymal marker VIM, in PAECs. Subsequent molecular docking, CETSA, and DARTS experiments demonstrated that LEO directly binds to SERPINB3 in vitro and inhibits downstream STAT1/3 phosphorylation. Silencing Serpinb3 attenuated the ability of LEO on neutrophil migration and S100A9 production and on PAEC dysfunction. LEO targeted SERPINB3 to inhibit S100A9 production in neutrophils, thereby attenuating endothelial dysfunction and vascular remodeling in HPH.