Study on the protective mechanism of Astragaloside IV by regulating the NETs pathway against ischemic stroke

Ischemic stroke (IS) triggers systemic inflammatory immune response, increasing blood neutrophil count. Inflammatory factors from ischemic area recruit neutrophils to form neutrophil extracellular traps (NETs), worsening injury. Astragaloside IV (AS-IV) exhibits proven neuroprotective effects against cerebral ischemia, potentially through suppressing NETs. This study examines how AS-IV prevents cerebral ischemic injury by blocking NETs formation (NETosis).

The Phorbol 12-myristate 13-acetate (PMA)-induced mouse bone marrow-derived neutrophils (BMNs) injury model was built to evaluate AS-IV's impact on NETosis, reactive oxygen species (ROS) levels, and marker proteins via scanning electron microscopy, flow cytometry, immunofluorescence techniques. AS-IV's protective action against NETs-damaged HT22 cells was elucidated via untargeted metabolomics. Targeted metabolomics for differential metabolites was carried out on a ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry. AS-IV's impact on neurological features, regional cerebral blood flow (CBF), and inflammatory infiltrates in transient middle cerebral artery occlusion (tMCAO) was evaluated by behavioral assessment, laser speckle contrast imaging, histological sectioning. Combined with peptidyl arginine deiminase 4 (PAD4) inhibitor Cl-amidine, NETs-associated proteins in cerebral tissue were detected by western blot (WB) and immunohistochemistry (IHC), and serum MPO-DNA and NE-DNA levels were tested using enzyme-linked immunosorbent assay (ELISA) to further elucidate AS-IV's mechanism against ischemic brain injury.

AS-IV dose-dependently inhibited NETs formation in PMA-induced mouse BMNs by reducing ROS levels and alleviating NETs-damaged HT22 cells. AS-IV reversed metabolic disorders in NETs-induced HT22 cells. Of the 14 differential metabolites detected, purine metabolism represented the predominantly altered metabolic pathway. Our in vivo data demonstrate that AS-IV improves neurological function, rescues impaired regional cerebral perfusion, and mitigates inflammatory cell infiltration. Mechanistically, these neuroprotective outcomes are largely attributed to the inhibition of PAD4-mediated NETosis..

AS-IV protects against ischemic brain injury by inhibiting PAD4-mediated NETosis.