Elucidating the mechanism of anti-ischemic stroke action of Irisflorentin:A combined approach using network pharmacology, molecular docking, and in vitro validation

Irisflorentin, an isoflavonoid isolated from Belamcanda chinensis (L.) Redouté, serves as a key bioactive molecule in Sagittaria, exhibiting antioxidative, hypoglycemic, and anti-inflammatory properties. Despite these attributes, the precise roles and mechanisms of Irisflorentin in mitigating cerebral ischemia/reperfusion injury are not well elucidated. This study aims to clarify the therapeutic targets and pathways of irisflorentin in ischemic stroke utilizing network pharmacology, molecular docking, molecular dynamics simulation and experimental validation. The predictions generated by network pharmacology and molecular docking concerning potential targets and pathways. Irisflorentin influences numerous biological processes, such as signaling, protein phosphorylation, the suppression of neuronal apoptosis and the amplification of the MAPK cascade. Key targets identified via molecular docking include ESR1, EGFR, HIF1A, HSP90AB1, CCND1, ERBB2, MTOR, ESR2, MAPK8, and MAPK1. Molecular dynamics simulations indicate that the Irisflorentin small molecule exhibits favourable binding interactions with the HIF1A and MTOR target proteins.Cellular studies showed that irisflorentin improves cellular morphology, boosts cell viability and mitochondrial membrane potential, and decreases LDH release, ROS production, Ca 2+ levels, and apoptosis rates. IFR enhances the expression of PI3K, AKT, mTOR, Bcl-2, BDNF, TRқB, ERK1, ERK2, p90RSK, and CREB, while suppressing FOXO1A, HIF1A, Bax, and Caspase-3.Ultimately, Irisflorentin promotes neuroprotection and lessens cerebral ischemia/reperfusion damage by activating the PI3K/AKT/FOXO1A, mTOR/HIF1A, and BDNF/TRқB/ERK1/2/p90RSK/CREB pathways, underscoring its potential as an adjunct therapy for ischemic stroke and providing a foundation for its further development and clinical utility. • Irisflorentin has antioxidative, anti-apoptotic, anti-inflammatory effects. It targets PI3K/AKT, FOXO, HIF-1, MAPK pathways to alleviate cerebral ischemia/reperfusion injury. • Molecular docking identifies key targets (ESR1, EGFR, HIF1A, HSP90AB1, MAPK1/8) mediating irisflorentin’s protective effects against ischemic stroke. • Irisflorentin improves cell viability and mitochondrial function, reduces oxidative stress (ROS, LDH, Ca 2+ ), and modulates apoptosis-related proteins (Bcl-2, Bax, Caspase-3). • The compound activates PI3K/AKT/FOXO1A, mTOR/HIF1A and BDNF/TRκB/ERK1/2/p90RSK/CREB pathways, providing a novel multi-pathway strategy for ischemic stroke adjunct therapy.