Integrative transcriptomic, pharmacological, and genetic evidence reveals that ginsenoside Rb1 protects against HIE via inhibition of MKK3/MAPK14-mediated ferroptosis

Background Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal mortality and long-term neurological disability, with limited effective therapies. Ginsenoside Rb1, a bioactive compound from Panax ginseng, has shown neuroprotective potential, but its mechanisms remain incompletely understood. Methods A neonatal HIE rat model was established to evaluate Rb1's neuroprotective effects. Network pharmacology, transcriptomic sequencing, molecular docking, molecular dynamics simulations, and two-sample Mendelian randomization were integrated to identify key targets. Rescue experiments using a MAPK14 agonist in vivo and siRNA-mediated MAPK14 knockdown in vitro were performed to validate pathway involvement. Results Rb1 dose-dependently improved neurobehavioral deficits, reduced cerebral edema and infarct volume, and reversed hippocampal neuronal damage. It upregulated GPX4 and ferritin, downregulated ACSL4, and normalized iron, MDA, GSH, and oxidative stress levels. Network pharmacology and transcriptomics identified MAPK14 and MKK3 as core hub genes, with molecular dynamics confirming stable Rb1-MAPK14 binding. Mendelian randomization provided supportive genetic evidence for a protective causal effect of the Rb1-regulated pathway on HIE. Notably, a MAPK14 agonist abolished Rb1's anti-ferroptotic and neuroprotective effects, while siMAPK14 knockdown largely recapitulated Rb1's protection. Conclusions Ginsenoside Rb1 attenuates HIE-induced brain injury, with mechanistic evidence supporting that this effect involves suppression of MKK3/MAPK14-mediated neuronal ferroptosis..