Procyanidin B2 ameliorates hypoxic-ischemic brain damage in neonatal rats by inhibiting pyroptosis via the TLR4/NF-κB signaling pathway

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal death and long-term neurodevelopmental impairment. Secondary neuroinflammation and inflammasome-mediated pyroptosis substantially contribute to progressive brain injury. Procyanidin B2 (PCB2), a plant-derived proanthocyanidin abundant in several traditional botanicals, exhibits anti-inflammatory activity, but its role in neonatal hypoxic-ischemic brain injury remains unclear. OBJECTIVE: To determine whether PCB2 protects against neonatal hypoxic-ischemic brain damage (HIBD) in rat and to clarify whether the TLR4/NF-κB pathway-driven pyroptosis is involved. METHODS: A Rice-Vannucci hypoxic-ischemic brain injury model was established in postnatal day 7 Sprague-Dawley rats. Brain injury was assessed by H&E, Nissl staining, ultrastructural analysis, and long-term behavioral tests at P28. Transcriptomic profiling and enrichment analyses were used to identify key pathways. qRT-PCR, Western blotting, and immunofluorescence were performed to evaluate TLR4/MyD88/NF-κB signaling and pyroptosis markers. TLR4 gain- and loss-of-function experiments were conducted in OGD-injured BV2 microglial cells. Molecular docking was applied to predict the potential PCB2-TLR4 interaction. RESULTS: PCB2 markedly reduced histopathological damage and improved P28 cognitive performance, accompanied by restoration of synaptic/neuronal markers. Transcriptomics identified enrichment of Toll-like receptor and cytokine-related inflammatory pathways among PCB2-reversed genes. PCB2 suppressed TLR4/MyD88/NF-κB activation and decreased NLRP3 inflammasome/pyroptosis proteins (NLRP3, ASC, caspase-1, GSDMD-N) and IL-1β/IL-18 (all P < 0.05). TLR4 overexpression attenuated, whereas TLR4 silencing enhanced, PCB2's anti-pyroptotic effects in vitro. Molecular docking predicted a potential interaction between PCB2 and TLR4. CONCLUSIONS: PCB2 alleviates neonatal hypoxic-ischemic brain injury by inhibiting TLR4/NF-κB-dependent pyroptosis, supporting its potential as a therapeutic candidate for neonatal HIBD.