Berberine alleviates early brain injury after subarachnoid hemorrhage by inhibiting GSK3β-mediated CASP1-dependent pyroptosis.

Abstract

BACKGROUND: Neuroinflammation and programmed cell death are two key pathogenic processes that contribute to poor patient outcomes in early brain injury (EBI) following subarachnoid hemorrhage (SAH). Neuronal pyroptosis in SAH has been explored in connection to berberine (BBR), a naturally occurring isoquinoline alkaloid having neuroprotective properties. However, its precise role and molecular mechanisms in this case remain unknown. OBJECTIVE: To investigate the protective effects of BBR on EBI following SAH and elucidate its potential molecular mechanisms. METHODS: Network pharmacology and molecular docking techniques were employed to identify core targets and pathways of BBR. In vivo experiments: A mouse SAH model was established via intravascular puncture. Groups included sham surgery, SAH model, BBR treatment, and BBR combined with GSK3β overexpression. Neurological function, cerebral edema, blood-brain barrier permeability, and key molecules in the pyroptosis pathway were assessed using neurological function scoring, brain water content measurement, immunofluorescence, and Western Blot techniques. In vitro experiments simulated SAH injury by exposing HT22 hippocampal neurons to oxygenated hemoglobin (OxyHb). BBR's effects were validated using CCK-8 assays, fluorescence analysis, qPCR, and Western Blot. Mechanistic rescue experiments employed GSK3β agonists. RESULTS: Network pharmacology predicted GSK3β as a key target of BBR, enriched in the pyroptosis pathway. In vivo experiments demonstrated that BBR significantly improved neurological deficits in SAH mice and reduced cerebral edema and blood-brain barrier disruption while simultaneously inhibiting GSK3β activation in cortical neurons and downstream Caspase-1 cleavage, GSDMD-N fragment generation, and IL-1β maturation. However, intracerebroventricular overexpression of GSK3β reversed these protective effects of BBR. In vitro experiments further confirmed that BBR concentration-dependently inhibits OxyHb-induced pyroptosis and inflammatory responses in HT22 neurons, while GSK3β overexpression significantly antagonizes its protective effects. CONCLUSION: BBR alleviates EBI following SAH by targeting GSK3β inhibition, thereby blocking the caspase-1/GSDMD-dependent neuronal pyroptosis pathway.