Fyn Promotes NLRP3 Inflammasome Activation in Microglia and Exacerbates Early Brain Injury Following Subarachnoid Hemorrhage

Background Subarachnoid hemorrhage (SAH) is a lethal stroke subtype with high mortality. Microglia-driven neuroinflammation plays a pivotal role in early brain injury (EBI) after SAH. Xanthotoxol (Xol), a natural furanocoumarin, exhibits anti-inflammatory properties and neuroprotective potential, yet its role in SAH remains unclear. This study investigates whether Xol alleviates SAH-induced neuroinflammation and its underlying mechanisms. Methods SAH was induced in male SD rats and Balb/c mice via endovascular perforation. Three weeks prior to the SAH surgery, AAV2/9-CX3CR1-ZsGreen-miR30-Fyn virus was injected into the medial prefrontal cortex for in vivo mechanistic investigations. In vitro SAH models were established using oxyhemoglobin-stimulated microglia and microglia-neuron co-cultures. Finally, the efficacy of Xol was investigated both in vivo and in vitro following SAH. Assessments included neurological and cognitive function evaluation, brain water content measurement, detection of apoptosis, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining, immunohistochemistry, and Nissl staining. Results Fyn was upregulated in microglia post-SAH and associated with neuroinflammatory and apoptotic pathways via proteomics. Its knockdown ameliorated neurological deficits, edema, neuronal injury, and NLRP3 activation. Mechanistically, Fyn promoted K48-linked ubiquitination and degradation of Sirt1 via E3 ligase C-cbl. Xol directly bound Fyn, inhibiting its kinase activity and NLRP3 activation, thereby mitigating brain injury. Conclusion This study revealed Fyn recruits E3 ligase C-cbl to mediate Sirt1-K48 ubiquitination/degradation, activating NLRP3 inflammasome to drive SAH-induced neuroinflammation and brain injury. Xol inhibits Fyn activation, thereby attenuating inflammatory injury. The Fyn/Sirt1 axis represents a novel therapeutic target, and Xol offers a promising natural strategy for SAH treatment.