Didymin protects against retinal ischemia–reperfusion injury by suppressing microglial pyroptosis and neuroinflammation via EGFR-mediated NLRP3/GSDMD signaling

PURPOSE: To investigate whether didymin confers neuroprotection against retinal ischemia-reperfusion (IR) injury by modulating microglial pyroptosis and to elucidate the underlying molecular mechanisms. METHODS: A mouse retinal IR injury model was used to evaluate the neuroprotective effects of didymin and its regulation of pyroptosis and inflammation in vivo. LPS/ATP-stimulated BV-2 microglia were used to further evaluate the inhibitory effect of didymin on microglial pyroptosis in vitro. Retinal structure and function were assessed via histology and visual behavioral tests. The regulation of pyroptosis and inflammation was detected by quantitative real-time PCR (qPCR), western blotting, and immunofluorescence. Network pharmacology, molecular docking, and cellular thermal shift assay (CETSA) were employed to identify and validate EGFR as a target of didymin. Pharmacological inhibition of EGFR was employed in vivo to verify its functional involvement. RESULTS: Didymin treatment significantly alleviated IR-induced retinal damage and preserved visual function. Mechanistically, didymin primarily targeted microglia, effectively suppressing NLRP3/GSDMD-dependent pyroptosis and neuroinflammation. Didymin was found to bind and activate EGFR. Crucially, pharmacological inhibition of EGFR with AG1478 abolished the anti-pyroptotic, anti-inflammatory, and neuroprotective effects of didymin, confirming that didymin acts by activating EGFR signaling. CONCLUSIONS: Our study demonstrates that didymin exerts potent neuroprotective effects in IR-induced retinal damage by activating EGFR signaling, thereby inhibiting microglial pyroptosis and neuroinflammation via the NLRP3/GSDMD pathway. Didymin may represent a potential immunomodulatory and neuroprotective candidate for ischemia-associated retinal neurodegenerative conditions.