The natural isoflavone puerarin mitigates cerebral ischemia-reperfusion injury by directly targeting CaMKⅡδ to block DLG4-Ser654 phosphorylation.

BACKGROUND: Synaptic collapse drives severe neurological deficits following cerebral ischemia-reperfusion (I/R) injury. Puerarin, a bioactive isoflavone from Pueraria lobata, exhibits neuroprotective properties, yet the precise kinase-substrate networks orchestrating its targeted efficacy remain elusive. PURPOSE: To systematically identify the pathogenic kinase networks in I/R injury and elucidate the underlying molecular mechanisms of puerarin's neuroprotection. METHODS: We adopted an integrated pipeline combining quantitative phosphoproteomics, ensemble deep learning-based virtual screening, molecular dynamics, in vitro LC-MS/MS kinase/phosphorylation assays, and in vivo/in vitro experimental validations. RESULTS: Phosphoproteomics and Kinase-Substrate Enrichment Analysis identified aberrant CaMKⅡδ activation, specifically via Thr287 autophosphorylation, as a key pathogenic mediator in I/R-injured rat hippocampi. Ensemble deep learning screening identified puerarin as a potent CaMKⅡδ modulator, while molecular dynamics, SPR, and in vitro kinase assays confirmed that puerarin directly anchors into the CaMKⅡδ ATP-binding pocket to effectively inhibit its kinase activity. Crucially, integrating bioinformatics and in vitro reconstitution, we uncovered Ser654 on the synaptic scaffold DLG4 (PSD-95) as a novel, direct phosphorylation target of CaMKⅡδ. Pathological DLG4-Ser654 phosphorylation is closely associated with the dissociation of AMPA receptors (GluR1/2) and subsequent mitochondria-dependent neuronal apoptosis. In both the NMDA-induced excitotoxicity model in PC-12 cells and MCAO rat models, puerarin suppressed CaMKⅡδ activation, blocked DLG4-Ser654 phosphorylation, restored DLG4-GluR1 interactions, and mitigated infarct volumes and neurological deficits. These protective effects were strictly occluded by CaMKⅡδ silencing or the specific inhibitor KN-93, confirming on-target efficacy. CONCLUSION: Our findings delineate a CaMKⅡδ-DLG4-AMPAR pathological cascade in ischemic stroke and highlight puerarin as a promising structure-based therapeutic candidate to rescue synaptic stability and prevent neuronal apoptosis.