ALPK1 signaling pathway activation by HMGB1 drives microglial pyroptosis and ferroptosis and brain injury after acute ischemic stroke

Pyroptosis and ferroptosis emerge as remarkable contributors to neuronal death and inflammation following ischemic stroke. High mobility group box 1 (HMGB1), a principal damage-associated molecular pattern (DAMP), is implicated in pyroptosis and ferroptosis post-stroke. Our previous research has demonstrated that alpha kinase 1 (ALPK1), a novel cytoplasmic pattern recognition receptor (PRR), plays an important role in mediating inflammatory damage following ischemic stroke. However, the interaction between ALPK1 and HMGB1, and their combined impact on pyroptosis and ferroptosis post-ischemic stroke remain unexplored, which is what this study aims to investigate. Initially, we observed that ALPK1 ablation attenuated ischemic brain injury of transient middle cerebral artery occlusion (tMCAO) mice. Moreover, recombinant HMGB1 (rHMGB1) stimulation induced the greatest upregulation of ALPK1 expression in microglia compared to astrocytes and neurons. Further investigation using co-immunofluorescence, co-immunoprecipitation, pull-down assay, and molecular docking revealed an interaction between HMGB1 and ALPK1. Additionally, the exacerbation of ischemic brain injury and the induction of microglial pyroptosis and ferroptosis by rHMGB1 treatment in tMCAO mice were significantly mitigated through ALPK1 deficiency by inhibiting the NLRP3/Caspase-1/GSDMD and JAK2/STAT3 signaling pathways. The inhibitory effects of ALPK1 deficiency on pyroptosis and ferroptosis induced by rHMGB1 in microglial cells were further substantiated. Finally, glycyrrhizic acid (GA), an inhibitor of HMGB1, exhibited significant neuroprotective effects in both tMCAO mice and BV2 cells subjected to oxygen-glucose deprivation/ reperfusion (OGD/R) by downregulating ALPK1 expression and inhibiting microglial pyroptosis and ferroptosis. Collectively, these findings suggest that HMGB1 may interact with ALPK1 to drive microglial pyroptosis and ferroptosis via the activation of the ALPK1/NF-kappa B/NLRP3/GSDMD and JAK2/STAT3 signaling pathways, thereby exacerbating brain injury following acute ischemic stroke.