Abstract
BACKGROUND: Ischemic stroke, a predominant cause of global mortality and disability, involves complex pathophysiological processes where neuroinflammation and pyroptosis play a crucial role. We aimed to investigate the role of the brain's major water channel Aquaporin-4 (AQP4) in regulating neuronal pyroptosis, a highly inflammatory form of cell death, following cerebral ischemia. METHODS: Utilizing integrated in vivo and in vitro approaches, we employed AQP4 knockout mice subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and neuron-astrocyte co-cultures under oxygen-glucose deprivation/reoxygenation (OGD/R) with AQP4 knockdown to investigate the association between AQP4 and neuronal pyroptosis. Also, the downstream pathway was studied via RNA sequencing analysis and the following validation experiment. RESULTS: Our results demonstrated that AQP4 deficiency significantly worsened neurological deficits, enlarged infarct volume, and intensified oxidative stress. Crucially, AQP4 loss markedly exacerbated neuronal pyroptosis in both the ipsilateral and contralateral cortices in vivo, and in cultured neurons in vitro. This was evidenced by the specific up-regulation of the NLRP1 inflammasome, increased cleaved caspase-1, and elevated expression of gasdermin D (GSDMD), alongside heightened release of pro-inflammatory cytokines (IL-1β, IL-18, IL-6, TNF-α). RNA sequencing analysis of AQP4-knockdown neurons revealed the nuclear factor-kappa B (NF-κB) signaling pathway as a key downstream target. Mechanistic validation showed that AQP4 deficiency down-regulated NF-κB inhibitor-alpha (IκBα, encoded by NFKBIA), leading to increased nuclear translocation and activity of the NF-κB p50/p65 heterodimer. Subsequent gain- and loss-of-function experiments confirmed that NFKBIA/IκBα mediated the anti-pyroptotic effect of AQP4. CONCLUSION: Our findings establish AQP4 as a critical suppressor of neuronal pyroptosis after ischemic stroke. It confers protection by enhancing IκBα expression to inhibit NF-κB signaling, thereby dampening NLRP1 inflammasome activation and the subsequent pyroptotic cascade. This study unveils a novel AQP4/IκBα/NF-κB axis in post-ischemic neuroinflammation and highlights AQP4's role in mitigating remote secondary injury, offering new insights for developing neuroprotective strategies targeting global brain resilience.