Targeting the Zbp1-Ripk3 axis attenuates ischemic neuronal injury by suppressing PANoptosis

Cerebral ischemia-reperfusion injury (CIRI) significantly exacerbates neuronal damage following ischemic stroke. Emerging evidence implicates PANoptosis, a novel inflammatory form of programmed cell death, in CIRI pathogenesis. However, the role of Z-DNA-binding protein 1 (Zbp1), a key regulator of PANoptosis and innate immune responses, remains poorly understood in this context. Here, we establish a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model and an oxygen-glucose deprivation/re-oxygenation (OGD/R) model in which HT-22 cells are used to simulate CIRI and investigate the effects of Zpb1. Western blot analysis is performed to assess Zbp1 and PANoptosis-related protein expressions. Cell viability and death are evaluated using Hoechst 33342 staining and Calcein AM/PI assays, and the cerebral infarct volume is quantified using 2,3,5-triphenyltetrazolium chloride staining. Immunoprecipitation and mass spectrometry identify Ripk3 as a potential downstream effector of Zbp1. Furthermore, Zbp1 and PANoptosis-related proteins are significantly upregulated following OGD/R treatment. <i>Zbp1</i> knockdown markedly reduces PANoptosis and cell injury in both models, decreases infarct volume and improves neurological outcomes in MCAO/R model mice. Conversely, Zbp1 overexpression exacerbates OGD/R-induced neuronal injury and PANoptosis in HT-22 cells. This effect is partially reversed by <i>Ripk3</i> knockdown, indicating that Ripk3 mediates Zbp1-induced neuronal damage. These findings highlight Zbp1 as a promising therapeutic target in ischemic stroke and underscore the need for further research into Zbp1-mediated PANoptotic pathways to aid in the development of novel neuroprotective strategies.