ZBP1-mediated PANoptosis: A possible novel mechanism underlying the therapeutic effects of penehyclidine hydrochloride on myocardial ischemia-reperfusion injury.

Although penehyclidine hydrochloride (PHC) has been identified to alleviate myocardial injury induced by ischemia/reperfusion (I/R), the regulatory molecules and related mechanisms are unknown. In this study, bioinformatics, molecular biology, and biochemistry methods were used to explore the molecular mechanisms and targets of PHC. In the myocardial ischemia-reperfusion injury (MIRI)-induced rat model, PHC pretreatment significantly improved cardiac function (p < 0.01). Multiple differentially expressed genes, including Z-DNA binding protein 1 (ZBP1), were identified through mRNA sequencing analysis of myocardial ischemic penumbra tissue in MIRI rats. The transduction of the ZBP1 adenovirus vector (Ad-Zbp1) in PHC-pretreated rats exhibited a reversible augmentation in myocardial infarct size (p < 0.01), pronounced pathological damage to the myocardial tissue, as well as a significant elevation of serum myocardial enzymes (p < 0.05). The interaction among ZBP1, fas-associating via death domain (FADD), and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) leads to a remarkable up-regulation of cleaved-Caspase-1 (Cl-Casp-1), N-terminal gasdermin D (N-GSDMD), phospho-mixed lineage kinase domain-like Ser358 (p-MLKL), and other regulatory proteins, thereby triggering pyroptosis, apoptosis, and necroptosis (PANoptosis) in cardiomyocytes of MIRI rats. Moreover, the transduction of Ad-Zbp1 in the oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced H9c2 cell model also dramatically augmented the number of cell deaths. However, the intervention of PHC considerably enhanced cell viability (p < 0.01), effectively mitigated the release of myocardial enzymes (p < 0.05), and markedly attenuated the expression levels of PANoptosis regulatory proteins through restraint of ZBP1 expression. Therefore, the therapeutic efficacy of PHC in improving MIRI might be attributed to targeting ZBP1-mediated PANoptosis.