HSYA targets the ZBP1–166R site to inhibit MAVS signaling: A potential therapeutic strategy for cerebral ischemia-reperfusion injury

Background and purpose: Z-DNA-binding protein 1 (ZBP1) is a marker of neuroinflammation caused by ischemic stroke. Hydroxylsafflower Yellow A (HSYA), the main active component of safflower, has neuroprotective effects in ischemic stroke. Whether HSYA targets ZBP1 to protect mitochondrial function against cerebral ischemia-reperfusion injury (CIRI) remains unclear. Methods: This study involves lateral ventricle injection of plasmid adeno-associated virus (AAV) targeting the ZBP1 locus in SD rats. The MCAO/R model is established using the line plug method to simulate CIRI. Laser speckle blood flow meter and TTC staining are used to evaluate the model. Behavioral, morphological, and imaging techniques were employed to assess the cerebro-protective effects of HSYA against CIRI. Protein-protein docking and co-immunoprecipitation (CO-IP) experiments confirmed the interaction between ZBP1 and MAVS. Subsequently, an oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in HT22 cells. The levels of inflammatory markers and ATP were measured using ELISA, and the expression of signaling pathway proteins was analyzed by Western blot. Results: The cerebral blood flow in rats with a successfully established model was significantly decreased. HSYA reduces neurobehavioral scores and cerebral indices, improves ZBP1 pathologic distribution, and inhibits cell apoptosis. HSYA significantly enhanced cell viability and markedly decreased the apoptosis rate of cells following oxygen-glucose deprivation/reoxygenation (OGD/R) treatment. Additionally, HSYA enhances mitochondrial function and suppresses inflammatory factor release by down-regulating ZBP1 and MAVS expression while up-regulating TBK1 expression, after MCAO/R injury. The protective effect of HSYA is weakened after ZBP1 site mutation. Moreover, ZBP1 and MAVS exhibit a significant interaction. Conclusion: Our study reveals the mechanism of HSYA resisting MCAO/R may be associated with ZBP1 - A166R locus, and to design new ZBP1 antagonist treatment of various diseases associated with ZBP1 provides a template.