Puerarin mitigates cerebral ischemia/reperfusion (CIR)-induced ferroptosis by suppressing Ser15 phosphorylation-mediated p53 activation.

Cerebral ischemic stroke is a primary contributor to global mortality and disability, with its prevalence rising alongside aging population. While the restoration of blood supply to the ischemic brain regions is crucial, the subsequent cerebral ischemia/reperfusion (CIR) injury can trigger a cascade of pathological events associated with cell death. Ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, plays a significant role in the pathophysiology of CIR injury and has emerged as a key target for improving the unfavorable prognosis following ischemic stroke. Puerarin, an antioxidant compound extracted from the Pueraria lobata root, has been widely used as an effective therapeutic agent for CIR injury. However, the underlying mechanisms remain unclear. Here, we comprehensively investigated the protective effects of puerarin against CIR-induced ferroptosis and elucidated its mechanisms. Our study revealed that puerarin alleviated CIR-induced ferroptosis in tMACO rats and HT22 cells. Through a comprehensive approach involving network pharmacological analysis, molecular docking, microscale thermophoresis (MST), and cellular thermal shift assay (CETSA), we identified p53 as a molecular target of puerarin. Mechanistically, puerarin physically binds to p53, inhibiting its Ser15 phosphorylation and preventing p53 activation, which consequently leads to the inhibition of ferroptosis. Moreover, the overexpression of wild-type p53, rather than mutant p53 (p53S15D), reversed the inhibitory effect of puerarin on ferroptosis in vitro. Subsequently, functional assays in vivo further validated that modulating p53 activity, either with the pharmacological inhibitor (Pifithrin-α) or activator (MeOIstPyrd), could influence the inhibitory effect of puerarin on CIR-induced ferroptosis. Collectively, our study demonstrates that puerarin effectively mitigates CIR injury by suppressing Ser15 phosphorylation-mediated p53 activation, thereby reducing ferroptosis. These findings provide novel mechanistic insights into therapeutic potential of puerarin for the treatment of CIR injury.