Puerarin alleviates cerebral ischemic injury by modulating PINK1-mediated mitophagy and NLRP3/GSDMD-dependent pyroptosis: an integrated in silico and in vitro study

CIRI is characterized by severe oxidative stress and neuroinflammation, fundamentally driven by the pathological interplay between defective mitophagy and robust pyroptosis. While the natural isoflavonoid puerarin exhibits neuroprotective potential, its precise atomistic interactions with targeted biological macromolecules remain elusive. In this study, we employed an integrated computational biophysics and in vitro approach to explore the multiscale mechanisms by which puerarin modulates PINK1-mediated mitophagy and NLRP3/GSDMD-dependent pyroptosis. ADMET profiling and DFT calculations initially confirmed puerarin’s favorable pharmacokinetics and intrinsic reactive oxygen species (ROS) scavenging capacity (Bond Dissociation Energy = 89.68 kcal/mol). Extensive 100 ns MD simulations and MM/GBSA analyses suggested that puerarin could act as a structural modulator. It was computationally predicted to anchor stably within the functional cavities of the PINK1, NLRP3, and GSDMD monomers and act as a steric hindrance, theoretically attenuating the binding free energies of the native NLRP3-ASC and Caspase-3-GSDMD macromolecular assemblies. These thermodynamic predictions were empirically supported by an in vitro OGD/R model using HT22 cells, with nimodipine included as a reference comparator. At the protein level, puerarin significantly upregulated markers of PINK1/Parkin-mediated mitophagy while profoundly suppressing NLRP3 inflammasome activation and subsequent GSDMD cleavage. Furthermore, the application of the autophagy inhibitor 3-MA exacerbated OGD/R-induced pyroptotic marker expression, experimentally validating the intrinsic biological crosstalk between defective mitophagy and pyroptosis. Collectively, our findings propose that puerarin may mitigate CIRI through a dual-pronged mechanism: the computationally predicted thermodynamic destabilization of pyroptotic machineries, coupled with the in vitro modulation of mitophagy-related proteins to potentially clear upstream oxidative triggers.