Amygdalin attenuates post-ischemic neuroinflammation by targeting TLR4/NF-κB signaling and NLRP3 inflammasome in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) is a major contributor to neuronal damage after ischemic stroke, and current therapies remain limited by a narrow therapeutic window and reperfusion-associated injury. Amygdalin (Amg) has anti-inflammatory and antioxidant activities, but its role in regulating microglial polarization, pyroptosis, and the TLR4/NF-κB/NLRP3 axis in CIRI remains unclear. In this study, rat MCAO/R and BV-2 cell OGD/R models were used to evaluate the protective effects of Amg in vivo and in vitro. Transcriptomics, untargeted metabolomics, integrated pathway analysis, and molecular docking were combined with qPCR, Western blotting, LPS/ATP stimulation, and inhibitor interventions using TAK-242 and MCC950. Amg enhanced BV-2 cell viability, reduced OGD/R-induced pro-inflammatory cytokine release, and restored antioxidant enzyme activities. In MCAO/R rats, Amg significantly improved neurological function and alleviated brain tissue damage. In addition, Amg attenuated inflammation and oxidative stress, suppressed microglial M1 polarization while promoting M2-related phenotypes, and reduced pyroptosis-related marker expression in vivo and in vitro. Integrated multi-omics analysis further narrowed the candidate mechanisms to core pathways including NF-κB, Toll-like receptor, and NOD-like receptor signaling, and identified Tlr4, Myd88, Nlrp3, and Caspase-1 as key candidate molecules; pharmacological dissection experiments showed that combined treatment with Amg and either a TLR4 inhibitor or an NLRP3 inhibitor did not produce a significant additional synergistic effect, suggesting that its protective action depends on regulation of the TLR4/NF-κB/NLRP3-signaling axis. These findings provide mechanistic evidence for the neuroprotective effects of Amg in CIRI and support its potential as a therapeutic candidate for ischemic stroke.