Tetramethylpyrazine alleviates cerebral ischemia-reperfusion injury via NRF2/HO-1/GPX4-mediated ferroptosis inhibition.
BACKGROUND Cerebral ischemia-reperfusion injury (CIRI) is a devastating neurological disorder involving autophagy, oxidative stress, and ferroptosis. Tetramethylpyrazine (TMP) has demonstrated potential pharmacological efficacy in antiplatelet aggregation, antithrombotic effects, vasodilation, and neuroprotection. PURPOSE This study aimed to determine the therapeutic efficacy of TMP in treating CIRI and to investigate whether TMP could improve CIRI by inhibiting ferroptosis through activation of the nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1)/glutathione Peroxidase 4 (GPX4) pathway in rats. METHODS We established MCAO/R rat models and OGD/R-treated HT22 cell models to evaluate the neuroprotective effects of TMP, employed network pharmacology and metabolomics to predict key signaling pathways, and assessed ferroptosis-related changes via biochemical assays, Western blotting, and transmission electron microscopy. RESULTS In vivo studies showed that TMP improved neurological scores, reduced infarct volume, and mitigated pathological features following MCAO/R. Network pharmacology and metabolomics revealed that TMP indirectly activated NRF2-mediated antioxidant responses while regulating glycerophospholipid metabolism and glycine/serine/threonine metabolism pathways. Mechanistically, TMP reduced oxidative stress markers, restored antioxidant capacity, and upregulated NRF2, HO-1, GPX4 and SLC7A11 expression, while the NRF2 inhibitor ML385 reversed these effects. These findings were further corroborated by in vitro experiments in OGD/R-exposed HT22 neurons, where TMP enhanced cell viability, attenuated lipid peroxidation and iron accumulation, and preserved mitochondrial ultrastructure, with ML385 partially reversing the protective effects. CONCLUSION TMP exerts neuroprotective effects in mitigating CIRI, which is associated with the regulation of ferroptosis and involves activation of the NRF2/HO-1/GPX4 pathway.