Tetramethylpyrazine attenuates cerebral ischemia-reperfusion injury by inhibiting ferroptosis via the AMPK / Nrf2 pathways.

Objectives: Ferroptosis is involved in the development and exacerbation of cerebral ischemia-reperfusion injury (CIRI), and its inhibition can alleviate CIRI. Tetramethylpyrazine (TMP) is used for the treatment of ischemic stroke. However, the mechanism by which TMP regulates ferroptosis in CIRI is yet to be explored. This study demonstrated the effects of TMP on ferroptosis and CIRI, including the roles of the adenosine 5'-monophosphate-activated protein kinase (AMPK)/nuclear factor erythroid-2-related factor 2 (Nrf2) signaling pathway.

Materials and methods: A Sprague-Dawley rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was generated. The extent of neuronal injury was measured using 2,3,5-triphenyl tetrazolium chloride staining and Garcia neurological scoring and behavior was evaluated using open-field tests. Ferroptosis-related indexes were examined and ferroptosis-related proteins were detected using western blotting. The binding modes of TMP and AMPK were evaluated using molecular docking and molecular dynamics simulations.

Results: MCAO/R rats showed a reduced cerebral infarct area and improved neurological function after TMP intervention. TMP reduced levels of Fe²⁺, 4-hydroxynonenal, malonaldehyde, and acyl-coenzyme synthetase long-chain family member 4 and increased levels of glutathione and glutathione peroxidase 4. Increased AMPK phosphorylation and Nrf2 expression were also detected. TMP bound tightly to the AMPKα subunit in silico, and the LEU157, VAL41, LEU33, VAL107, and TYR106 residues were important for binding.

Conclusions: Our results indicate that TMP can alleviate CIRI by inhibiting ferroptosis via the activation of the AMPK/Nrf2 pathway, providing a theoretical basis for the clinical use of TMP in treating CIRI.