Naoqing formula alleviates acute ischaemic stroke-induced ferroptosis via activating Nrf2/xCT/GPX4 pathway

Backgrounds Ferroptosis is a form of regulated cell death. The accumulation of iron in the brain is linked to trigger ferroptosis after an ischaemic stroke (IS). Naoqing formula (NQ) is a traditional Chinese medicine metabolites with the clinical function of activating blood circulation, which is applied to treat IS clinically in China. Methods Mice and SH-SY5Y cells were utilized to investigate the protective effects and the underlying mechanism of NQ against middle cerebral artery occlusion (MCAO) induced acute ischaemic stroke (AIS) and neuronal cellular ferroptosis caused by ferroptosis inducer Erastin in vitro and in vivo. Utilizing molecular biological techniques, transcriptomics, and proteomics analyses, the role of NQ in Nrf2 regulation and ferroptosis was evaluated through the pharmacologic inhibition of Nrf2. Results NQ attenuated AIS-induced neuronal damage and cerebral infarction by increasing cortical blood flow (CBF). Transcriptomics and proteomics analyses revealed that NQ might regulate lipid and iron metabolism through Nrf2 pathway. Additionally, NQ can protect AIS from ferroptosis by reducing oxidative stress and iron overload. Meanwhile, Nrf2, solute carrier family 7 member 11 (SLC7A11; also known as xCT) and glutathione peroxidase 4 (GPX4) were upregulated in NQ-treated AIS mice. Consistent with the results in vivo, NQ led to ferroptosis resistance upon exposure to a ferroptosis-inducing compound through activation of Nrf2/xCT/GPX4 pathway in vitro. Notably, in vivo inhibition of Nrf2 expression by ML385 aggravated the ferroptotic events and weakened the neuroprotective effect of NQ as well as subsequently reduced the expression of xCT and GPX4. Conclusion This study demonstrated that NQ protected against AIS via suppression of ferroptosis and oxidative stress, which were largely dependent on the upregulation of Nrf2 pathway.