Angong Niuhuang Wan inhibit ferroptosis on ischemic and hemorrhagic stroke by activating PPARγ/AKT/GPX4 pathway

Angong Niuhuang Wan (AGNHW) is a prescription from traditional Chinese medicine (TCM) that has been used for centuries to treat ischemic stroke (IS) and hemorrhagic stroke (HS). According to a recent study, targeting ferroptosis might be effective in the management of IS and HS. However, the ferroptosis-related effects and mechanisms of AGNHW have not yet been reported. This research examines the anti-ferroptosis mechanisms of AGNHW in the treatment of IS and HS. A system pharmacological approach including in vivo experiment, UHPLC-Q-Orbitrap HRMS, network pharmacology, molecular docking, microscale thermophoresis, and in vitro experiment was utilized to study the anti-ferroptosis mechanisms of AGNHW against IS and HS. In vivo experiments indicated that AGNHW enhanced nerve function, decreased cerebral infarct volume, ameliorated histological brain injuries, improved the structural integrity of the blood-brain barrier, ameliorated the mitochondrial dysfunction and morphology disruption, and inhibits ROS, LPO and Fe2+ accumulations in IS and HS rats. Using UHPLC-Q-Orbitrap HRMS, the key ingredients of AGNHW-containing serum were identified as bilirubin, berberine, baicalin, and wogonoside. According to the network pharmacology analyses, AGNHW could inhibit ferroptosis by modulating the PPAR and PI3K/AKT signaling pathways. The core targets are PPARγ, AKT, and GPX4. Molecular docking and microscale thermophoresis experiments further revealed that the key ingredients have strong interactions with ferroptosis-regulating core proteins. Moreover, in vitro experiment results showed that AGNHW alleviated ferroptosis injury induced by erastin in PC12 cells, increased cell viability, reduced the LPO and Fe2+ levels, and up-regulated mRNA expressions of PPARγ, AKT, and GPX4. AGNHW also up-regulated protein expressions of PPARγ, p-AKT/AKT, and GPX4 in IS and HS rats. AGNHW attenuated ferroptosis in treating IS and HS by targeting the PPARγ/AKT/GPX4 pathway. This work reveals AGNHW's anti-ferroptosis mechanism against IS and HS, but it also develops an integrated approach to demonstrate the common characteristics of drugs in treating different diseases.