Cryptotanshinone alleviates cerebral ischemia reperfusion injury by regulating ferroptosis through the PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathway

Ethnopharmacological relevance The Cryptotanshinone (CT) is a kind of Chinese medicine extracted from salvia miltiorrhiza, which has various pharmacological activities and is widely used in the treatment of diseases. Aim of the study The objective is to delve into the mechanism by which cryptotanshinone (CT) exerts its effects on rats with the middle cerebral artery occlusion/reperfusion (MCAO/R) model. Additionally, it aims to further assess the interplay between inflammation and oxidative stress, along with the underlying mechanism of CT's anti-ferroptosis function. Materials and methods We constructed the middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats. The effects of cryptotanshinone (CT) were evaluated using 2,3,5 - triphenyltetrazolium chloride (TTC) staining, behavioral assays, immunofluorescence, hematoxylin - eosin (HE) staining, and Nissl staining. Additionally, in vitro, cell viability was assessed by the Cell Counting Kit - 8 (CCK - 8) assay following experimental dosing. Oxygen - glucose deprivation/oxidation (OGD/R) models were established in PC12 and BV2 cells. Flow cytometry was employed to detect cellular reactive oxygen species (ROS) expression. The activities of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-px), andMitochondrial Membrane Potential Assay Kit with JC-1(JC-1) were measured using biochemical methods. Inflammatory factor levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. Immunoblotting was used to detect the levels of rat phosphatidylinositol 3 - kinase (PI3K), phosphorylated-PI3K (P-PI3K), protein kinase B (AKT), phosphorylated - AKT (P-AKT), nuclear factor erythroid 2related factor 2 (Nrf2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4). Results In rats with the MCAO/R model, CT demonstrated the ability to decrease ROS levels, enhance the activity of glutathione (GSH), mitigate inflammation, augment the activity of glutathione peroxidase 4 (GPX4), inhibit ferroptosis, safeguard neurons, and facilitate the restoration of nerve function. Results from network pharmacology indicated that the action of CT might be mediated via the PI3K/Akt signaling pathway. Simultaneously, in-vivo investigations revealed that CT curbs ferroptosis through the PI3K/AKT/Nrf2 and SLC7A11/GPX4 signaling pathways. Conclusion CT can inhibit ferroptosis by inhibiting the vicious cycle between oxidative stress and inflammation, protect neurons and promote motor function recovery.