Metrnl/C-KIT Axis Attenuates Early Brain Injury Following Subarachnoid Hemorrhage by Inhibiting Neuronal Ferroptosis

Abstract

Background and purpose: Ferroptosis is a distinct form of cell death characterized by iron-dependent lipid peroxidation and plays a crucial role in the early brain injury (EBI) following subarachnoid hemorrhage (SAH). As a newly discovered endogenous ligand for the C-KIT receptor tyrosine kinase, meteorin-like protein (Metrnl) exerts regulatory functions in oxidative stress and protects against various diseases. However, the specific role of the Metrnl/C-KIT axis in neuronal ferroptosis during EBI following SAH remains to be elucidated.

Methods: Sprague Dawley rats were used to establish the SAH model through endovascular perforation. r-Metrnl was administered intranasally 1 h after SAH. Metrnl shRNA, C-KIT inhibitor ISCK03, AMPK inhibitor dorsomorphin, and Nrf2 inhibitor ML385 were administered intracerebroventricularly or intraperitoneally before r-Metrnl treatment to explore the underlying mechanisms. Neurobehavioral assessments, immunofluorescence, western blot, ELISA, Fluoro-Jade C staining, transmission electron microscopy, and Nissl staining were conducted to evaluate the effects. Additionally, primary neuron culture with hemoglobin (Hb) stimulation was used for in vitro studies.

Results: Phosphorylated C-KIT and endogenous Metrnl levels were upregulated after SAH. Knockdown of Metrnl aggravated neurobehavioral deficits and neuronal ferroptosis, whereas r-Metrnl treatment showed a protective effect. Mechanistically, r-Metrnl significantly increased the protein levels of SLC7A11, GPX4, FTH, FSP1, and GSH, whereas it decreased the levels of ACSL4, 4HNE, and MDA in the ipsilateral hemisphere 24 h after SAH. Also, r-Metrnl reduced mitochondrial shrinkage, increased mitochondrial crista, and decreased membrane density. However, the beneficial effects of r-Metrnl were partially reversed by ISCK03, dorsomorphin, or ML385 treatment both in vivo and in vitro.

Conclusions: Our study demonstrated that r-Metrnl reduced neuronal ferroptosis and improved neurological outcomes after SAH by modulating the C-KIT/AMPK/Nrf2 signaling pathway.

Keywords: C‐KIT; Metrnl; ferroptosis; subarachnoid hemorrhage.