CKLF1 Upregulation Exacerbates Secondary Brain Injury after Intracerebral Hemorrhage by Promoting Neuronal Ferroptosis and Oxidative Stress.
TL;DR
CKLF1 is identified as a previously unrecognized regulator of neuronal ferroptosis following ICH through the CCR5/p38/p53 signaling pathway and is highlighted as a promising therapeutic target for precision treatment of hemorrhagic stroke.
Abstract
Background
Secondary brain injury (SBI) following spontaneous intracerebral hemorrhage (ICH) is a critical determinant of neurological outcome. It is closely associated with neuronal death, and inflammatory responses. However, current research on SBI after ICH remains limited, and effective therapeutic targets are still lacking.
Methods
In this study, a collagenase-induced intracerebral hemorrhage (ICH) model was employed, and experiments were conducted 24 h after hemorrhage induction. For in vitro experiments, differentiated PC12 cells were stimulated with hemin and the CKLF1 agonist peptide C27 to evaluate the potential of CKLF1 to induce neuronal ferroptosis. Mechanistically, the CKLF1 antagonist peptide C19, the CCR5 inhibitor Maraviroc (MVC), and a p38 MAPK inhibitor were applied, in combination with immunoprecipitation (Co-IP) assays and overexpression of p53 site mutation, to elucidate the molecular mechanisms underlying CKLF1-induced ferroptosis in PC12 cells. In vivo, the effects of CKLF1 knockdown on secondary brain injury after ICH were assessed using quantitative real-time PCR (qPCR), Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) analyses.
Results
Mechanistic studies revealed that CKLF1 binds to its receptor CCR5 to activate p38 MAPK, which promotes ferroptosis by regulating p53 phosphorylation and nuclear translocation, while the mutation of p53 partially reversed the effect. This signaling cascade leads to the downregulation of ferroptosis-suppressing proteins (SLC7A11 and GPX4) and the upregulation of pro-ferroptotic proteins (COX2 and ACSL4), thereby exacerbating lipid peroxidation and oxidative stress. Conversely, AAV-mediated knockdown of CKLF1 significantly suppressed this ferroptotic signaling pathway in vivo/vitro, reduced iron deposition and neuronal ferroptosis in perihematomal tissue, improved blood-brain barrier (BBB) integrity, inhibited microglial activation, and enhanced both neurological function scores and 7-day survival rates in ICH rats.
Conclusion
The present study identifies CKLF1 as a previously unrecognized regulator of neuronal ferroptosis following ICH through the CCR5/p38/p53 signaling pathway. These findings provide new mechanistic insights into ICH pathogenesis and highlight CKLF1 as a promising therapeutic target for precision treatment of hemorrhagic stroke.