CCDC50 Alleviates Cerebral Ischemia-Reperfusion Injury via Enhancing Autophagy to Inhibit STING/IRF3-Mediated Microglial Polarization.

Ischemia-reperfusion injury (IRI) is a major reason for adverse prognosis in ischemic stroke, with multiple signaling pathways involved in this process. This study aims to explore the role of the novel autophagy receptor CCDC50 in cerebral IRI and its molecular mechanism. Cellular oxygen-glucose deprivation/reoxygenation model and middle cerebral artery occlusion (MCAO) animal model were adopted in this study. Quantitative real-time polymerase chain reaction, Western blot, immunofluorescence, and immunohistochemistry assays were applied to detect the expression and subcellular localization of key molecules including LL37, CCDC50, STING, IRF3, Rspondin3, and autophagy-related proteins. Co-immunoprecipitation was performed to explore protein-protein interactions. Enzyme-linked immunosorbent assay and flow cytometry were utilized to determine the levels of inflammatory cytokines and the M1/M2 polarization phenotypes of microglia. Additionally, 2,3,5-triphenyltetrazolium chloride staining was carried out to measure cerebral infarct volume. Results revealed that LL37 directly bound to CCDC50 and facilitated its ubiquitination and degradation. This process disrupted the interaction between CCDC50 and STING, thereby triggering the activation of the STING/IRF3 signaling pathway. Conversely, CCDC50 enhanced autophagy and restrained the STING/IRF3 pathway activation. It further drove microglial polarization toward the anti-inflammatory M2 phenotype, blocked pro-inflammatory M1 phenotype transition and inflammatory cytokine secretion, and ultimately alleviated neuronal damage. The above in vitro molecular mechanisms were also validated in the MCAO model. By enhancing autophagy, CCDC50 suppresses the STING/IRF3 pathway and facilitates M2 polarization of microglia, ultimately mitigating cerebral ischemia-reperfusion injury. In contrast, LL37 counteracts this protective effect by mediating CCDC50 degradation. This study identifies novel potential therapeutic targets for ischemic stroke.