Abstract

Intracerebral hemorrhage (ICH) induces neuroinflammation and neuronal damage, partially driven by microglial necroptosis and M1 polarization. Recent evidence implicates red blood cell-derived exosomes (RBC-Exos) in inflammatory pathologies, yet their role in ICH remains unexplored. This study investigates how hemorrhagic RBC-Exos (ICH-Exos) regulate microglial dysfunction and neuropathology through complement signaling. RBC-Exos were isolated and validated using flow cytometry (AnnexinV + CD235 + ), transmission electron microscopy, nanoparticle tracking analysis, and western blot (CD63/TSG101). In vitro, hemin-treated microglia were exposed to Normal-Exos or ICH-Exos. Murine ICH models assessed neuropathology via histology (H&E, Nissl staining), adhesion molecule expression (ICAM-1/VCAM-1), and neurobehavioral tests. Proteomics identified exosomal protein cargo, complemented by C5 monoclonal antibody (mAb) blocking experiments to dissect mechanistic pathways. ICH-Exos exacerbated hemin-induced microglial necroptosis, marked by upregulated phosphorylated RIPK1, RIPK3, and MLKL, and amplified M1 polarization (elevated CD86, iNOS, CCL2; suppressed CD163, ARG1). In ICH mice, ICH-Exos aggravated cerebral hemorrhage, neuronal loss, and neurobehavioral deficits while elevating pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Proteomics revealed C5 enrichment in ICH-Exos, correlating with elevated C5a levels in serum and brain tissues. C5 mAb neutralized ICH-Exos effects, rescuing microglial viability, restoring M1/M2 equilibrium, and attenuating necroptosis. In vivo, C5 inhibition reduced hemorrhage volume, restored neuronal Nissl bodies, suppressed ICAM-1/VCAM-1, and mitigated MAPK pathway activation (p-ERK1/2, p-p38). In conclusion, ICH-Exos drive neuroinflammation and neuronal injury post-ICH by promoting C5-dependent microglial necroptosis and M1 polarization. Targeting the C5/C5a axis counteracts these effects, suggesting a novel therapeutic strategy to ameliorate I