Complement Molecule C3a Exacerbates Early Brain Injury After Subarachnoid Hemorrhage by Inducing Neuroinflammation Through the C3aR-ERK-P2X7-NLRP3 Inflammasome Signaling Axis.

An important aspect of the pathophysiology of early brain damage (EBI) after subarachnoid hemorrhage (SAH) is inflammasome-mediated neuroinflammation. It has been demonstrated that C3aR activation exacerbates neuronal damage in a number of neurological disorders. This study aims to explore the role of C3a in activating the NLRP3 inflammasome and exacerbating neuroinflammation after SAH. Preprocessing of RNA-seq transcriptome datasets using bioinformatics analysis, and screening of differentially expressed genes between SAH patients and healthy individuals from the GEO database. Internal carotid artery puncture was performed to establish SAH models in rats and mice. SAH grading, neurological scoring, brain water content, behavioral analysis, and assessments using ELISA, Western blot, immunofluorescence, and immunohistochemistry were conducted. An in vitro model of SAH was induced in BV-2 cells treated with heme (200 μM). The mechanism of C3a in post-SAH neuroinflammation was studied by interfering with and inhibiting C3aR. Results showed that the expression of C3aR was upregulated in the GEO dataset (serum of SAH patients) and identified as a key differential gene in SAH. Further, elevated levels of C3a were found in the cerebrospinal fluid of clinically collected SAH patients. In the cerebral cortex and/or serum of SAH rats, expression of C3a, IL-1β, IL-6, TNF-α, CD11b, and Ki67 were significantly increased, while IL-10 was significantly decreased. Correlation analysis revealed that C3a showed negative correlation with IL-10 and positive correlation with IL-1β, IL-6, TNF-α, CD11b, and Ki67. After stimulation with heme, protein levels of C3a increased in BV-2 cells. Interfering with C3aR significantly reduced LDH release, IL-1β secretion, Caspase1 activation, levels of NLRP3 expression and ASC oligomerization, and ATP release after heme stimulation in BV-2. Subsequently, the addition of inhibitors of ERK1/2 phosphorylation demonstrated that C3a promotes ATP efflux by activating ERK1/2 phosphorylation, thereby activating P2X7. Further addition of JNJ-55308942 (a P2X7R antagonist) revealed that C3a activated the NLRP3 inflammasome via P2X7. Finally, administering SB290157 (a C3aR inhibitor) in vivo effectively alleviated brain edema, reduced mortality, improved Garcia score, ameliorated motor dysfunction, and suppressed inflammation and NLRP3 inflammasome activation in mice after SAH. Overall, C3a exacerbates EBI-associated NLRP3 inflammasome and neuroinflammation via the C3aR-ERK-P2X7 pathway after SAH. Inhibiting C3aR may serve as a one possible treatment approach to alleviate SAH after EBI.