MFG-E8 attenuates neuro-inflammation in subarachnoid hemorrhage through driving microglial M2 polarization via modulating Integrin β3/SOCS3/STAT3 signaling pathway

Abstract

Background Increasing evidence suggests microglial polarization plays an important role in the pathological processes of neuro-inflammation following subarachnoid hemorrhage (SAH). Previous studies indicated that milk fat globule-EGF factor-8 (MFG-E8) has the potential in anti-apoptosis and anti-inflammation in cerebral ischemia. However, the effects of MFG-E8 on microglial polarization have not been evaluated after SAH. Therefore, the aim of this study was to explore the role of MFG-E8 on anti-inflammation, and its potential mechanism on microglial polarization following SAH. Methods We established the SAH model via prechiasmatic cistern Blood injection in mice. Double-immunofluorescence staining, Western blotting and quantitative real-time polymerase chain reaction (q-PCR) were performed to investigate the expression and cellular distribution of MFG-E8. Two different dosages (1 μg and 5 μg) of recombinant human MFG-E8 (rhMFG-E8) were injected intracerebroventricular (i.c.v.) at 1 h after SAH. Brain water content, neurological scores, beam-walking score, Fluoro-Jade C (FJC) and terminal deoxynucleotidyl transferase dUTP nick endlabeling staining (TUNEL) were measured at 24 h. Intervention of MFG-E8, integrin β3 and phosphorylation of STAT3 were achieved by specific siRNAs (500 pmol/5 µl) and STAT3 inhibitor Stattic (5 µM). The potential signal pathway and microglial polarization were measured by immunofluorescence labeling and Western blotting. Results SAH induction increased the levels of inflammation mediators, the proportion of M1 and caused neuronal apoptosis in mice at 24 h. Treatment with rhMFG-E8 (5 µg) remarkably decreased brain edema, improved neurological functions, reduced the levels of pro-inflammation factors, and promoted microglia shifted to M2 phenotype. However, knockdown MFG-E8 and integrin β3 via siRNA abolished the effects of MFG-E8 on anti-inflammation and M2 phenotype polarization. STAT3 inhibitor Stattic further clarified the role of rhMFG-E8 on microglial polarization through regulating the protein levels of integrin β3/SOCS3/STAT3 pathway. Conclusions rhMFG-E8 inhibits neuron-inflammation through transformation microglial phenotype towards M2 after SAH, which may be mediated by modulation of the integrin β3/SOCS3/STAT3 signaling pathway, and highlighting rhMFG-E8 as a potentially therapeutic target for the treatment of SAH patients.