Role of BMEC-Derived Exosomal miR-144-3p in Microglial Polarization: Unveiling the NLRP3-GSDMD Pathway in Intracerebral Hemorrhage.

BACKGROUND

Intracerebral hemorrhage (ICH) often triggers a cascade of inflammatory responses, leading to secondary brain injury. The present work elucidated the regulatory mechanisms of NLRP3 inflammasome pathway activation in exosomal vesicle secretion by brain microvascular endothelial cells (BMECs)-derived exosomes and its influence on the polarization and recruitment of microglial cells (GCs).

METHODS

In an in vitro model, BMECs were pretreated with hemin to mimic the ICH environment and then cocultured with GCs in a replaced medium. NLRP3 inhibitor MCC950 and exosome release inhibitor GW4869 were used to observe the effects of NLRP3 inflammation and exosomes in BMECs. Gene expression was detected using western blotting (WB), immunofluorescence (IF), and real-time quantitative PCR. M1 polarization and migration of GCs were evaluated using biomarker expression, flow cytometry, and Transwell assays. The cellular mechanisms were further explored using an in vivo ICH rat model.

RESULTS

Hemin pretreatment activated NLRP3 inflammasomes and upregulated gasdermin D (GSDMD-N) production in BMECs, which further contributed to enhanced migration and M1 polarization of cocultured GCs. MCC950 and GW4869 significantly inhibited the effects of BMECs on GCs migration and M1 polarization. Mechanically, the NLRP3/GSDMD pathway promoted the release of exosomes from BMECs, which were absorbed by GCs and resulted in changes in their migration ability and polarization. Furthermore, miR-144-3p was identified as an important regulator in BMEC-derived exosomes and mediated the effect of BMECs on GSDMD. Knockdown of GSDMD in BMECs weakened the enhanced migration and M1 polarization of GCs by inhibiting the release of BMEC-derived exosomes, while miR-144-3p overexpression in GCs abolished these effects. An in vivo study illustrated that MCC950 decreased pro-inflammatory cytokine and CD86 levels, which were recovered by miR-144-3p overexpressing adeno-associated virus (AAV) injection.

CONCLUSIONS

This investigation revealed that the NLRP3/GSDMD pathway promotes the secretion of BMEC-derived exosomes, which transfer miR-144-3p from BMECs to GCs, further inducing the migration and M1 polarization of GCs.