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Updated: May 28, 2026
Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Published on: September 4, 2013
Role of BMEC-Derived Exosomal miR-144-3p in Microglial Polarization: Unveiling the NLRP3-GSDMD Pathway in
Jun Ma1, Zhongying Ran1, Tao Luo1
1Department of Neurosurgery, Guizhou Provincial People's Hospital, Guiyang, China, 5055.cn.
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.
Insights
The NLRP3 inflammasome pathway in brain endothelial cells drives exosome release, promoting microglial activation and migration after brain injury. This pathway involves GSDMD and transfers miR-144-3p, influencing neuroinflammation.
Area of Science:
- Neuroscience
- Cell Biology
- Immunology
Background:
- Intracerebral hemorrhage (ICH) causes secondary brain injury via inflammatory responses.
- The NLRP3 inflammasome pathway is implicated in ICH pathogenesis.
- Brain microvascular endothelial cells (BMECs) and microglial cells (GCs) play key roles in neuroinflammation.
Purpose of the Study:
- To elucidate the regulatory mechanisms of NLRP3 inflammasome activation in exosome secretion by BMECs.
- To investigate the influence of these exosomes on microglial cell polarization and recruitment.
- To identify the role of specific molecular mediators in this process.
Main Methods:
- In vitro models using BMECs and GCs co-culture, mimicking ICH conditions with hemin.
- Pharmacological inhibition of NLRP3 (MCC950) and exosome release (GW4869).
- Molecular analyses including western blotting, qPCR, immunofluorescence, flow cytometry, and Transwell assays.
- In vivo ICH rat model to validate findings.
Main Results:
- Hemin activated NLRP3 inflammasomes and GSDMD in BMECs, enhancing GC migration and M1 polarization.
- NLRP3/GSDMD pathway promoted exosome release from BMECs, which altered GC behavior.
- miR-144-3p within exosomes mediated the effects of BMECs on GCs, regulating GSDMD.
- In vivo studies confirmed MCC950 reduced inflammation, an effect reversed by miR-144-3p restoration.
Conclusions:
- The NLRP3/GSDMD pathway in BMECs promotes exosome secretion.
- These exosomes transfer miR-144-3p to GCs, inducing their migration and M1 polarization.
- This mechanism contributes to secondary brain injury following ICH.
