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Updated: Sep 17, 2025
Three-dimensional Confocal Analysis of Microglia/macrophage Markers of Polarization in Experimental Brain Injury
Published on: September 4, 2013
MiR-223-3p promote microglia "M2" polarization by targeting FOXO3a in subarachnoid hemorrhage
Xiaolong Yao1, Bizhou Bie2, Zhizhong Wang2
1Department of Neurosurgery, Renmin Hospital of Wuhan University,Wuhan 430000, Hubei, China; Department of Neurosurgery, The Third People's Hospital of Hubei Province, Wuhan 430000, Hubei, China; Hubei Cerebrovascular Association, Wuhan 430000, Hubei, China.
Objective:
Exosome-derived micorRNAs (miRs) play important role in regulation the inflammatory response in subarachnoid hemorrhage (SAH). However, the ability of miR-223-3p, which is enriched in astrocyte-derived exosomes, to regulate FOXO3a in microglia is still unclear.
Methods:
MiR-223-3p in CSF from SAH patients was measured by qRT-PCR. Rats and BV2 cells were used to establish SAH model. Neurological function was evaluated by the mNSS, rotarod test, and Morris water maze. QRT-PCR and enzyme-linked immunosorbent assay (ELISA) were used to analyze oxidative stress and inflammatory factors interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α). The levels of FOXO3a, TLR4, NLRP3, NF-κB, iNOS, Cox2, Nrf2 and HO-1 were detected by WB. The exosomes were labeled with KPH67, and uptaken by microglia, detecting by IF.
Results:
Among the miRs involved in SAH, miR-223-3p exhibited one of the greatest change. MiR-223-3p in the brain tissue of SAH rats was upregulated. Moreover, the upregulation of miR-223-3p significantly improved neurological deficits and reduced brain edema. Meanwhile, miR-223-3p reduced the inflammatory factors like IL-1β, IL-6, TNF-α, and decreased oxidative stress, inhibited the activation of NF-κB, TLR4/NLRP3 in microglia by targeting Foxo3a. The "M1" polarization marker, including iNOS, Cox2, TLR4, NLRP3 and NF-κB, in microglia decreased markedly after the overexpression of miR-223-3p. Moreover, miR-223-3p targets FOXO3a and inhibits it expression no matter in vitro or in vivo. In vitro, both miR-223-3p mimics and astrocyte-derived exosomes obviously increased the expression of miR-223-3p in microglia.
Conclusion:
In SAH, astrocyte-derived exosomes rich in miR-223-3p may regulate the activation and phenotype of microglia by targeting FOXO3a, resulting in the inhibition of inflammatory injury.
Insights
Astrocyte-derived exosomes carrying miR-223-3p reduce brain inflammation after subarachnoid hemorrhage (SAH). This microRNA targets FOXO3a, improving neurological function and mitigating inflammatory responses in microglia.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- MicroRNAs (miRs) are crucial regulators of inflammatory responses in subarachnoid hemorrhage (SAH).
- Astrocyte-derived exosomes are known to contain miRs that influence the brain microenvironment.
- The specific role of miR-223-3p in regulating microglial function following SAH remains largely uncharacterized.
Purpose of the Study:
- To investigate the role of miR-223-3p, enriched in astrocyte-derived exosomes, in regulating microglial response in subarachnoid hemorrhage (SAH).
- To determine if miR-223-3p targets FOXO3a in microglia and modulates the inflammatory cascade.
- To evaluate the therapeutic potential of miR-223-3p in improving neurological outcomes after SAH.
Main Methods:
- Quantification of miR-223-3p in cerebrospinal fluid (CSF) of SAH patients using qRT-PCR.
- Establishment of SAH models in rats and BV2 microglial cells.
- Assessment of neurological function via mNSS, rotarod, and Morris water maze tests.
- Analysis of inflammatory and oxidative stress markers (IL-1β, IL-6, TNF-α) using qRT-PCR and ELISA.
- Detection of key protein levels (FOXO3a, TLR4, NLRP3, NF-κB, iNOS, Cox2, Nrf2, HO-1) via Western blot (WB).
- Exosome tracking using fluorescent labeling (KPH67) and immunofluorescence (IF) to confirm microglial uptake.
Main Results:
- miR-223-3p was significantly upregulated in SAH brain tissue and improved neurological deficits and reduced brain edema.
- Overexpression of miR-223-3p decreased inflammatory factors (IL-1β, IL-6, TNF-α) and oxidative stress.
- miR-223-3p inhibited microglial activation by targeting FOXO3a, suppressing the NF-κB and TLR4/NLRP3 pathways.
- Exogenous miR-223-3p, delivered via mimics or astrocyte-derived exosomes, effectively increased miR-223-3p levels in microglia.
Conclusions:
- Astrocyte-derived exosomes carrying miR-223-3p play a protective role in subarachnoid hemorrhage.
- miR-223-3p regulates microglial activation and phenotype by targeting FOXO3a, thereby inhibiting inflammatory injury.
- This pathway represents a potential therapeutic target for managing SAH-induced brain damage.
