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Published on: March 15, 2024
Exosomes derived from M2-type microglia ameliorate oxygen-glucose deprivation/reoxygenation-induced HT22 cell injury
1Department of Neurology, Shanghai University of Medicine & Health Sciences Affiliated Zhoupu Hospital, No. 1500 Zhouyuan Road, Pudong New District, Shanghai, 201318, China.
Background:
Although it has been reported that miRNA carried by M2 microglial exosomes protects neurons from ischemia-reperfusion brain injury, the mechanism of action remains poorly understood. This study aimed to explore the miRNA signaling pathway by which M2-type microglia-derived exosomes (M2-exosomes) ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cytotoxicity in HT22 cells.
Methods:
BV2 microglia were induced by M2 polarization. Then, M2-exosomes were identified via transmission electron microscopy and special biomarker detection and co-cultured with HT22 cells. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Intracellular concentrations of reactive oxygen species (ROS), Fe2+, glutathione (GSH), and malondialdehyde (MDA) were determined using dichlorofluorescein fluorescence and biochemical determination. miR-124-3p levels were determined using qRT-PCR, and protein expressions were examined via western blotting.
Results:
OGD/R suppressed the proliferation and induced the accumulation of Fe2+, ROS, and MDA and reduction of GSH in mouse HT22 cells, suggesting ferroptosis of HT22 cells. OGD/R-induced changes in the above mentioned indexes was ameliorated by M2-exosomes but restored by the exosome inhibitor GW4869. M2-exosomes with (mimic-exo) or without miR-124-3p (inhibitor-exo) promoted and suppressed proliferation and ferroptosis-associated indexes of HT22 cells, respectively. Moreover, mimic-exo and inhibitor-exo inhibited and enhanced NCOA4 expression in HT22 cells, respectively. NCOA4 overexpression reversed the protective effects of miR-124-3p mimic-exo in OGD/R-conditioned cells. NCOA4 was targeted and regulated by miR-124-3p.
Conclusions:
M2-exosome protects HT22 cells against OGD/R-induced ferroptosis injury by transferring miR-124-3p and NCOA4 into HT22 cells, with the latter being a target gene for miR-124-3p.
Insights
M2 microglial exosomes protect neurons from brain injury by delivering miR-124-3p, which targets NCOA4 to reduce ferroptosis. This study reveals a key mechanism for exosome-mediated neuroprotection against oxygen-glucose deprivation/reoxygenation injury.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- MicroRNAs (miRNAs) within M2 microglial exosomes are known to protect neurons from ischemia-reperfusion brain injury.
- The precise molecular mechanisms underlying this neuroprotective effect remain largely unexplored.
Purpose of the Study:
- To investigate the miRNA signaling pathway through which M2-type microglia-derived exosomes (M2-exosomes) mitigate cytotoxicity induced by oxygen-glucose deprivation/reoxygenation (OGD/R) in HT22 cells.
- To elucidate the role of miR-124-3p and its downstream targets in this process.
Main Methods:
- M2 polarization of BV2 microglia and isolation of M2-exosomes.
- Assessment of HT22 cell proliferation (CCK-8 assay) and ferroptosis markers (ROS, Fe2+, GSH, MDA) after co-culture with M2-exosomes.
- Quantification of miR-124-3p levels (qRT-PCR) and protein expression (Western blotting), including NCOA4.
Main Results:
- OGD/R induced ferroptosis in HT22 cells, characterized by reduced proliferation and altered oxidative stress markers.
- M2-exosomes ameliorated OGD/R-induced ferroptosis, an effect reversed by the exosome inhibitor GW4869.
- miR-124-3p delivered by M2-exosomes suppressed ferroptosis by targeting and downregulating NCOA4 expression.
Conclusions:
- M2-exosomes protect HT22 cells from OGD/R-induced ferroptosis.
- This protection is mediated by the transfer of miR-124-3p, which targets NCOA4, thereby inhibiting ferroptosis.
- The findings highlight a novel therapeutic pathway involving M2-exosome-derived miR-124-3p for treating brain injury.
