Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviate M1 Microglial Activation in Brain Injury of Mice With Subarachnoid Hemorrhage via microRNA-140-5p Delivery

Abstract

Background: It is documented that mesenchymal stem cells (MSCs) secrete extracellular vesicles (EVs) to modulate subarachnoid hemorrhage (SAH) development. miR-140-5p expression has been detected in MSC-derived EVs, while the mechanism of MSC-derived EVs containing miR-140-5p in SAH remains unknown. We aim to fill this void by establishing SAH mouse models and extracting MSCs and MSC-EVs.

Methods: After ALK5 was silenced in SAH mice, neurological function was evaluated, neuron apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling with NeuN staining, and expression of serum inflammatory factors (interleukin-6, interleukin-1β, and tumor necrosis factor-α) was determined by enzyme-linked immunosorbent assay. The effect of ALK5 on NOX2 expression was assessed by western-blot analysis. Targeting the relationship between miR-140-5p and ALK5 was evaluated by dual luciferase assay. Following extraction of MSCs and MSC-EVs, EVs and miR-140-5p were labeled by PKH67 and Cy3, respectively, to identify the transferring of miR-140-5p by MSC-EVs. SAH mice were treated with EVs from miR-140-5p mimic/inhibitor-transfected MSCs to detect effects of MSC-EV-miR-140-5p on brain injury and microglial polarization.

Results: ALK5 silencing increased the neurological score and reduced neuron apoptosis and neuroinflammation in SAH mice. ALK5 silencing inhibited M1 microglia activation by inactivating NOX2. ALK5 was a target gene of miR-140-5p. MSC-derived EVs contained miR-140-5p and transferred miR-140-5p into microglia. MSC-EV-delivered miR-140-3p reduced ALK5 expression to contribute to repression of brain injury and M1 microglia activation in SAH mice.

Conclusions: MSC-derived EVs transferred miR-140-5p into microglia to downregulate ALK5 and NOX2, thus inhibiting M1 microglia activation in SAH mice.

Keywords: ALK5; M1 microglia activation; NOX2; brain injury; extracellular vesicles; mesenchymal stem cells; microRNA-140-5p; subarachnoid hemorrhage.