miR-666-3p Mediates the Protective Effects of Mesenchymal Stem Cell-derived Exosomes Against Oxygen-glucose Deprivation and Reoxygenation-induced Cell Injury in Brain Microvascular Endothelial Cells via Mitogen-activated Protein Kinase Pathway

Background: Previous studies have reported that mesenchymal stem cell (MSC)-derived exosomes can protect primary rat brain microvascular endothelial cells (BMECs) against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. Objective: The aim was to identify the key factors mediating the protective effects of MSC-derived exosomes. Methods: Primary rat BMECs were either pretreated or not pretreated with MSC-derived exosomes before exposure to OGD/R. Naive cells were used as a control. After performing small RNA deep sequencing, quantitative reverse transcription polymerase chain reaction was performed to validate microRNA (miRNA) expression. The effects of rno-miR-666-3p on cell viability, apoptosis, and inflammation in OGD/R-exposed cells were assessed by performing the Cell Counting Kit 8 assay, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Moreover, the role of rno-miR-666-3p in regulating gene expression in OGD/R-exposed cells was studied using mRNA deep sequencing. Lastly, to evaluate whether mitogen-activated protein kinase 1 (MAPK1) was the target of rno-miR-666-3p, western blotting and the dual-luciferase assay were performed. Results: MSC-derived exosomes altered the miRNA expression patterns in OGD/R-exposed BMECs. In particular, the expression levels of rno-miR-666-3p, rno-miR-92a-2-5p, and rnomiR-219a-2-3p decreased in OGD/R-exposed cells compared with those in the control; however, MSC-derived exosomes restored the expression levels of these miRNAs under OGD/R conditions. rno-miR-666-3p overexpression enhanced cell viability and alleviated the apoptosis of OGD/R-exposed cells. Moreover, rno-miR-666-3p suppressed OGD/R-induced inflammation. mRNA deep sequencing revealed that rno-miR-666-3p is closely associated with the MAPK signaling pathway. Western blotting and the dual-luciferase assay confirmed that MAPK1 is the target of rnomiR-666-3p. Conclusion: MSC-derived exosomes restore rno-miR-666-3p expression in OGD/R-exposed BMECs. Moreover, this specific miRNA exerts protective effects against OGD/R by suppressing the MAPK signaling pathway.