SHED-Exo delivery of miR-708-5p activates PI3K/AKT signaling pathway to alleviate glucocorticoid-induced osteogenic inhibition in BMSCs.

Steroid-induced osteonecrosis of the femoral head (SONFH) is the most prevalent type of non-traumatic femoral head necrosis. Exosomes (Exo) secreted from stem cells of human exfoliated deciduous teeth (SHED) play crucial roles in biological processes, particularly in tissue regeneration and repair. Previous studies have demonstrated that exosomes from SHED (SHED-Exo) can alleviate dexamethasone (Dex)-induced osteogenic inhibition in bone marrow mesenchymal stem cells (BMSCs). This study aims to further investigate the molecular mechanism by which SHED-Exo rescues BMSCs osteogenic inhibition. Dex-induced BMSCs were used to construct the SONFH model, and SHED-Exo were used for treatment. Differentially expressed miRNAs (DE miRNAs) were identified by miRNA-seq and miRNA target genes were predicted using the database. Regulation was assessed by transfection of miRNA inhibitor and mimics. Alizarin red staining was used to verify osteogenic differentiation capacity, while qPCR and western blotting were employed to analyze osteogenesis-related proteins (OCN, OPN, and RUNX2), EFNA1, and PI3K/AKT pathways. Modulation using PI3K/AKT inhibitors and EFNA1 small interfering RNAs (siRNA) was performed to probe the molecular mechanism of SHED-Exo therapy. miRNA-seq results identified miR-708-5p as a DE miRNA. qPCR confirmed that its expression was down-regulated after Dex treatment and elevated after SHED-Exo treatment. CNTFR was validated as a miR-708-5p target gene through four database predictions and dual luciferase reporter gene results. Transfection of SHED with miR-708-5p inhibitor and mimics demonstrated that miR-708-5p derived from SHED-Exo negatively regulates CNTFR to promote osteogenesis in BMSCs and increases EFNA1 expression, thereby activating the PI3K/AKT pathway. It was further demonstrated that CNTFR is upstream of EFNA1 and negatively regulates the EFNA1/PI3K/AKT pathway by inhibiting PI3K/AKT and interfering with EFNA1 expression. SHED-Exo carries miR-708-5p into BMSCs, promoting EFNA1 expression by negatively regulating CNTFR, which in turn activates the PI3K/AKT pathway and enhances the osteogenic capacity of BMSCs.