Exosomal microRNA-223 from neutrophil-like cells inhibits osteogenic differentiation of PDLSCs through the cGMP-PKG signaling pathway.

Abstract

Neutrophils-derived exosomes have been shown to cause tissue inflammation in many diseases, but their role in periodontitis, a neutrophil-mediated disease, is unknown. Here, we investigated the effect of neutrophil-like cells derived exosomes on osteogenic dysfunction of periodontal ligament stem cells (PDLSCs) in periodontitis. Neutrophil-like cells were derived from HL-60 cells by dimethylsulfoxide stimulation. Exosomes were isolated by ultracentrifugation and characterized using transmission electron microscopy, nanoflow cytometry and western blot. MicroRNA-223 (miR-223) expression were analyzed by real-time PCR. Western blot, alkaline phosphatase (ALP), and alizarin red staining were conducted to assess whether exosomes could affect the osteogenic differentiation of PDLSCs. The expression of miR-223 was inhibited in PDLSCs by transfecting with miR-223 inhibitor. Cyclic guanosine monophosphate (cGMP) expression was determined by enzyme-linked immunosorbent assay. We found that miR-223 was significantly increased in neutrophils and neutrophil-like cells derived exosomes. Treatment with exosomes derived from neutrophil-like cells upregulated miR-223 expression and inhibited the osteogenic differentiation of PDLSCs, while transfection with miR-223 inhibitor significantly promoted PDLSCs osteogenic differentiation. In addition, co-treatment with KT5823, a cGMP-PKG pathway inhibitor, markedly abrogated the rescue effects of miR-223 inhibitor on the osteogenic differentiation of PDLSCs. Our findings suggest that neutrophil-like cells derived exosomes might inhibit osteogenic differentiation of PDLSCs by transporting miR-223 and regulating the cGMP-PKG signaling pathway.