Suppression of osteogenic differentiation and mitochondrial function change in human periodontal ligament stem cells by melatonin at physiological levels

Abstract

N-Acetyl-5-methoxytryptamine (melatonin, MT) at pharmacological concentrations promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; however, its role at physiological concentrations (1 pM-10 nM) remains unclear. We explored the effects of 1 pM-1 μM MT on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its underlying mitochondrial dynamics-mediated mechanisms. T he PDLSC phenotype was detected by flow cytome- try and evaluated for three-line differentiation. Alkaline phosphatase activity assay and Alizarin red staining were used to evaluate osteogenic differentiation. Osteogenesis- related gene and protein expression levels were measured by quantitative reverse transcription -polymerase chain reaction and western blotting. Mitochondrial function assays were performed using reactive oxygen species, ATP and NAD⁺/NADH kits and molecular mechanisms of mitochondrial dynamics-related proteins were assessed by western blotting. Our results have shown that physiological MT concentrations induced differentiation of hPDLSCs and down-regulated osteopontin (OPN) and osteocalcin (OCN) expression levels, which were restored or even up-regulated by 1 μM MT (lowest pharmacological concentration). Compared to the osteogenic induction alone, this treatment decreased the intracellular ATP content, whereas the intracellular reactive oxygen species level and NAD+/NADH ratio were increased. Mitochondrial function- and dynamics-related protein expression levels were consistent with those of osteogenic genes following osteogenic induction and MT treatment of hPDLSCs at various physiological concentrations. Physiological MT concentrations inhibited the osteogenic differentiation of hPDLSCs and simultaneously altered mitochondrial function. These findings provide insights into the stem cell tissue engineering and functions of MT.