Melatonin promotes osteogenic differentiation of rat adipose derived stem cells via the p38/MAPK signaling pathway

Background Adipose-derived stem cells (ADSCs) are an ideal therapeutic strategy for bone defect repair, but their osteogenic capacity is limited. Melatonin can promote osteogenic differentiation of various stem cells, but there are rarely studies on melatonin promoting osteogenic differentiation of ADSCs in rats; therefore, the aim of this study was to explore the effects of melatonin on osteogenic differentiation of ADSCs in rats.Methods The effects of different concentrations of melatonin on cell morphology, growth density and proliferative activity of rat ADSCs were investigated. The effects of different concentrations of melatonin on osteogenic differentiation, expression of osteogenic marker genes and marker proteins in rat ADSCs were determined by alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red staining (ARS), RT-qPCR, Western-blot, and immunofluorescence. The optimal concentration of melatonin for promoting rat ADSCs osteogenic differentiation was screened out. The molecular mechanism of melatonin promoting osteogenic differentiation of rat ADSCs was analyzed by RNA sequencing, MAPK signaling pathway inhibitor blocking assay and p38 siRNA interference assay.Results Morphological observation and CCK-8 assay displayed that 0-100 melatonin did not affect the morphology, growth density and proliferative activity of rat ADSCs. The results of ALP staining, ALP activity assay and ARS showed that melatonin could promote the osteogenic differentiation of rat ADSCs. Moreover, the results of RT-qPCR, Western-blot and immunofluorescence showed that melatonin could upregulate the expression of osteogenic marker genes and proteins in rat ADSCs.