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.

背景：脂肪来源干细胞（Adipose-derived stem cells, ADSCs）是骨缺损修复的理想治疗策略，但其成骨能力有限。褪黑素可促进多种干细胞的成骨分化，但目前关于褪黑素促进大鼠脂肪来源干细胞成骨分化的研究较为匮乏，因此本研究旨在探讨褪黑素对大鼠脂肪来源干细胞成骨分化的影响。方法：本研究考察了不同浓度褪黑素对大鼠ADSCs的细胞形态、生长密度及增殖活性的影响。采用碱性磷酸酶（alkaline phosphatase, ALP）染色、ALP活性检测、茜素红染色（alizarin red staining, ARS）、实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹（Western-blot）及免疫荧光实验，测定不同浓度褪黑素对大鼠ADSCs成骨分化、成骨标志物基因及标志物蛋白表达的影响，筛选出促进大鼠ADSCs成骨分化的最优褪黑素浓度。通过RNA测序、丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路抑制剂阻断实验及p38小干扰RNA（small interfering RNA, siRNA）干扰实验，分析褪黑素促进大鼠ADSCs成骨分化的分子机制。结果：形态学观察及CCK-8实验结果显示，0~100 μmol/L褪黑素不会影响大鼠ADSCs的细胞形态、生长密度及增殖活性。ALP染色、ALP活性检测及ARS结果表明，褪黑素可促进大鼠ADSCs的成骨分化。此外，RT-qPCR、Western-blot及免疫荧光实验结果显示，褪黑素可上调大鼠ADSCs中成骨标志物基因及蛋白的表达。