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KMID : 1377020220190010189
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Tissue Engineering and Regenerative Medicine
2022 Volume.19 No. 1 p.189 ~ p.202
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The Neuropeptide Spexin Promotes the Osteoblast Differentiation of MC3T3-E1 Cells via the MEK/ERK Pathway and Bone Regeneration in a Mouse Calvarial Defect Model
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Assefa Freshet
Kim Ju-Ang
Lim Ji-Won
Nam Sang-Hyeon
Shin Hong-In
Park Eui-Kyun
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Abstract
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BACKGROUND: The neural regulation of bone regeneration has emerged recently. Spexin (SPX) is a novel neuropeptide and regulates multiple biological functions. However, the effects of SPX on osteogenic differentiation need to be further investigated. Therefore, the aim of this study is to investigate the effects of SPX on osteogenic differentiation, possible underlying mechanisms, and bone regeneration.
METHODS: In this study, MC3T3-E1 cells were treated with various concentrations of SPX. Cell proliferation, osteogenic differentiation marker expressions, alkaline phosphatase (ALP) activity, and mineralization were evaluated using the CCK-8 assay, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), ALP staining, and alizarin red S staining, respectively. To determine the underlying molecular mechanism of SPX, the phosphorylation levels of signaling molecules were examined via western blot analysis. Moreover, in vivo bone regeneration by SPX (0.5 and 1 μg/μl) was evaluated in a calvarial defect model. New bone formation was analyzed using micro-computed tomography (micro-CT) and histology.
RESULTS: The results indicated that cell proliferation was not affected by SPX. However, SPX significantly increased ALP activity, mineralization, and the expression of genes for osteogenic differentiation markers, including runt-related transcription factor 2 (Runx2), Alp, collagen alpha-1(I) chain (Col1a1), osteocalcin (Oc), and bone sialoprotein (Bsp). In contrast, SPX downregulated the expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). Moreover, SPX upregulated phosphorylated mitogen-activated protein kinase kinase (MEK1/2) and extracellular signal-regulated kinase (ERK1/2). In vivo studies, micro-CT and histologic analysis revealed that SPX markedly increased a new bone formation.
CONCLUSION: Overall, these results demonstrated that SPX stimulated osteogenic differentiation in vitro and increased in vivo bone regeneration via the MEK/ERK pathway.
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KEYWORD
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Neuropeptide, Spexin, Bone regeneration, MEK/ERK pathway
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