OBJECTIVE: To explore whether MOTS-c could improve osteoporosis by promoting osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs) via transforming growth factor-β (TGF-β)/Smad pathway.
MATERIALS AND METHODS: Rat BMSCs were isolated and cultured, followed by osteogenic and lipid differentiation. CCK-8 (cell counting kit-8) assay was performed to detect the highest treatment dose of MOTS-c that did not affect cell proliferation. Expressions of osteogenesis-related genes (ALP, Bglap, and Runx2) were detected by qRT-PCR (quantitative Real-Time Polymerase Chain Reaction) and Western blot, respectively. Alizarin red staining and alkaline phosphatase (ALP) cytochemical staining were carried out to evaluate the effect of MOTS-c on BMSCs osteogenesis. TGF-β/Smad pathway-related genes (TGF-β1, TGF-β2, and Smad7) in BMSCs treated with MOTS-c were detected. Finally, TGF-β1 was knocked down to investigate the regulatory effect of MOTS-c on BMSCs osteogenesis.
RESULTS: BMSCs exhibited an elongated morphology and was identified with a high purity by flow cytometry. After osteogenic differentiation, alizarin red staining and ALP staining were all positive. MOTS-c treatment could remarkably stimulate the formation of calcified nodules in BMSCs. Besides, TGF-β/Smad pathway-related genes were significantly upregulated after BMSCs were treated with MOTS-c. Promoted osteogenesis by MOTS-c treatment was reversed by the TGF-β1 knockdown.
CONCLUSIONS: MOTS-c promotes cell differentiation of BMSCs to osteoblasts via TGF-β/Smad pathway.