Regulatory effect of uncarboxylated osteocalcin on osteogenic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions / 中国组织工程研究

ABSTRACT

BACKGROUND: The method of promoting osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions to inhibit adipogenic differentiation can provide prevention and treatment ideas for the treatment of bone metabolic diseases such as diabetic osteoporosis. OBJECTIVE: To explore the effects of uncarboxylated osteocalcin on adipogenic and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions so as to reveal the action mechanism of uncarboxylated osteocalcin on the differentiation of bone marrow mesenchymal stem cells. METHODS: Mouse bone marrow mesenchymal stem cells were cultured by whole bone marrow culture and adherent purification. Cells were treated with uncarboxylated osteocalcin at different concentrations (0, 1, 3, 10, and 30 μg/L). Cell proliferation was detected by cell counting kit-8 to determine the best mass concentration. Passage 3 bone marrow mesenchymal stem cells were incubated with adipogenic (or osteogenic) differentiation medium, and assigned to four groups control group, high glucose group, uncarboxylated osteocalci n group, and high glucose + uncarboxylated osteocalcin group. Corresponding groups received the addition of 25.5 mmol/L exogenous glucose and 3 μg/L uncarboxylated osteocalcin. Lipid droplets and calcium nodules were detected by oil red and alizarin red staining. Quantitati ve reverse transcription-polymerase chain reaction was used to detect the relative expression levels of adipogenic marker genes (Fabp4, PPARγ, Adipsin and FAS) and osteogenic differentiation marker genes (Runx2, Osx, alkaline phosphatase, and type I collagen). Kits were used to detect alkaline phosphatase activity and type I collagen levels. The relative expression levels of P-Erk and P-AMPKα were detected using signal pathway specific inhibitors (PD98059 and BML) and western blot assay. RESULTS AND CONCLUSION: (1) Uncarboxylated osteocalcin 3 μg/L promoted cell proliferation (P < 0.01). (2) Uncarboxylated osteocalcin promoted the formation of calcium nodules (P < 0.01) in bone marrow mesenchymal stem cells under high-glucose conditions but inhibited the formation of lipid droplets (P < 0.05), down-regulating the relative expression levels of adipogenic marker genes (PFabp4 < 0.01; PPPARγ < 0.05; PAdipsin < 0.01; PFAS < 0.01), but increasing the relative expression levels of osteogenic differentiation marker genes (PRunx2 < 0.05; POsx < 0.05; PALP < 0.01; PCOLI < 0.01). Uncarboxylated osteocalcin increased alkaline phosphatase activity (P < 0.01) and type I collagen level (P < 0.05). (3) Uncarboxylated osteocalcin up-regulated the expression levels of P-Erk (P < 0.01) and P-AMPKα (P < 0.01) under high-glucose conditions. (4) These results indicate that uncarboxylated osteocalcin promoted osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions through Erk/AMPKα signaling pathway and inhibited adipogenic differentiation.