ABSTRACT
BACKGROUND:Our previous studies have found that polygonatum sibiricum polysaccharide (PSP) promotes osteogenic differentiation of bone marrow mesenchymal stem cel s (BMSCs) by Wnt/β-catenin signaling pathway, but the molecular mechanism is unclear.OBJECTIVE:To investigate the effect of PSP promoting the osteogenic differentiation via Wnt signaling pathways in BMSCs after LRP5 silencing. METHODS:LRP5 interference vectors were constructed and then transfected into C57BL/6 mouse BMSCs cultured in vitro. The transfection efficiency of cel s was calculated under fluorescence inverted microscope and the expression of LRP5 protein was detected by western blot assay. The osteogenic potential of BMSCs after LRP5-siRNA transfection was analyzed by alkaline phosphatase staining, alizarin red staining and western blot assay. Effect of PSP on the osteogenic differentiation of LIRP5-silenced mouse BMSCs was detected by real-time PCR and dual luciferase assay. RESULTS AND CONCLUSION:Compared with the control group, the mineralization ability, the mRNA expressions of Runx2 and Osterix, and the protein expression of LRP5 were significantly decreased in the LRP5-siRNA group (P<0.05). PSP could promote LRP5-siRNA transfected mouse BMSCs differentiating into osteoblasts and significantly upregulated the expressions ofβ-catenin and Osterixin, and also induced the high expression of luciferase reporter gene (TOPFlash) containing wild type TCF binding sites (P<0.05). To conclude, LRP5 plays an important role in the process of mouse BMSCs differentiating into osteoblasts. PSP can promote the osteogenic differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway independent on LRP5.
ABSTRACT
BACKGROUND:Bone marrow mesenchymal stem cels (BMSCs) have the ability of multi-directional differentiation. Polygonatum sibiricum polysaccharide can promote osteogenetic differentiation of mouse BMSCs by activating Wnt/β-catenin signaling pathway, which is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE:To investigate the effects of Polygonatum sibiricum polysaccharide on Wnt/β-catenin signaling pathway in the osteogenic differentiation of mouse BMSCs. METHODS:The mouse BMSCs were cultured and induced in osteoblast medium containing final concentrations (5, 10, 25, 50mg/L) of Polygonatum sibiricum polysaccharide. The mouse BMSCs treated without Polygonatum sibiricum polysaccharide was set as the negative control group. The morphological changes of cels were observed under an inverted microscope. Alkaline phosphatase (ALP) activity assay was performed by PNPP method. The mineralization nodules were observed and stained with alizarin red S and the number and area fraction were recorded under an inverted microscope. The mRNA expressions of osteogenesis-related genes ALP, Runx2, and osteocalcin were evaluated by quantitative real-time PCR (qRT-PCR). qRT-PCR and western blot were used to determine the expression level of β-catenin. The downstream β-catenin/TCF transcriptional activity was evaluated with the Dual-Luciferase Reporter Assay System. RESULTS AND CONCLUSION: Compared with the control group, polygonatum sibiricum polysaccharide significantly enhanced the alkaline phosphatase activity, the mineralization ability of cels, and the mRNA expression of ALP, Runx2 and osteocalcin in the differentiated BMSCs in a dose dependent manner (P <0.05). After induction, the mRNA expression of β-catenin was the highest on the 3rd day. Polygonatum sibiricum polysaccharide significantly increased the expression of β-catenin (P < 0.05) in the process of promoting the differentiation of BMSCs into osteoblasts, and also promoted the high-level expression of luciferase reporter gene (TOPFlash) which contains wild type TCF binding sites (P < 0.05). These results demonstrate that Polygonatum sibiricum polysaccharide can promote the osteoblast differentiation of mouse BMSCs by activating the Wnt/β-catenin signaling pathway.