| Background and Objective:Osteoporosis is a progressive systemic metabolic bone disease, characterized by bone mass decrease and microarchitectural deterioration of bone tissue, leading to increased bone fragility and risk of fracture. Currently about 200 million people suffer from osteoporosis, the incidence rate has leapt to the seventh place of common and frequently-occurring disease. At present, most of anti-osteoporosis drugs had no curative and long-term effects, showed a lack of security and leading to poor patient compliance, although to a certain extent they can alleviate clinical symptoms of patients, improve bone mineral density and reduce the risk of fracture. Our previous studies suggest that polygonatum sibiricum polysaccharide (PSP), the main active component of rhizoma polygonati, can inhibit bone absorption, improve bone loss and prevent osteoporosis in ovariectomized rats model. But its mechanism is not yet clear. In this study, we aimed to investigate the effect of PSP on osteoblastic differentiation of mouse bone marrow mesenchymal stem cells (BMSCs) in vitro and its possible mechanism by wnt/β-catenin signaling pathway.Methods:(1) The BMSCs were cultured in vitro and the change of cell morphology and the cellular growing state were observed under an inverted microscope. (2) MTT assay was performed to determine the effect of cell proliferation on BMSCs treated with different concentrations of PSP. (3) BMSCs of the experimental groups were cultured and induced in osteoblast medium containing final concentrations (5,10,25,50, 100mg/L) of PSP. The other cells treated with same amount of serum-free medium replaced PSP were set as the negative control group. (4) After treatment for 14 d, PNPP method was used to detect alkaline phosphatase activity. (5) The mineralization nodules were observed and stained with Alizarin red S at 4 weeks, and the number and area fraction were recorded under an inverted microscope. (6) The mRNA expressions of osteogenesis-related genes COL Ⅰ, ALP, Runx2, and osteocalcin were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). (7) The qRT-PCR was performed to determine the gene expression level of β-catenin, and Western Blot was performed to determine the expression level of β-catenin in total protein extracts, nuclear and cytoplasmic protein extracts. (8) After induction for 72h, the distribution of β-catenin in cell was detected by immunofluorescence assay. (9) The downstream β-catenin/TCF transcriptional activity was evaluated with the Dual-Luciferase Reporter Assay System.Results:(1) Normal BMSCs, with long, spindle-shaped, fibroblast-like morphology, appeared variable in cell volume with a short spindle shape after induced osteogenesis. (2) There was no statistically significant difference between the OD values, indicating that PSP at doses (5-100mg/L) had no inhibitory or cytotoxic effects. (3) Compared with the control group, the cell ALP activity was significantly improved by PSP in a dose-dependent manner (P <0.05), of which 25mg/L showed the most obvious effect. (4) The number and area fraction of mineralization nodules were significantly higher than that of control group (P< 0.05). There was no significant difference number between the treatment groups, however, area fraction increased in a dose-dependent manner. (5) The mRNA expression of osteogenesis-relate genes (COL I, ALP, Runx2, and osteocalcin) were significantly increased (P< 0.05). (6) The gene expression level of P-catenin increased along with extending the induction time, and the highest at 72h. The 10-,25- and 50-mg/L concentration groups were significantly higher than the control group. (7) The Western Blot results showed that PSP treatment in 5-50 mg/L could significantly increase the total and nuclear amount of P-catenin in a dose-dependent manner, peaking at 25mg/L, The β-catenin level in cytoplasmic protein extracts, however, has no obvious difference between groups. (8) The immunofluorescence results showed that, compared with the control group, the 25mg/L PSP group appeared to accumulate the β-catenin in the cytoplasm and especially in the nucleus. (9) The 10-,25- and 50-mg/L PSP treatment groups significantly enhanced TOPFlash luciferase activity compared with the control group, of which the 25mg/L group had a maximum effect, increased by 3 times.Conclusions:(1) Polygonatum sibiricum polysaccharide could promote the osteoblast differentiation of mouse BMSCs, indicating that PSP had a potentially anti-osteoporosis effect. (2) PSP increased the expression of P-catenin and promoted its nuclear translocation, and enhanced β-catenin/TCF transcriptional activity. Therefore, PSP could accelerate the osteoblast differentiation of BMSCs maybe through activating the Wnt/β-catenin signaling pathway. |
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