Polygonatum sibiricum polysaccharide attenuates bone marrow-derived macrophages to differentiate into osteoclasts and protects against lipopolysaccharide-induced osteolysis in vivo / 中国组织工程研究

ABSTRACT

BACKGROUND: Bone marrow-derived mononuclear cells (BM-MNCs) hold the potential of differentiating into osteoclasts. Polygonatum sibiricum polysaccharide (PSP) may inhibit the differentiation of BM-MNCs into osteoclasts and it is expected to become a new drug for the treatment of osteoporosis. OBJECTIVE: To investigate the effect of PSP on the differentiation of mouse BM-MNCs into osteoclasts induced by receptor activator of nuclear factor kappa-B ligand (RANKL) and bone resorption in vivo. METHODS: Mouse bone marrow-derived macrophages cultured in vitro, the effect of macrophage colony stimulating factor and PSP (5, 10, 20, 40, 80,160, 320, 640, 1280, 2560 mg/L) on the proliferation of mouse BM-MNCs was detected by cell counting kit-8 assay to determine the PSP concentration range; the mouse BMMs were cultured and induced in DMEM medium containing macrophage colony stimulating factor, RANKL and 5, 10, 20, 40, 80,160, 320, 640 mg/L PSP, respectively; those cultured without PSP served as control group. The morphological changes of cells were observed under an inverted microscope.; the number of osteoclasts was detected by tartrate-resistant acid phosphatase staining; the mRNA expression levels of osteoclast-related genes including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 were evaluated by quantitative real-time PCR. A mouse model of calvarial osteolysis induced by lipopolysaccharide was established to receive PSP intervention, and then micro CT scanning, three-dimensional reconstruction and relevants software were used for quantitative analysis of bone volume/volume percentage, trabecular number, trabecular bone spacing and thickness. The number of osteoclasts was identified by tartrate-resistant acid phosphatase staining and quantitative analysis of bone resorption area was conducted. RESULTS AND CONCLUSION: Compared with the control group, the concentration of PSP below 640 mg/L showed no significant effect on the proliferation of BMMs (P > 0.05). Different concentrations of PSP (40-640 mg/L) significantly reduced the number of osteoclasts, osteoclast differentiation and maturation, and the mRNA expression levels of tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, and nuclear factor of activated T cells c1 TRAP, MMP-9, CtsK and NFATc1 (P < 0.05). Compared with lipopolysaccharide, PSP could effectively alleviate the lipopolysaccharide-induced calvarial osteolysis, and the bone volume/volume percentage, trabecular number, and trabecular bone spacing were significantly decreased (P < 0.05); additionally, the number of osteoclasts and the area of bone resorption were decreased significantly (P < 0.01). To conclude, PSP can inhibit the differentiation and maturation of mouse BMMs to osteoclasts and alleviate lipopolysaccharide-induced calvarial osteolysis.