Effects Of Geraniin On Osteogenic Differentiation And Wnt/β-catenin Signaling Pathway In Rabbit With Osteoporotic Fracture BMSCs

Objectives:To investigate the effects of geraniin on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem eells(BMSCs)in osteoporotic fractures,explore the regulation of geraniin on the expression of key factors β-catenin,Wnt5a and Lef-1 in Wnt/β-catenin signaling pathway.Methods:1.Rabbit OPF model was duplicated:The rabbits were resected bilaterally with ovaries and the oviducts were ligated.bone mineral density(BMD)and bone mineral content(BMC)were measured after 3 months of normal feeding.Hematoxylin-eosin(HE)confirmed that the successful of replication osteoporosis model;bone saw wire was used to excision the bone of the middle segment of radius about 5 mm to duplicate the fracture model.2.Isolation and identification of BMSCs:BMSCs were isolated by gradient centrifugation after two weeks,cultured to the third generation,BMSCs were identified by observation of cell morphology and Flow cytometry.3.Effect of geraniin on proliferation of BMSCs:Different concentrations(10-11～10-8 mol/L)of geraniin were incubated with BMSCs,and the cell proliferation was detected by CCK-8 method.4.Effect of geraniin on osteogenic differentiation of BMSCs:BMSCs were incubated with geraniin at different concentrations(10-11～10-8 mol/L),cell alkaline phosphatase stain was used to detect the effect of geraniin on osteogenic differentiation of BMSCs.5.Effects of geraniin on expression of β-catenin,Lef-1 and Wnt5a:Q-PCR was used to detect mRNA expression level;Western blotting was used to detect protein expression level.Results:1.OPF model was duplicated successfully:Castrated rabbits were kept for 3 months the bone mineral density and bone content of the whole body were significantly reduced in model animals.The HE staining model group was sparsely trabecular,and the chondrocyte layer became thin and disordered.X-rays after fracture showed obvious bone defect at the bone biting site.2.The isolated cells were BMSCs:The cultured cells had typical morphology of BMSCs,Flow cytometry revealed that surface antigens CD44 and CD90 were highly expressed,while CD34 and CD45 were poorly expressed.3.Test results of CCK-8 showed that:Geraniin has no obvious promoting effect on the proliferation of BMSCs in rabbits with osteoporotic fracture.4.The results of cell alkaline phosphatase stain were shown:geraniin can significantly increase the number of calcium nodules and ALP activity in BMSCs.5.Q-PCR and Western blotting experimental results were shown:geraniin could significantly up-regulate the expression of β-catenin and Lef-1 mRNA and protein and down-regulate the expression of Wnt5a mRNA and protein.Conclusions:1.Geraniin had no significant effect on BMSCs proliferation in rabbits with osteoporotic fracture.2.Geraniin significantly up-regulated the expression of β-catenin,Lef-1 protein and mRNA in Wnt/β-catenin signaling pathway,and down-regulated the expression of Wntsa protein and mRNA,and signifieantly promotes osteogenie differentiation of rabbit BMSCs in osteoporotic fractures.