Effects Of Epimedium Flavonoids On Wnt/β-catenin Signaling Pathway In SAMP6 Mouse Osteoblasts

Objective: To investigate the effects of total flavonoids of epimedium(TFE)on signaling targets of Wnt/β-catenin,a key signaling pathway in SAMP6 mouse osteoblasts,and to explore the role of TFE in prevention of osteoporosis(osteoporosis,The mechanism of action of OP).METHODS: The skull of newborn SAMP6 mice was isolated by mechanical separation.SAMP6 mouse osteoblasts were cultured by enzyme digestion method.The morphology of SAMP6 mouse osteoblasts was observed by alkaline phosphatase staining and R-J staining.Alizarin red staining showed calcified nodules to identify SAMP6 mouse osteoblasts.After successful identification,the third generation osteoblasts were divided into control group and experimental group(containing TFE1ug/ml,10ug/ml,20ug/ml).TFE culture medium was added to the corresponding concentration for 48 hours.RT-PCR and Western Blot were used to detect the expression of β-catenin,Runx2,and cyclin D1 m RNA and protein,respectively.Results: The growth curve of osteoblasts in SAPM6 mice: 1-3 days is the latent adaptation period of osteoblasts in SAMP6 mice,logarithmic growth phase of cells in3-7 days,and then becomes plateau phase.The SAMP6 mouse osteoblast cultured in vitro was stained by R-J and observed by microscope.The cytoplasm showed blue particles,and the nucleus was stained with purple-brown,with an average of 1-3nucleoli.ALP stained microscopy showed that the distribution of purple particles in the cytoplasm,alizarin red staining can be seen in the naked eye and the calcified orange nodules,indicating that isolation and culture of SAMP6 mouse cells are experimental osteoblasts.The study found that different concentrations of TFE can promote the proliferation of osteoblasts in SAMP6 mice.The OD values of experimental osteoblasts were higher than those of control osteoblasts,and the difference was statistically significant(P<0.01).The concentration of 20ug/ml promoted the most significant proliferation.The results of RT-PCR showed that the expression levels of β-catenin,Runx2,and cyclin D1 in osteoblasts of SAMP6 mice in each group were significantly increased compared with the control group,and the difference was statistically significant(P<0.01).The results of Western Blot showed that the protein content ofβ-catenin,Runx2,and cyclin D1 in osteoblasts of SAMP6 mice was significantly higher than that of the control group,and the difference was statistically significant(P<0.01).Conclusions: The SAMP6 mouse cells cultured in vitro have the characteristics of common osteoblasts after identification.TFE can promote the proliferation of SAMP6 mouse osteoblasts,and promote the expression of β-catenin,Runx2 and cyclin D1 m RNA and protein,with the most significant effect of TFE drug concentration20ug/ml.It is speculated that TFE may promote the proliferation of osteoblasts in SAMP6 mice through the Wnt/β-catenin signaling pathway.