Objective: To investigate the effects of Epimedium on proliferation, function and apoptosis of mouse osteoblasts in vitro.
Methods: Primary osteoblasts were obtained by sequential digestion of mouse calvaria with collagenase and hyaluronidase. The identification of derived cells was done by histochemical staining of alkaline phosphatase (ALPase) and immunohistochemical staining of type I collagen, bone sialoprotein and osteopontin. MTT assay was employed to examine the proliferation of osteoblasts after treatment with Epimedium. The alkaline phosphatase activity level of mouse osteoblasts was also determined through an enzyme dynamical method. Apoptosis of osteoblasts was induced by dexamethasone and flow cytometry was utilized to examine the effects of Epimedium on the dexamethasone-induced apoptosis of osteoblasts.
Results: Five populations of bone cells were obtained by sequential digestion. Osteoblasts were purely obtained by discarding the first two populations and identified by the positive staining of ALPase, type I collagen, bone sialoprotein, and osteopontin. The alkaline phosphatase activity level of osteoblasts was significantly increased by the addition of Epimedium at 0.1 - 10 g/L, with the most significant increase at 1 g/L. On the other hand, the proliferation of osteoblasts was not affected after different doses of Epimedium added into the culture medium. Determined by flow cytometry, apoptosis of osteoblasts were induced by treatment with dexamethasone for 72 h. However, simultaneous administration of 1 g/L Epimedium had no effects on dexamethasone-induced apoptosis in osteoblasts.
Conclusion: Epimedium did not affect the cell proliferation and cell survival of mouse osteoblasts, but could significantly increase alkaline phosphatase activity of the cells. The increase of alkaline phosphatase activity by Epimedium in osteoblasts may be one of the important mechanisms by which Epimedium can effectively prevent osteoporosis.