The Effects of Epimedium on Proliferation, Differentiation, and Apoptosis of Cultured Mouse Osteoblasts in vitro

Objective: To study the effects of Epimedium on proliferation, differentiation and apoptosis of cultured 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), immunohistochemical staining of type I collagen, bone sialoprotein and osteopontin. MTT assay was employed to observe the proliferation of osteoblasts after different doses of Epimedium added into culture medium. The levels of alkaline phosphatase activity of mouse osteoblasts were determined by enzyme dynamical method. Different doses of dexamethasone were added to produce a system of apoptosis in osteoblasts. Flow cytometry was utilized to observe the effect of Epimedium on osteoblasts' apoptosis rate. Results: Purified osteoblasts were obtained by discarding the first two populations. The derived cells were identified to be osteoblastic cells by the criteria of ALPase, type I collagen, bone sialoprotein and osteopontin. The alkaline phosphatase activity level was increased obviously by Epimedium at 0.1mg/ml~10mg/ml(P<0.01), with the most marked increase at 1mg/ml. No marked proliferation was observed after different doses of Epimedium added into culture medium(P.>0.05). Dexamethasone treatment for 72h produced a dose-dependent increase in the number of apoptotic cells, determined by Flow cytometry, with a maximal response at 1000nmol/L dexamethasone in primary mouse osteoblasts. Simultaneous administration of 0.1mg/ml Epimedium had no effects on apoptosis in osteoblasts. Conclusion: Epimedium can markedly increase alkaline phosphatase activity level of mouse osteoblasts. It is one of the mechanisms by which Epimedium can effectively prevent and treat osteoporosis. Epimedium is a Chinese herb medicine used for many years in treatment of a great number of diseases. It has been proved in the prevention and treatment of osteoporosis and alveolar residual ridge resorption by our animal experiments.

Association of China Science Foundation (39830430) sported this paper.