Icariin Promote Bone Formation Dependent on cAMP-PKA Signaling Pathway

OBJECTIVE To study the role of cAMP-PKA signaling pathway in the osteogenic differentiation process of rat calvarial osteoblasts(OB) induced by icariin. METHODS Osteoblasts were obtained by enzyme digestion from the segregated neonatal SD rat skull. Serial subcultivation was proceeded when cells covered with 80% culture dish. OB and MCF-7 cells were treated by 1×10^-5 mol·L^-1 icariin separately, nuclear translocation of estrogen receptor α (ERα) in cells was detected by immunofluorescence. Icariin and 2’,3’-dideoxyadenosine (DDA) were supplemented into the culture separately at 1×10^-5 mol·L^-1. After further incubation for different time, cAMP level in the cultured cells was determined using a cAMP enzyme immune assay system in accordance with the manufacturer’s instructions. Cells were treated with icariin (1×10^-5 mol·L^-1) and KT5720 (1×10^-6 mol·L^-1) separately. The alkaline phosphatase ALP activity was determined at the 3rd and 6th day. Total RNA was isolated and the gene expression of ALP, collagen I and Runx-2 were investigated by real-time PCR. Finally total protein was also isolated and the secretion of collagen I and Runx-2 was examined by Western blot. RESULTS ERα nuclear translocation were found in MCF-7 treated by icariin after 4 h, but this phenomenon coundn’t be detected in OB cells. Elevation of intracellular cAMP concentration was consistently noted in all cultured cells following icariin treatment. The increasing of cAMP was blocked after the treatment of DDA. icariin significantly improved ALP activity, the level of factors related to the osteogenic differentiation included collagen I, Runx-2 and ALP also enhanced. The secretion of collagen and Runx-2 also promoted significantly. But all of these effects could be inhibited by KT5720 which could specifically inhibit the activity of protein kinase A(PKA). CONCLUSION Icariin can elevate intracellular cAMP concentration and promote bone formation dependent on cAMP-PKA signaling pathway.