Abstract
Icariin, a prenylated flavonol glycoside isolated from Epimedium, has been considered as a potential alternative therapy for osteoporosis. The present study aimed to clarify the detailed molecular mechanisms of action of icariin on osteoblast function, using bone marrow-derived mesenchymal stem cells (BM‑MSCs). BM-MSCs were first stimulated by icariin. Then, gene and protein expression of cAMP/ PKA/CREB signaling molecules were analyzed by RT-PCR and western blotting (WB), and alkaline phosphatase (ALP) was analyzed in cell lysates by ELISA. MTT assays indicated that icariin did not have significant effects on cell viability up to 1 μM. Icariin showed a dose-dependent effect on the alkaline phosphatase activity of BM-MSCs. WB analysis showed that icariin treatment of BM-MSCs significantly enhanced the protein expression of protein kinase A (PKA) and cAMP-responsive element binding protein (CREB), while RT-PCR results showed that icariin dose-dependently increased the mRNA levels of PKA and CREB. Icariin induced BM-MSC differentiation by BMP2, Smad1, and Runx2. RT‑PCR and WB results indicated that icariin significantly increased the expression of BMP2, Smad1, and Runx2 in BM‑MSCs. These results suggest that icariin is an agonist of the cAMP/PKA/CREB pathway in BM-MSC differentiation, raising the possibility that it could be used in the treatment of osteoporosis.
Highlights
Osteoporosis is characterized by low bone mineral density (BMD) and loss of the structural and biomechanical properties that are required to maintain bone homeostasis (Ivanova et al, 2015)
Hormones that stimulate the synthesis of cAMP regulate many cell type-specific processes including gene transcription, cell proliferation, differentiation, FIGURE 2 - Icariin actives the cAMP/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signaling pathway in BM-Mesenchymal stem cells (MSCs). (A) Icariin showed a dose-dependent effect on alkaline phosphatase (ALP) activity in bone marrow-derived mesenchymal stem cells (BM-MSCs). (B) western blotting (WB) analysis showed that icariin treatment of BM-MSCs significantly enhanced protein expression of PKA and CREB. (C)(D) RT-PCR results showed that icariin dose-dependently increased the mRNA levels of PKA and CREB in BM-MSCs. vs. treatment with 0 μM, *P < 0.05, **P < 0.01
Previous studies have shown that activation of the PKA pathway synergistically participates in BMP-2induced osteoblastic differentiation, possibly by mediating the CREB and/or Ras/MAPK pathways, and activation of the PKA pathway may be one of the key Bone morphogenesis protein-2 (BMP-2)-activated signaling events that lead to osteogenic differentiation
Summary
Osteoporosis is characterized by low bone mineral density (BMD) and loss of the structural and biomechanical properties that are required to maintain bone homeostasis (Ivanova et al, 2015). Bone morphogenesis protein-2 (BMP-2), a member of the BMP family, is one of the best-characterized inducers of osteochondrogenesis and can increase the expression of markers of both osteo- and chondrogenic lineage differentiation of progenitor cells, such as MSCs. It has recently been shown that protein kinase A (PKA) activation. Icariin is a bone anabolic agent that may exert its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating expression of Cbfa1/Runx, OPG, and RANKL. This effect may contribute to its effect on the induction of osteoblast proliferation and differentiation, resulting in bone formation (Hsieh et al, 2010). The plates were read on a micro-plate reader at a wavelength of 492 nm
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