Icariside II promotes the osteogenic differentiation of canine bone marrow mesenchymal stem cells via the PI3K/AKT/mTOR/S6K1 signaling pathways.

The aim of the present study was to investigate the osteogenic effects of icariside II (ICSII) on canine bone marrow mesenchymal stem cells (BMSCs) and the pathways by which these effects were induced. BMSCs were cultured and expanded to the fourth passage. The proliferative effects of ICSII were assessed using the cell counting kit-8 (CCK-8) assay. The osteogenic response to ICSII in BMSCs in vitro was examined by alkaline phosphatase (ALP) activity assays and Alizarin red staining. Using Western blot assays and real-time PCR (RT-PCR), we examined the expression of osteogenetic proteins/genes. We also evaluated changes in Akt and S6K1 phosphorylation levels, along with changes in the expression of osteogenesis proteins/genes after pretreatment with wortmannin (an inhibitor of phosphatidylinositol 3-kinase; PI3K) or rapamycin [a specific inhibitor of mammalian target of rapamycin (mTOR)] in the presence or absence of ICSII. Our results show that ICSII promotes the proliferation of BMSCs, while inhibiting ALP activity. We also found that calcium nodules form after BMSCs are treated with ICSII and osteogenetic medium for 21 days. ICSII significantly increased the expression of osteogenesis proteins/genes and elevated the phosphorylation levels of Akt and S6K1. Treatment with wortmannin or rapamycin attenuated the expression of p-Akt, p-S6K1, and osteogenesis proteins/genes. These results suggest that ICSII promotes the osteogenic differentiation of canine BMSCs via the PI3K/AKT/mTOR/S6K1 signaling pathways.