Icariin stimulates the osteogenic differentiation of rat bone marrow stromal cells via activating the PI3K-AKT-eNOS-NO-cGMP-PKG.

Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10(-5)M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K-AKT-eNOS-NO-cGMP-PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10(-6)M (a physiologically achievable concentration in vivo) also activated this signal pathway.

Zhai YK ，Guo XY ，Ge BF ，Zhen P ，Ma XN ，Zhou J ，Ma HP ，Xian CJ ，Chen KM ... -  《-》

Icariin promotes osteogenic differentiation of rat bone marrow stromal cells by activating the ERα-Wnt/β-catenin signaling pathway.

Icariin, the main effective component of Herba Epimedii, has been identified to regulate the osteogenic differentiation of rat bone marrow stromal cells (rBMSCs) and have potential application in preventing bone loss and promoting bone regeneration. However, the underlying signaling pathway of its osteogenic effect remains unclear. Here, we investigated whether icariin could promote osteogenesis of rBMSCs through modulating the estrogen receptor alpha (ERα) and Wnt/β-catenin signaling pathways, which implicated in rBMSCs osteogenesis. rBMSCs were cultured in osteogenic induction medium and treated with icariin or together with ICI 182780 or DKK1, the effects on the expression of osteogenic genes and Wnt/β-catenin signaling were detected. Results indicated that icariin (0.1μM) markedly enhanced the proliferation of rBMSCs and alkaline phosphatase (ALP) activity. Additionally, icariin (0.1μM) significantly up-regulated the expression of osteogenic genes (Runx2, osteopotin, DLX5, osteocalcin, collagen type I, and ERα) and Wnt signal members (β-catenin, Lef1, TCF7, c-jun, c-myc, and cyclin D). Furthermore, icariin stimulated the activation of β-catenin as evidenced by increased total β-catenin protein and nuclear translocation. These osteogenesis-potentiating effects of icariin were blocked by ICI 182780 or DKK1. Taken together, these results suggest that the osteogenic effects of icariin on rBMSCs involves the ERα-Wnt/β-catenin signaling pathway.

Wei Q ，Zhang J ，Hong G ，Chen Z ，Deng W ，He W ，Chen MH ... -  《-》

Osteogenesis and angiogenesis induced by porous β-CaSiO(3)/PDLGA composite scaffold via activation of AMPK/ERK1/2 and PI3K/Akt pathways.

As a potential bioactive material, β-calcium silicate (β-CS) has attracted particular attention in the field of bone regeneration. In this study, porous β-CS/Poly-D,L-Lactide-Glycolide (PDLGA) composite scaffolds were developed with the goals of controlling the degradation rate and improving the mechanical and biological properties. The compressive strength and toughness were significantly enhanced by PDLGA modification of porous β-CS ceramic scaffolds. The effects of the ionic extract from β-CS/PDLGA composite scaffolds on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs), proliferation of human umbilical vein endothelial cells (HUVECs) and the related mechanisms were investigated. It was shown that bioactive ions from β-CS/PDLGA scaffolds could enhance cell viability, alkaline phosphatase (ALP) activity, calcium mineral deposition, and mRNA expression levels of osteoblast-related genes of rBMSCs without addition of extra osteogenic reagents. The activation in AMP-activated protein kinase (AMPK), extracellular signal-related kinases (ERK) 1/2 and RUNX-2 were observed in rBMSCs cultured in the extract of β-CS/PDLGA, and these effects could be blocked by AMPK inhibitor Compound C. The extracts of β-CS/PDLGA composites stimulated HUVECs proliferation that was associated with phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) as well as an increase in nitric oxide (NO) production and secretion of vascular endothelial growth factor (VEGF). The inductions were abolished by the addition of phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. The composite scaffolds were implanted in critical sized rabbit femur defects (6 × 10 mm) for 4, 12 and 20 weeks with β-tricalcium phosphate (β-TCP) as controls. Sequential histological evaluations and radiographs revealed that β-CS/PDLGA dramatically stimulated new bone formation and angiogenesis. The biodegradation rate of the β-CS/PDLGA scaffolds was lower than that of β-TCP at each time point examined, and matched the new bone formation rates. These data suggest that β-CS/PDLGA could promote bone regeneration in vivo, which might be ascribed to the enhanced osteogenic differentiation of mesenchymal stem cells (MSCs) and increased angiogenic activity of endothelial cells (ECs).

Wang C ，Lin K ，Chang J ，Sun J ... -  《-》

Glimepiride promotes osteogenic differentiation in rat osteoblasts via the PI3K/Akt/eNOS pathway in a high glucose microenvironment.

Ma P ，Gu B ，Xiong W ，Tan B ，Geng W ，Li J ，Liu H ... -  《PLoS One》

Calycosin stimulates the osteogenic differentiation of rat calvarial osteoblasts by activating the IGF1R/PI3K/Akt signaling pathway.

Calycosin has been reported to have a strong osteogenic activity and a positive correlation with anti-osteoporosis effects. However, its precise mechanism of action remains unclear. Since insulin-like growth factor 1 receptor (IGF1R) signaling and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling have been shown to play a pivotal role in regulating osteogenesis, we hypothesized that the osteogenic activity of calycosin is mediated by these signaling pathways. Rat calvarial osteoblasts (ROBs) were cultured in osteogenic medium containing calycosin with or without GSK1904529A (GSK) or LY294002 (LY) (inhibitors of IGF1R and PI3K, respectively). The effects on cell proliferation, alkaline phosphatase (ALP) activity, calcified nodules, mRNA or protein expression of osteogenic genes [alkaline phosphatase (Alpl), collagen type I (Col1a1), runt-related transcription factor 2 (Runx2), Osterix, and bone morphogenetic protein 2 (Bmp2)], and phosphorylation of IGF1R and Akt were examined. The present results showed that calycosin enhanced cell proliferation, ALP activity and Alizarin Red-S staining in a dose-dependent manner in the range of 10-8 -10-6  M, while an inhibitory effect was observed at 10-5  M. Treatment at the optimal concentration (10-6  M, a physiologically achievable concentration) increased mRNA levels of osteogenic genes and phosphorylation of IGF1R and Akt. Furthermore, treatment with GSK or LY partly reversed the effects of calycosin on ROBs, as indicated by the decreases in calycosin-induced ALP activity, calcified nodules and osteogenic gene expression. These results suggest that the osteogenic effect of calycosin partly involves the IGF1R/PI3K/Akt signaling pathway.

Fang Y ，Xue Z ，Zhao L ，Yang X ，Yang Y ，Zhou X ，Feng S ，Chen K ... -  《-》