Osthol, a coumarin isolated from common cnidium fruit, enhances the differentiation and maturation of osteoblasts in vitro.

The effect of osthol on osteoblasts was investigated in primary osteoblastic cells isolated from newborn Wistar rats. Osthol was supplemented into cultured medium at 10⁻7, 10⁻6, 10⁻5 and 10⁻4 mol/l, respectively. No stimulating effect was found on cell proliferation, but 10⁻5 mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. The results showed that the ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. Results of ALP histochemical staining and mineralized bone nodule assays both showed that the number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen. p38MAPK protein and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control. The results of these studies support the conclusion that osthol significantly enhances the osteogenic differentiation of cultured osteoblasts. The results also indicated that osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway and that osthol may be used as an important compound in the development of new antiosteoporosis drugs.

[The changes of iNOS and NO in the osteogenic differentiation process of rat bone marrow stromal cells promoted by icariside II].

This study is to investigate the effects on the expression of iNOS and production of NO in the osteogenic differentiation process of rat bone marrow stromal cells (rBMSCs) by icariside II. rBMSCs were cultured by adherence screening method. When the culture dishes were covered with 80% cells, the osteogenic induced cultures were adopted. Icariside II was supplemented into the culture at 1 x 10(-5) mol x L(-1). The activity of iNOS, content of NO and osteogenic differentiation markers including alkaline phosphatase (ALP) activity, CFU-FALP and mineralized bone nodules were compared among the icariside II-supplemented group, L-NMAE group, icariside II + L-NAME group and the control. Total RNA was isolated and the gene expression of iNOS, Osterix and Runx-2 was investigated by real-time PCR. Total protein was also isolated and the secretion of iNOS and collagen I was examined by Western blotting. Icariside II can significantly improved ALP activity, CFU-FALP amount and mineralized nodules. Besides, the mRNA level of factors related to the osteogenic differentiation includes Osterix and Runx-2 also enhanced. The secretion of collagen I also promoted significantly. But all of these effects can be inhibited by L-NAME which can specifically inhibit the activity of iNOS. Icariside II enhances the osteogenic differentiation of rBMSCs significantly, but if the activity of iNOS was blocked by L-NAME, the osteogenic differentiation markers decrease accompanied with iNOS and NO decrease, suggesting that icariside II stimulates the osteogenic differentiation via enhancing the activity of iNOS and promoting the generation of NO.

[Effects of icariin on the proliferation, differentiation and maturation of rat calvarial osteoblasts in vitro].

OBJECTIVE: To investigate the effects of icariin on the proliferation, differentiation and maturation of rat calvarial osteoblasts (ROB). METHODS: Segregated neonatal SD rat skull,enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Icariin was added into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol/L respectively. MTT method was adopted in proliferation analysis. The activity of ALP was assayed after 9 days' induced. Optimal concentration icariin was added into the medium, then the osteogenic differentiation markers including mineralized bone nodules, CFU-F(ALP) were compared between the icariin-added group and the control. Total RNA was isolated and the gene expressions of Runx-2 and Osterix were investigated by Real Time RT-PCR. Total protein was also isolated and the secretion of collagen I was examined by Western-blot. RESULTS: The ROB proliferation was inhibited by icariin in a dose-dependent manner. But it evidently led to osteogenic process and maturation. 1 x 10(-5) mol/L was the best concentration. Icariin improved the secretion of collagen I, CFU-F(ALP) amounts and mineralized nodules significantly. It also enhanced the mRNA level of Runx-2 and Osterix. CONCLUSION: The icariin with final concentration of 1 x 10(-5) mol/L can enhance the osteogenic differentiation and maturation of ROB significantly, suggesting that icariin has the activity of inducing bone formation, it has the potential to be developed into a new drug of anti-osteoporosis.