[Icariin enhances differentiation and maturation of rat calvarial osteoblasts in collagen hydrogel three-dimensional culture].

OBJECTIVE: To investigate the effects of icariin on the differentiation and maturation of rat calvarial osteoblasts(ROB) in collagen hydrogel three-dimensional culture. METHODS: ROB were obtained by enzyme digestion from the segregated neonatal SD rats skull and were embedded in 2 mg/mL rat tail collagen for three-dimensional culture. The growth state of ROB was observed by FDA/PI staining, HE staining and scanning electron microscopy. ROB were treated with icariin at the concentration of 1 × 10⁻4, 1 × 10⁻5, 1 × 10⁻6 and 1 × 10⁻7 mol/L respectively. The activity of alkaline phosphatase(ALP) was detected after 3, 6, 9 d of icariin treatment. Three-dimensional cultured ROB were treated with optimal concentration icariin for 12, 24, 36, 48 h and total RNA was extracted and the mRNA expressions of bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX-2) and Osterix were detected by real time RT-PCR. The protein expression of BMP-2, RUNX-2 and Osterix were examined by Western-blotting. RESULTS: ROB were cultured in collagen hydrogel successfully. FDA/PI staining, HE staining, and scanning electron microscopy showed that ROB adhered with collagen tightly and distributed homogeneously. Icariin at final concentration of 1 × 10⁻5, 1 × 10⁻6 and 1×10⁻7 mol/L all enhanced the activity of ALP of collagen hydrogel three-dimensional cultured ROB, and 1 × 10⁻6 mol/L was the optimal concentration. Besides, icariin (1 × 10⁻6 mol/L) increased mRNA and protein expression of BMP-2、RUNX-2 and Osterix compared to control group. CONCLUSION: Icariin can enhance the expression of osteogenic markers of ROB in collagen hydrogel three-dimensional culture significantly.

Desmodeleganine, a new alkaloid from the leaves of Desmodium elegans as a potential monoamine oxidase inhibitor.

Desmodeleganine (1), a new potential monoamine oxidase inhibitor, along with three known alkaloids, bufotenin (2), hydroxy-N, N-dimethyltryptamine N(12)-oxide (3), 2-(5-methoxy-1H-indol-3-yl)-N, and N-dimethylethylamine (4) were isolated from the leaves of Desmodium elegans. Their structures were elucidated by IR, MS, 1D and 2D NMR spectra. 1 showed strong monoamine oxidase inhibitory activity with IC50 value of 13.92 ± 1.5 µM, when the IC50 value of iproniazid as a standard was 6.5 ± 0.5 µM. The molecular modeling was also performed to explore the binding mode of compounds 1, 2 at the active site of MAO-A and MAO-B.

Higher Bioavailability of Organic Bound Zinc from High Zinc-Enriched Fungi.

The organic forms of trace elements are considered more bioavailable than the inorganic forms. Although yeast can enrich metal elements and convert inorganic zinc to organic species, its tolerability and transforming capacity are limited. It would therefore be very interesting to look for higher conversion and accumulation in zinc fungi to obtain organic bound zinc from the natural environment. In this paper, potato dextrose agar (PDA) medium containing 800 µg/mL zinc was used for initial screening, with twenty-two fungal strains that tolerated high zinc isolated from the natural environment, and one strain (No.LZ-1108) growing well at a zinc (II) concentration of 10,000 µg/mL. According to morphological analysis, 18S rDNA sequence analysis, and biophysical and biochemical characteristics, No.LZ-1108 was tentatively identified as Fusarium oxysporum. Using atomic absorption spectrometry, the zinc content in the No.LZ-1108 cells was found to be 6.7 mg/g dry cell. After oral administration to rats at a dose of 10 mg Zn (II)/kg body weight, the area under the plasma concentration-time curve (AUC) and the maximum zinc blood concentration (Cmax) of No.LZ-1108 and zinc gluconate were 8.10 g/L.min and 4.28 g/L.min, 23.72 µg/mL and 6.23 µg/mL, respectively. The AUC of No.LZ-1108 was significantly higher than those of zinc gluconate (P<0.05), and the mean relative bioavailability of AUC(test)/AUC(zinc gluconate) was 190 %, which showed that the bound zinc in No.LZ-1108 was more bioavailable than zinc gluconate. The present study reports an interesting alternative to developing zinc-based supplements from a natural source of zinc.

[Effect of osthol on apoptosis and bone resorption of osteoclasts cultured in vitro].

This study is to investigate the effect of osthol on osteoclasts' activity, bone resorption as well as apoptosis in vitro, and explore the mechanism of osthol in preventing osteoporosis. Osteoclasts were separated from long-limb bones of new born rabbits, cultured in 24-well plate with glass slices and bone slices, and treated by 1 x 10(-5) mol x L(-1) osthol. Osteoclasts were identified by observing live cells with phase contrast microscope, HE staining, TRAP staining and toluidine blue staining of bone resorption pits. The numbers of bone resorption pits were counted as well as the surface area of bone resorption on bone slice. Osteoclasts were stained with acridine orange to detect the cell apoptosis. The ratio of apoptotic osteoclasts was observed under fluorescence microscope. The gene expression of RANKL, OPG, TRAP and p-JNK1/2 protein expression were examined using real time PCR and Western blotting, respectively. Comparing with the control group without osthol, the rates of apoptotic osteoclasts increased obviously and the number and area of bone resorption pits decreased evidently with 1 x 10(-5) mol x L(-1) osthol. There is significant difference between control group and experiment group treated by 1 x 10(-5) mol x L(-1) osthol. Therefore, the osthol through RANK+RANKL/TRAF6/Mkk/JNK signal pathway inhibits the osteoclasts activity, enhances osteoclasts apoptotic and inhibits the bone resorption.