Modern pharmacological studies have shown that SWT extract has anti pruritic and anti inflammatory effects, and protects against radiation induced bone marrow damage in an animal model. Previous stud ies have shown that anti inflammatory selleck Bortezomib and anti oxidant agents have the potential to treat osteoporosis by increas ing bone formation andor suppressing bone resorption. However, the effect of SWT on bone cell function has not yet been reported. In the current study, we report that SWT extract increases ALP BMP 2, and OPN expres sion and bone mineralization. Furthermore, we show that the phosphatidylinositol Inhibitors,Modulators,Libraries 3 kinase, Akt, and NF ��B signaling pathways are involved in the SWT mediated in crease in gene expression and bone mineralization. Finally, treatment of mice with SWT extract prevented bone loss induced by ovariectomy in vivo.

Our data, therefore, sug gest that SWT may be used to stimulate bone formation for the treatment of osteoporosis. Methods SWT extract and materials SWT extract was kindly provided by Timing Pharmaceut ical Company. The extraction and isolation of SWT were performed as previously de scribed. Rabbit polyclonal antibodies for BMP 2, OPN, p p85, p85, p Akt, Inhibitors,Modulators,Libraries Akt, p p65, and p65 were purchased from Santa Cruz Biotechnology. The osteopontin BMP 2 ELISA kit was purchased from Inhibitors,Modulators,Libraries Biosource Technology. The C terminal telopeptides of type I collagen ELISA kit was obtained from Cross Laps. p85 and Akt siRNAs were purchased from Santa Cruz Biotechnology. All other reagents were obtained from Sigma Aldrich. Cell culture The murine osteoblast cell line MC3T3 E1 was purchased from American Type Culture Collection.

Cells were cultured in 5% CO2 with MEM supplemented with 20 mM HEPES and 10% heat inactivated fetal calf serum, 2 mM glutamine, penicillin, and streptomycin. Measurement of mineralized nodule formation Levels of mineralized nodule formation were evaluated as previously described. Briefly, osteoblasts Inhibitors,Modulators,Libraries were cultured in medium containing Inhibitors,Modulators,Libraries vitamin C and B glycerophosphate for 2 wks, and the medium was changed every 3 d. After incubation with SWT extract for 12 d, cells were washed twice with 20 mM Tris buffered saline containing 0. 15 M NaCl, fixed in ice cold 75% ethanol for 30 min, and air dried. Calcium deposition was determined using alizarin red S staining. Briefly, ethanol fixed cells and matrix were stained for 1 h with 40 mM alizarin red S and rinsed extensively with water.

The bound stain was eluted with 10% cetylpyridinium chlor ide, and alizarin red S in the samples was quantified by measuring absorbance at 550 nm and comparing to a standard curve. One mole of alizarin red S selectively binds approximately 2 moles of calcium. Quantitative real time PCR Total RNA was extracted from osteoblasts using a TRIzol KPT-330 chemical structure kit. Reverse transcription was performed using 2 ug of total RNA and oligo primers.