ABSTRACT
<p><b>OBJECTIVE</b>To explore the effect of total flavonoids of Herba Epimedium (FHE) on BMP-2/RunX2/OSX signaling pathway in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).</p><p><b>METHODS</b>Passage 3 BMSCs were randomly divided into the control group, the experimental group, and the inhibitor group. BMSCs in the control group were cultured in 0.2% dimethyl sulfoxide + Osteogenuxic Supplement (OS) fluid + DMEM/F12 culture media. BMSCs in the experimental group were intervened by 20 microg/mL FHE. BMSCs in the inhibitor group were intervened by 20 microg/mL FHE and 1 microg/mL NOGGIN recombinant protein. At day 9 alkaline phosphatase (ALP) activity was measured. Calcium nodules were stained by alizarin red staining and the density was observed. The transcription expression of osteogenic differentiation-related proteins (type I collagen, osteocalcin, and osteopontin) and related factors of BMP-2/RunX2/OSX signaling pathway was assayed by RT-PCR.</p><p><b>RESULTS</b>Compared with the control group, ALP activities were enhanced and the density of calcium nodules significantly increased; type I collagen, osteocalcin, and osteopontin expression levels were increased in the experimental group. The expression of osteogenesis-related transcription factor was also increased in the experimental group. Noggin recombinant protein inhibited FHE promoting BMSCs osteogenesis in the inhibitor group. Compared with the experimental group, ALP activity decreased (P < 0.05), the density of calcium nodules was lowered, expression levels of type I collagen, osteocalcin, osteopontin significantly decreased (P < 0.05) in the inhibitor group.</p><p><b>CONCLUSION</b>20 microg/mL FHE promoted osteogenic differentiation process of BMSCs by BMP-2/RunX2/OSX signaling pathway.</p>
Subject(s)
Humans , Bone Morphogenetic Protein 2 , Metabolism , Cell Differentiation , Cells, Cultured , Collagen Type I , Metabolism , Core Binding Factor Alpha 1 Subunit , Metabolism , Drugs, Chinese Herbal , Pharmacology , Epimedium , Chemistry , Flavonoids , Pharmacology , Mesenchymal Stem Cells , Cell Biology , Osteocalcin , Metabolism , Osteogenesis , Osteopontin , Metabolism , Signal Transduction , Sp7 Transcription Factor , Transcription Factors , MetabolismABSTRACT
A sensitive and selective LC-MS/MS method for determination of citalopram in human plasma was established to study the bioequivalence of different formulations containing citalopram. The samples were simply pretreated by protein precipitation using acetonitrile, and then analyzed on a Zorbax Extend C8 column. The mobile phase consisted of acetonitrile-water-formic acid (60:40:0.2), at a flow-rate of 0.5 mL x min(-1). A Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring using the precursor to product ion combinations of m/z 325 --> m/z 109 and m/z 265 --> m/z 167 was performed to quantify citalopram and the internal standard, respectively. The pharmacokinetic parameters of citalopram in different formulations were calculated by non-compartment model. The linear calibration curves were obtained in the concentration range of 0.10-100 microg x L(-1). The lower limit of quantification was 0.10 microg x L(-1). The intra- and inter-day relative standard deviation (RSD) over the entire concentration range was less than 5.2%. Accuracy determined at three concentrations (0.25, 8.00 and 90.0 microg x L(-1) for citalopram) ranged from -4.7% to 1.3%. Each plasma sample was chromatographed within 3.0 min. The method was successfully used in bioequivalence study of citalopram in human plasma after oral administration of 20 mg citalopram. Calculated with AUC(0-120 h), the bioavailability of two formulations was (102.1 +/- 10.9)%. The method is rapid, selective, robust and is proved to be suitable for bioequivalence evaluation of different formulations containing citalopram.