OBJECTIVETo investigate the effect of 1,25-dihydroxyvitamin D3 (1,25VD3) on house dust mites (HDM)-induced expression of thymic stromal lymphopoietin (TSLP) in human airway epithelial cells in vitro.

METHODSHuman airway epithelial 16HBE cells were incubated with 200, 400, and 800 U/L in the absence or presence of 1,25VD3 (10(-8) mol/L) for 6 h and 24 h, and TSLP mRNA and protein expressions in the cells were assessed using quantitative PCR and ELISA.

RESULTS16HBE cells incubated with HDM at 200, 400, and 800 U/L showed significantly increased TSLP mRNA and protein expressions (P<0.05). Pretreatment of the cells with 1,25VD3 obviously lowered 400 U/L HDM-induced TSLP expressions (P<0.05), but 1,25VD3 added along with HDM in the cells did not produce significant effects on TSLP expressions (P=0.58).

CONCLUSIONBoth 1,25VD3 and HDM can induce TSLP expression and release in 16HBE cells, but pretreatment with 1,25VD3 can decrease HDM-augmented TSLP expression in the cells.

Animals ; Bronchi ; cytology ; Calcitriol ; pharmacology ; Cell Line ; Cytokines ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Pyroglyphidae

OBJECTIVETo establish a convenient and efficient model for investigating the expression of CYP3A4 and drug metabolism in vitro.

METHODS1alpha,25-dihydroxyvitamin D3 was utilized as an inducer to enhance CYP3A4 expression in HepG2 cells. 0.1, 0.25, 0.35 micromol/L 1alpha,25-dihydroxyvitamin D3 were added to the cell culture media, and cells were harvested after 24, 48, 72 and 96 hours. Cell proliferation was determined with MTT assay. CYP3A4 mRNA level was analyzed with RT-PCR and expressions of CYP3A4 protein were measured by Western blot.

RESULTS1alpha,25-dihydroxyvitamin D3 in 3 concentrations, namely 0.10, 0.25 and 0.35 micromol/L, did not show obvious toxicity to HepG2 cells. At 24 h of the cultivation, the expression of CYP3A4 mRNA was not increased significantly, but CYP3A4 mRNA expression significantly increased by 120%, 134%, 200% at 48 h, by 174%, 254%, 420% at 72 h, and by 258%, 450%, 370% at 96 h, respectively under the three concentrations. Similar results were observed in the induction of CYP3A4 protein expression. At 48, 72 and 96 hours after treatment with 0.25 micromol/L and 0.35 micromol/L 1alpha,25-dihydroxyvitamin D3, CYP3A4 protein increased in various folds in the controls (1.2 and 2.2 after 48 h, 3.4 and 6.5 after 72 h, 6.1 and 7.2 after 96 h), while 0.10 micromol/L 1alpha,25-dihydroxyvitamin D3 only induced protein expression at 72 h and 96 h (1.8 and 4.1 folds, respectively).

CONCLUSION1alpha,25-dihydroxyvitamin D3 could induce the expression of CYP3A4 mRNA as well as CYP3A4 protein in HepG2, which provides a convenient and efficient in vitro system for investigation of CYP3A4 and drug interaction.

Calcitriol ; pharmacology ; Cytochrome P-450 CYP3A ; drug effects ; genetics ; metabolism ; Hep G2 Cells ; Humans ; Transcription, Genetic

OBJECTIVETo evaluate the effect of 25-hydroxyvitamin D(3) on the expression and distribution of vitamin D receptor in normal human bronchial epithelial cells.

METHODSMTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-h exposure to different concentrations of 25-hydroxyvitamin D(3). Real-time quantitative PCR, Western blotting, and immunofluorescence assay were used to observe the expression and distribution of vitamin D receptor in the cells following the exposure.

RESULTSCompared with the control cells, 16HBE cells exposed to different concentrations of 25-hydroxyvitamin D(3) exhibited no significantly increase in the expression or distribution of vitamin D receptor.

CONCLUSIONThe influence of 25-hydroxyvitamin D(3) on bronchial epithelial cells might be independent of the expression and translocation of vitamin D receptor.

Bronchi ; cytology ; Calcifediol ; pharmacology ; Cell Line ; Epithelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Receptors, Calcitriol ; genetics ; metabolism

OBJECTIVETo investigate the effect of 1,25 (OH)(2)D(3) on cell proliferation and the expression of tumor necrosis factor-α (TNF-α ) in human gastric carcinoma SGC-7901 cells.

METHODSSGC-7901 cells were treated with 1×10(-6), 1×10(-7), 1×10(-8), and 1×10(-9) mol/L 1,25 (OH)(2)D(3) for 24, 48, 72 and 96 h. The cell proliferation was measured by MTT assay, and the cell cycle changes were analyzed using flow cytometry. RT-PCR and Western blotting were used to determine the expression of TNF-α mRNA and protein, respectively.

RESULTS1,25 (OH)(2)D(3) significantly inhibited SGC-7901 cell proliferation (P<0.05) in a time- and dose-dependent fashion. Treatment with 1,25 (OH)(2)D(3) for 72 h caused significant cell cycle arrest at G(0)/G(1) phase (F=9.81, P<0.05) and dose-dependently inhibited the expression of TNF-α at both mRNA and protein levels in SGC-7901 cells (P<0.05).

CONCLUSIONThe inhibitory effect of 1,25 (OH)(2)D(3) on SGC-7901 cell proliferation is probably associated with the down- regulation of TNF-α expression.

Calcitriol ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Stomach Neoplasms ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVE1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a potent inducer of differentiation in myeloid leukemia cells, but its clinical use is limited due to its hypercalcaemic effect and resistance. Carnosic acid is a plant-derived polyphenol food preservative with chemoprotective effects against carcinogens. Recent research has shown that carnosic acid potentiates the effects of 1,25(OH)2D3 on differentiation of human leukemia cells. This study examined the effects of 1,25(OH)2D3 in combination with carnosic acid on monocytic differentiation as well as intracellular reactive oxygen species (ROS) and Ca2+ levels in human leukemia HL-60 cells.

METHODSHL-60 cells were randomly treated with 1 nmol/L 1,25(OH)2D3, 100 nmol/L 1,25(OH)2D3, 10 micromol/L carnosic acid, a combination of 1 nmol/L 1,25(OH)2D3 and 10 micromol/L carnosic acid or placebo. Cell growth was observed by MTT assay for 72 hrs at an interval of 24 hrs. Cells were harvested after 72 hrs of culture. Morphologic features of the cells were observed by microscopy. Flow cytometry was used to detect cell cycle, monocytic differentiation marker CD14 expression, and intracellular ROS and Ca2+ levels.

RESULTSA combination of 1 nmol/L 1,25(OH)2D3 and 10 micromol/L carnosic acid resulted in greater proliferation inhibition (Ab: 0.56 0+/- 0.020 vs 1.482 +/- 0.327; P <0.01), mature monocytic features, G0 /G1 cell arrest, higher CD14 expression (57.62 +/- 0.817% vs 2.76 +/- 0.828%; P <0.01), lower intracellular ROS levels (52.67 +/- 10.76% vs 86.46 +/- 40.52%; P <0.01 and similar intracellular Ca2+ levels in HL-60 cells when compared with the placebo group. The ability of a combination of 1 nmol/L 1,25(OH)2D3 and 10 micromol/L carnosic acid to inhibit the proliferation and induce the differentiation of HL-60 cells was similar to that of 100 nmol/L 1,25(OH)2D3, while the intracellular Ca2+ level (115.64 +/- 17.74 nmol/L vs 185.75 +/- 27.38 nmol/L) was significantly lower than that in the 100 nmol/L 1,25(OH)2D3 group.

CONCLUSIONSLow concentration of 1,25(OH)2D3 combined with 10 micromol/L carnosic acid can produce enhanced differentiation, proliferation inhibition and antioxidant effects of HL-60 cells. The combination of the two inducers dose not increases intracellular Ca2+ levels.

Calcitriol ; pharmacology ; Calcium ; metabolism ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Diterpenes, Abietane ; pharmacology ; HL-60 Cells ; Humans ; Lipopolysaccharide Receptors ; analysis ; Plant Extracts ; pharmacology

Animals ; Bone Density Conservation Agents ; pharmacology ; Calcitriol ; pharmacology ; Calcium Channel Agonists ; pharmacology ; Humans ; Pulmonary Fibrosis ; metabolism ; Signal Transduction ; drug effects ; Transforming Growth Factor beta ; metabolism ; Wnt Proteins ; metabolism

OBJECTIVETo study the effects of 1, 25-dihydroxyvitamin D(3) (VD(3)) on the expression of vitamin D receptor (VDR), receptor activator of nuclear factor-kappa B ligand (RANKL) and osteoprotegerin (OPG) in human periodontal ligament cells (hPDLC) populations and to analyze the potential mechanisms.

METHODSTwelve hPDLC populations were primarily established from 12 donors individually. Two samples of each hPDLC population of passage three were treated respectively with 10(-8) mol/L VD(3) (V D(3) group) or 0.1% absolute ethyl alcohol as controls (control group). Six days later, the mRNA expression levels of VDR, RANKL and OPG in the samples were determined with real-time quantitative RT-PCR. The DNA base sequences upstream to the transcription start site of RANKL gene were also analyzed.

RESULTSCompared with the control group, the mRNA expression level of VDR increased significantly in the VD(3) group (P = 0.003), averagely (3.04 +/- 1.06) times of that in the control group; the mRNA expression level of RANKL was also up-regulated by VD(3) (P = 0.001), 9.82 (0.75-119.18) times of that in the control group; the OPG expression level was (94.48 +/- 39.15)% of the controls (P = 0.136); OPG/RANKL ratio was down-regulated in the VD(3) group to averagely 10.36% (1.01%-138.00%) of the controls (P = 0.003). No mutation was found in the DNA fragments upstream to the transcription start site in the RANKL gene and the genotypes of the polymorphism at -1832 (rs7984870, C/G) were not shown to be significantly related to the RANKL mRNA expression level.

CONCLUSIONSIn hPDLC, VD(3) can significantly increase the mRNA expression level of VDR; VD(3) can increase RANKL mRNA expression level to decrease OPG/RANKL ratio, but it has little effect on OPG mRNA expression. The big differences of the RANKL mRNA regulation in response to VD(3) treatment among hPDLC populations may not be associated with the DNA sequences upstream to the transcription start site in the RANKL gene.

Adolescent ; Calcitriol ; pharmacology ; Cells, Cultured ; Down-Regulation ; Female ; Humans ; Male ; Osteoprotegerin ; metabolism ; Periodontal Ligament ; drug effects ; metabolism ; RANK Ligand ; metabolism ; Receptor Activator of Nuclear Factor-kappa B ; metabolism ; Receptors, Calcitriol ; metabolism ; Up-Regulation ; Young Adult

OBJECTIVEThis study aimed at investigating the effects of vitamin D analogue EB1089 on the proliferation and apoptosis of hepatic carcinoma cells.

METHODSHepatic carcinoma cell strain G(2) (Hep-G(2)) in which prominent vitamin D receptor (VDR) mRNA could be expressed and the cell strain T (HCC-T) negative in VDR gene expression were incubated in culture media with 100 nmol/L, 10 nmol/L and 1 nmol/L EB1089 for 2 d, 4 d and 6 d, respectively. Survival and proliferation of the cells were detected by blue tetrazolium colorimetric test and plate clone-forming test, the VDR mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and apoptosis of the cells was detected by flow cytometry (FCM) and electron microscopy.

RESULTSEB1089 could inhibit the proliferation of hepatocellular cell line Hep-G(2) that expressed prominent vitamin D receptor mRNA, the inhibitory rate is 17.5% approximately 72.1%. On the other hand, EB1089 had no anti-proliferative effect on hepatocellular cell line HCC-T in which the gene expression of vitamin D receptors was negative. The electron microscope results showed that EB1089 could induce apoptosis of hepatocarcinoma cells and the percentages of apoptotic cells measured by flow cytometer was 21.4%. Cell cycle progression was blocked at G(1) phase with EB1089.

CONCLUSIONEB1089 could inhibit proliferation of human Hep-G(2), probably through VDR, and induce apoptosis of the cells.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Calcitriol ; analogs & derivatives ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Division ; drug effects ; Humans ; Liver Neoplasms ; pathology ; Tumor Cells, Cultured

It is well known that estrogen can inhibit bone absorption, decrease bone turnover and preserve bone mass. Some studies indicated that the effect of estrogen on calcium and bone is relative to vitamin D system, while others also reported that this effect of estrogen is independent of vitamin D. The genomic effect of 1alpha, 25(OH)(2)D(3)is mediated by the nuclear vitamin D receptor (VDR) in a ligand-dependent manner. Hypocalcemia, hyperparathyroidism and osteomalacia are developed in VDR gene knockout mice. To determine whether the effect of estrogen on calcium and bone is dependent on VDR, this study examined the effect of exogenous estrogen on calcium and bone homeostasis in VDR gene knockout mice. Male and female wild type (WT) and VDR gene knockout heterozygous mice were mated each other and the genotyping of their offsprings were determined by PCR. At age of 21-day, WT and knockout mice were weaned and treated by one of three different regimens: (1) WT-vehicle group: the WT mice were injected with normal saline; (2) VDR KO-vehicle group: the VDR gene knockout mice were injected with normal saline; (3) VDR KO-E group: the VDR gene knockout mice were subcutaneously injected with estradiol, 0.2 mug per mouse, once daily for 1 month. The bone mineral density (BMD) of mice was measured using dual-energy X-ray absorptiometry. All mice were sacrificed at age of 50-day. Blood was taken by heart puncture under anesthesia and serum calcium was measured by autoanalyser.Tibiae were removed, fixed and embedded with the methylmethacrylate (MMA), and undecalcified sections were cut. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue. Static histomorphometric analyses were performed on those stained sections. The results showed that the serum calcium level was (2.10+/-0.37) mmol/L in the VDR KO-vehicle mice and rose to (2.80+/-0.41) mmol/L in the VDR KO-E mice although it was still lower than WT-vehicle mice [(3.10+/-0.48) mmol/L]. BMD and mineralized trabeculer volume were increased significantly in VDR KO-E group compared with that in VDR KO-vehicle group. These results suggest that exogenous estrogen can improve calcium absorption and skeletal mineralization in a VDR-independent manner.
Animals ; Bone Density ; Calcification, Physiologic ; drug effects ; Calcium ; metabolism ; Estrogens ; pharmacology ; Female ; Gene Knockout Techniques ; Homeostasis ; Mice ; Mice, Knockout ; Receptors, Calcitriol ; genetics

Animals ; Calcitriol ; pharmacology ; Diabetes Mellitus, Experimental ; complications ; Diabetes Mellitus, Type 2 ; complications ; Liver ; drug effects ; Liver Diseases ; complications ; drug therapy ; Male ; Rats ; Rats, Sprague-Dawley