To investigate the expressions of mucosal barrier proteins in colon cell line DLD-1 under hypoxic environment and its mechanism. Methods After DLD-1 cells were treated separately with hypoxia(l% O),vitamin D(100 nmol/L),or vitamin D plus hypoxia for 48 hours,the expressions of vitamin D receptor(VDR),tight junction proteins zonula occludens-1(ZO-1),occludin,Claudin-1,and adherent junction protein(E-cadherin)were determined by Western blot.Stable VDR knock-down(Sh-VDR)DLD-1 cell line and control DLD-1 cell line were established by lentivirus package technology and the protein expressions after hypoxia treatment were detected. Results Compared with control group,the expressions of occludin,Claudin-1,and VDR increased significantly after hypoxia treatment(all <0.001).In addition to the protein expressions of occludin,Claudin-1 and VDR,the expressions of ZO-1 and E-cadherin were also obviously higher in vitamin D plus hypoxia group than in single vitamin D treatment group(all <0.001).After hypoxia treatment,Sh-VDR cell line showed significantly decreased expressions of ZO-1(<0.001),occludin(<0.05),Claudin-1(<0.01)and E-cadherin(<0.001)when compared with untreated Sh-VDR cell line. Conclusion VDR acts as a regulator for the expressions of intestinal mucosal barrier proteins under hypoxia environment in DLD-1 colon cell line,indicating that VDR pathway may be another important protective mechanism for gut barrier in low-oxygen environment.
Antigens, CD ; metabolism ; Cadherins ; metabolism ; Cell Hypoxia ; Cell Line ; Claudin-1 ; metabolism ; Colon ; cytology ; Humans ; Occludin ; metabolism ; Receptors, Calcitriol ; metabolism ; Tight Junctions ; Vitamin D ; pharmacology ; Zonula Occludens-1 Protein ; metabolism

PURPOSE: This study aimed to investigate whether Mullerian inhibiting substance (MIS) in combination with calcitriol modulates proliferation and apoptosis of human ovarian cancer (OCa) cell lines (SKOV3, OVCAR3, and OVCA433) and identify the signaling pathway by which MIS mediates apoptosis. MATERIALS AND METHODS: OCa cell lines were treated with MIS in the absence or presence of calcitriol. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay and apoptosis was evaluated by DNA fragmentation assay. Western blot and enzyme-linked immunosorbent assay were used to determine the signaling pathway. RESULTS: The cells showed specific staining for the MIS type II receptor. Treatment of OCa cells with MIS and calcitriol led to dose- and time-dependent inhibition of cell growth and survival. The combination treatment significantly suppressed cell growth, down-regulated the expression of B-cell lymphoma 2 (Bcl-2), and up-regulated the expressions of Bcl-2 associated X protein, caspase-3, and caspase-9 through the extracellular signal-regulated kinase signaling pathway. CONCLUSION: These results, coupled with a much-needed decrease in the toxic side effects of currently employed therapeutic agents, provide a strong rationale for testing the therapeutic potential of MIS, alone or in combination with calcitriol, in the treatment of OCa.
Anti-Mullerian Hormone/*pharmacology ; Apoptosis/*drug effects ; Calcitriol/*pharmacology ; Caspase 3/metabolism ; Caspase 9/metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/*drug effects ; Cell Survival/drug effects ; DNA Fragmentation/*drug effects ; Enzyme-Linked Immunosorbent Assay ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Female ; Growth Inhibitors/metabolism/pharmacology ; Humans ; Ovarian Neoplasms/*drug therapy/metabolism/*pathology ; Receptors, Peptide ; Receptors, Transforming Growth Factor beta ; Signal Transduction/*drug effects

OBJECTIVETo study the effects of 1,25-(OH)(2)D(3) on airway remodeling and expression of high mobility group box 1 (HMGB1) and IL-17 in asthmatic mice.

METHODSFifty female mice were randomly divided into 5 groups: control, asthma, low-dose, middle-dose, and high-dose intervention groups (n=10 each). Asthma was induced by intraperitoneal injections of ovalbumin (OVA) and aerosol inhalation of OVA solution. The low-dose, middle-dose, and high-dose intervention groups were administered with 1,25-(OH)(2)D(3) solution at the dosage of 1, 4 and 10 μg/kg respectively by intraperitoneal injections before asthma challenge. The airway structural changes were assessed by hematoxylin and eosin staining. mRNA expression levels of HMGB1 and IL-17 in the lung tissues were evaluated by RT-PCR. The protein levels of HMGB1 and IL-17 in the lung tissues were observed by immunohistochemistry.

RESULTSThe airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 were higher in the untreated asthma group than in the control group (P<0.05). The airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 were lower in the middle-dose and low-dose intervention groups than in the untreated asthma group, and the middle-dose intervention group demonstrated lower airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 than in the low-dose intervention group (P<0.05). However, the airway wall thickness, protein and mRNA expression levels of HMGB1 and IL-17 in the high-dose intervention group were higher than in the untreated asthma group (P<0.05).

CONCLUSIONSHMGB1 and IL-17 may be involved in the airway remodeling process in asthmatic mice. A moderate amount of HMGB1 and IL-17 may be involved in the airway remodeling process in asthmatic mice. A moderate amount of 1,25-(OH)(2)D(3) can improve the airway remodeling, but a higher dose of 1,25-(OH)(2)D(3) may affect adversely the airway remodeling process.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; metabolism ; pathology ; Calcitriol ; pharmacology ; Dose-Response Relationship, Drug ; Female ; HMGB1 Protein ; analysis ; genetics ; physiology ; Interleukin-17 ; analysis ; genetics ; physiology ; Lung ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C

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 investigate the effects of 1,25-(OH)(2)D(3) on the airway remodeling and expression of high-mobility group box 1 (HMGB1) and Toll-like receptor 4 (TLR4) in the lungs among asthmatic mice.

METHODSThirty female mice (BALB/c strain) were randomly divided into control, asthma and 1,25-(OH)(2)D(3) intervention groups. An asthmatic mouse model was established by intraperitoneal injection and aerosol inhalation of ovalbumin. The intervention group was given 1,25-(OH)(2)D(3) by intraperitoneal injection 0.5 hour before each aerosol inhalation, while the control group used normal saline instead. The hematoxylin-eosin staining was used to observe the mouse airway structural changes. The mRNA and protein expression of HMGB1 and TLR4 was measured by RT-PCR and immunohistochemistry, respectively. Pearson correlation analysis was performed.

RESULTSThe asthma group had a significantly increased airway wall thickness compared with the control group (P<0.05); the intervention group had a significantly lower increase in airway wall thickness than the asthma group (P<0.05). The mRNA and protein expression of HMGB1 and TLR4 was significantly higher in the asthma group than in the control group (P<0.05); the mRNA and protein expression of HMGB1 and TLR4 in the intervention group was significantly lower than that in the asthma group, but still higher than that in the control group (P<0.05). A positive correlation was found between the protein expression of HMGB1 and TLR4 (P<0.01), and so was their mRNA expression (P<0.01).

CONCLUSIONSHMGB1 and TLR4 may be involved in asthmatic airway remodeling. 1,25-(OH)(2)D(3) can reduce the airway remodeling in asthmatic mice, which may be related to the downregulation of HMGB1 and TLR4 expression in the lungs of asthmatic mice.

Airway Remodeling ; drug effects ; Animals ; Asthma ; drug therapy ; metabolism ; Calcitriol ; pharmacology ; therapeutic use ; Female ; HMGB1 Protein ; genetics ; Lung ; metabolism ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis ; Toll-Like Receptor 4 ; genetics

OBJECTIVETo investigate the correlation between regulatory T (Treg) cells and postmenopausal osteoporosis and the antiosteoporotic effect of 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] in relation to Treg cells.

METHODSFifty female BALB/c mice were randomly divided into five groups: the basal control (BAS), Sham, ovariectomy (OVX), OVX+diethylstilbestrol (OVX+DES), and OVX+1,25(OH)₂D₃. Tibias were harvested and processed with decalcification for quantitative bone histomorphometry. Femurs were stained by immunohistochemistry to detect Foxp3 protein expression. Spleens were used to detect Treg and Foxp3 gene expression by flow cytometry and quantitative RT-PCR, respectively.

RESULTSIn comparison with the Sham group, a significant decrease was found in the OVX group in such indices as trabecular bone volume/total tissue area (BV/TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th). 1,25(OH)₂D₃and DES partly prevented the decrease in BV/TV, Tb.N, Tb.Th in OVX mice. Treg cell number, Foxp3 mRNA expression in spleen and Foxp3 protein expression in femur significantly decreased in the OVX-treated group compared with those in the sham group. 1,25(OH)2D₃and DES significantly increased Treg cell number and Foxp3 expression. Treg cells and Foxp3 gene expression were related to bone histomorphometric parameters.

CONCLUSIONThe decrease in Treg cell numbers is relevant to the postmenopausal osteoporosis. The antiosteoporosis of 1,25(OH)₂D₃is related to regulatory T cells.

Animals ; Bone Density Conservation Agents ; pharmacology ; therapeutic use ; Calcitriol ; pharmacology ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Mice ; Mice, Inbred BALB C ; Osteoporosis ; drug therapy ; Ovariectomy ; T-Lymphocytes, Regulatory ; drug effects

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Acid Phosphatase/metabolism ; Animals ; Blotting, Western ; Calcitriol/*pharmacology ; Calcium Channel Agonists/pharmacology ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Enzymologic/*drug effects ; Isoenzymes/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Mice ; Osteoclasts/*cytology/*enzymology ; Tetrazolium Salts ; Thiazoles

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 establish a mouse model of asthmatic airway remodeling and investigate the effects of 1,25-(OH)2D3 on airway structure and T cell immunoglobulin mucin protein-4 (TIM-4) expression in asthmatic mice.

METHODSThirty female mice (BALB/c strain) were randomly divided into control, asthma and 1,25-(OH)2D3 intervention groups. An asthmatic mouse model was induced using ovalbumin. Lung tissue of the mice was collected, mRNA expression of TIM-4 was evaluated by RT-PCR and airway remodeling and protein expression of TIM-4 were observed by hematoxylineosin staining and immunohistochemistry.

RESULTSTypical airway remodeling was found in the asthma group, and TIM-4 expression in this group was significantly higher than in the control group (105±9 vs 42±5; P<0.05). Compared with the asthma group, the 1,25-(OH)2D3 intervention group showed improvement in airway remodeling and a decrease in TIM-4 expression (78±6) (P<0.05).

CONCLUSIONSTIM-4 may be involved in the airway remodeling of mice. As a new type of immunoregulator, 1,25-(OH)2D3 can downregulate expression of TIM-4 in the lungs and improve airway remodeling in asthmatic mice.

Airway Remodeling ; Animals ; Asthma ; metabolism ; Calcitriol ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Lung ; metabolism ; Membrane Proteins ; analysis ; genetics ; physiology ; Mice ; Mice, Inbred BALB C ; RNA, Messenger ; analysis

OBJECTIVETo study the effect of 1,25-(OH)2D3 on lipopolysaccharide (LPS)-induced expression of interleukin-13 (IL-13) and interleukin-17 (IL-17) in cord blood CD4(+)T cells, providing theoretical basis for clinical reasonable application of vitamin D and prevention of asthma and allergic diseases.

METHODSMononuclear cells (MNCs) were isolated from umbilical cord blood (50 mL) of 12 normal eutocia term newborns by gravity centrifugation. CD4(+)T cells were isolated using magnetic beads, which was cultured with following three kinds of stimulus for 72 hours: natural state (blank group), LPS (10 μg/mL)stimulation alone and LPS(10 μg/mL)+1,25-(OH)2D3 (10(-8) mmol/L)stimulation. Levels of IL-13 and IL-17 in the culture supernatant and mRNA expressions in cord blood CD4(+)T cells were detected using ELISA and real Time-PCR respectively.

RESULTSCompared with the blank group, levels of IL-13 and IL-17 in the culture supernatant and mRNA expression of IL-13 and IL-17 in the cord blood CD4(+)T cells increased in the LPS stimulation alone group (P<0.01). When co-stimulation of 1,25-(OH)2D3 with LPS, levels of IL-13 and IL-17 in the culture supernatant and mRNA expression of IL-13 and IL-17 in the cord blood CD4(+)T cells decreased compared with LPS-stimulated alone group (P<0.05), but remained higher than the blank group (P<0.01).

CONCLUSIONSLPS can promote expression of IL-13 and IL-17 in cord blood CD4(+)T cells. 1,25-(OH)2D3 inhibits the expression of IL-13 and IL-17, suggesting that vitamin D intake may provide protective effects in the development of atopy-predisposing immune responses in early life.

Asthma ; drug therapy ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; Calcitriol ; pharmacology ; Female ; Fetal Blood ; immunology ; Humans ; Infant, Newborn ; Interleukin-13 ; blood ; genetics ; Interleukin-17 ; blood ; genetics ; Lipopolysaccharides ; pharmacology ; Male ; RNA, Messenger ; blood