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BACKGROUND: 1,25(OH)2D3 plays an important regulatory role in the development of diabetic nephropathy. OBJECTIVE: To explore the effect of 1,25(OH)2D3 on the expression of microRNA-130b and transforming growth factor pi in kidney of diabetic nephropathy rats. METHODS: The study was approved by the Laboratory Animal Ethical Committee of Laboratory Animal Center of Xinjiang Medical University. Twenty-five clean Sprague-Dawley rats were randomly divided into normal control group, diabetic nephropathy+1,25(OH)2D3 group and diabetic nephropathy+peanut oil group. The latter two groups were given calcitriol (1,25(OH)2D3, active vitamin D3, 0.03 jg/kg
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Tumor cells along with a small proportion of cancer stem cells exist in a stromal microenvironment consisting of vasculature, cancer-associated fibroblasts, immune cells and extracellular components. Recent epidemiological and clinical studies strongly support that vitamin D supplementation is associated with reduced cancer risk and favorable prognosis. Experimental results suggest that vitamin D not only suppresses cancer cells, but also regulates tumor microenvironment to facilitate tumor repression. In this review, we have outlined the current knowledge on epidemiological studies and clinical trials of vitamin D. Notably, we summarized and discussed the anticancer action of vitamin D in cancer cells, cancer stem cells and stroma cells in tumor microenvironment, providing a better understanding of the role of vitamin D in cancer. We presently re-propose vitamin D to be a novel and economical anticancer agent.
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Objective It is not yet clear whether 1,25-(OH)2D3acts on endoplasmic reticulum stress(ERS)and autoph-agy in pulmonary fibrosis(PF).This study aimed to investigate the roles of ERS and autophagy in the development and progression of pulmonary fibrosis in rats and the effects of 1,25-(OH)2D3on the expressions of ERS-related molecules and autophagy-related gene 12 (ATG12). Methods Ninety male SD rats were randomly divided into a control, a PF model and a treatment group of equal number.Bleomycin was injected into the tracheas of the latter two groups of rats to induce PF.On the second day after modeling, the rats of the treatment group were injected intraperitoneally with 1,25-(OH)2D3at 2 μg/kg,those of the PF model group with 1,25-(OH)2D3solvent at 200 μL,and those of the control group with iso-tonic saline at 200 μL,all once 2 days.Then the mRNA expressions of PERK,ATF4 and ATG12 were measured by real-time PCR and the protein expressions of PERK and ATF4 detected by immunohistochemistry. Results At 14, 21 and 28 days after treat-ment,the expression levels of PERK were significantly higher in the PF model group(2.30±0.19, 3.59±0.27, and 4.63±0.19) and treatment group(1.44±0.34,1.92±0.17,and 2.52±0.15)than in the control(1.01±0.23,1.05±0.09,and 1.04±0.08)(P<0.05), and so were the expression levels of ATF4 in the PF models(2.10±0.12, 3.91±0.14, and 6.20±0.28)and treated rats (1.49±0.27,2.52±0.42, and 4.02±0.31)than in the controls(1.04±0.07,1.05±0.08,and 1.03±0.10)(P<0.05).Compared with the control group,the PF model and treatment groups showed markedly increased expression levels of ATG 12 mRNA at 14 days (P<0.05), but decreased at 21 and 28 days(P<0.05).Both the expressions of PERK and ATF4 proteins were remarkably higher in the model and treatment groups than in the control at 14,21 and 28 days(P<0.05), increasing in a time-dependent manner. Conclusion By suppressing the PERK -eIF2α-ATF4 signaling pathway,1,25-(OH)2D3inhibits the development and progression of pulmonary fibrosis.
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Objective To investigate the effects of 1,25-dihydroxy vitamin D3 [1,25 (OH)2D3] on immune mechanism of RBC and spleen in colitis model mice.Methods Thirty mice were randomly grouped as follows:blank control group,model group and 1,25(OH)2D3 group.The animal models were established.Then the general condition and colon pathological changes in mice were observed,the changes of the RBC immune compound(RCIC) and RBC surface C3b receptor(RC3bR) wreath were detected by the yeast wreath method,the weight and length of spleen were measured,the positive rates of spleen immune cells were detected by flow cytometry.Results Compared with the model group,RBC-C3bR in the 1,25(OH)2D3 group and blank control group was significantly increased and RBC-ICR was significantly decreased (P＜0.01).Compared with the blank control group,the positive rates of CD3,CD4,CD8 and CD45R in spleen mononuclear cell were significantly increased (P＜0.01);after the 1,25 (OH) 2D3 intervention,the positive rates of CD3,CD4,CD8 and CD56R in spleen mononuclear cells were significantly decreased compared with the model group(P＜0.01).Conclusion 1,25(OH)2D3 has the immune regulatory effect on RBC and peripheral spleen lymphocytes in chronic colitis mice.
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Objective To investigate the effects of 1,25-dihydroxy vitamin D3 [1,25 (OH)2D3] on immune mechanism of RBC and spleen in colitis model mice.Methods Thirty mice were randomly grouped as follows:blank control group,model group and 1,25(OH)2D3 group.The animal models were established.Then the general condition and colon pathological changes in mice were observed,the changes of the RBC immune compound(RCIC) and RBC surface C3b receptor(RC3bR) wreath were detected by the yeast wreath method,the weight and length of spleen were measured,the positive rates of spleen immune cells were detected by flow cytometry.Results Compared with the model group,RBC-C3bR in the 1,25(OH)2D3 group and blank control group was significantly increased and RBC-ICR was significantly decreased (P＜0.01).Compared with the blank control group,the positive rates of CD3,CD4,CD8 and CD45R in spleen mononuclear cell were significantly increased (P＜0.01);after the 1,25 (OH) 2D3 intervention,the positive rates of CD3,CD4,CD8 and CD56R in spleen mononuclear cells were significantly decreased compared with the model group(P＜0.01).Conclusion 1,25(OH)2D3 has the immune regulatory effect on RBC and peripheral spleen lymphocytes in chronic colitis mice.
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Objective To investigate the impact of 1,25(OH)2D3 on histological changes and activation of STAT3 in BLM?induced pulmonary fibrosis mice. Methods 30 male C57BL/6 mice were randomly divided into control group ,BLM group and BLM+VD group. Mice in BLM group and BLM+VD group received intratracheal injection of BLM(3 U/kg). Control group were intratracheally injected equal volume of sterile saline. From the first day after the surgery,mice in BLM+VD group received intraperitoneal injection of VD (5μg/kg·d). After 21 days, H&E and Masson′s trichrome staining were carried out. Aschroft score were used to evaluate histological changes in lungs. IL?6,IL?4 and INF?γin BALF were assessed by Elisa. p?STAT3,α?SMA and Collagen I were detected by western blot (WB) and immunohistochemistry. Results Fibrosis score and level of α?SMA,Collagen I in BLM group were significantly higher than that in control group (P < 0.05). However ,treatment with VD effectively at?tenuated fibrosis (P<0.05). IL?6 and IL?4 increased while INF?γwas decreased in BALF of BLM group (P<0.05). VD could ameliorate these changes. Upregulation and neuclear translocation of p?STAT3 were observed in BLM group,while VD intervention could inhibit phosphorylation of STAT3. Conclusions VD attenuate BLM?induced pulmonary fibrosis and regulate inflammatory cytokines probably by blocking STAT3 activation.
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Objective To evaluate the relationship of high mobility group box 1 (HMGB1) and TLR4 with airway inflammation and the role of vitamin D.Methods Totally 24 BALB/c mice were randomly divided into control group,asthma group,and 1,25-(OH)2D3 group,each having 8 mice.The pathological changes in lung tissue of the mice were observed by hematoxylin-eosin (HE) staining,bronchial wall thickness was measured with computer pathological image analysis system software.The expressions of HMGB1 and TLR4 in lung tissue were detected by immunohistochemical method.Bronchoalveolar lavage fluid (BALF) was collected for cytological examination;the contents of HMGB1,TLR4,IL-4 and IFN-γ in BALF and the peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA).Results The expressions of HMGB1 and TLR4 in lung tissue were stronger in asthma group,but weaker in intervention group.The total number of leukocytes as well as the percentages of eosinophils,neutrophils and lymphocytes increased significantly in BALF in asthma group,but significantly decreased in intervention group (all P ＜ 0.05).The ratio of monocyte/macrophage significantly decreased in asthma group,but increased significantly in intervention group (P＜0.05).The contents of HMGB1,TLR4 and IL-4 in BALF and the peripheral blood were significantly higher in asthma group than in control and intervention groups,whereas IFN-γ level was significantly lower than that in control and intervention groups (all P＜0.05).HMGB1 and TLR4 contents had a positive correlation with the total number of cells and IL-4 concentration in BALF,respectively (r1=0.796,0.730;r2=0.695,0.648;all P＜0.05).Conclusion HMGB1 and TLR4 were associated with airway inflammation and immune disorders.An appropriate amount of 1,25-(OH)2D3 can relieve airway inflammation,which may be associated with regulating Th1/Th2 cells balance.
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Objective Idiopathic pulmonary fibrosis ( IPF) is a chronic inflammatory disease with unknown etiology and is lack of effective therapy. The aim of this study is to explore the function of Ca2+ and PI3K/AKT/mTOR pathway in the pathogenesis of IPF, and the impact of 1,25?( OH) 2 D3 on Ca2+ and PI3K/AKT/mTOR pathway in type Ⅱalveolar epithelial cells of rat with IPF. Methods 150 male SD rats were randomly divided into 2 groups: prevention group ( control groupⅠ, model groupⅠ, medication groupⅠ) and treatment group ( control groupⅡ, model groupⅡ, medication groupⅡ) . The tracheal exposure surgery was operated in control groupⅠ/Ⅱ, and then 200μL sterile physiological saline was administered by intraperitoneal injection of each rats 2 days and 14 days after surgery, separately. Bleomycin(BLM)(5 mg/kg) was in?jected into the trachea of model groupⅠ/Ⅱ, and then vitamin D3 solvent(0.1%ethanol and 99.9%glycol propylene, 1μL/g) was ad?ministered by intraperitoneal injection 2 days and 14 days after surger?y, separately. Bleomycin( BLM) ( 5 mg/kg) was injected into the tra?chea of medication groupⅠ/Ⅱ, and then 1,25?( OH) 2 D3( 2μg/kg) was administered by intraperitoneal injection 2 days and 14 days after surgery, separately. IPF model was built by injecting Bleomycin into the trachea of rats, 1,25?(OH)2D3(2μg/kg) was used to prevent and treat IPF by intraperitoneal injection in medication group. The hydroxyproline content of lung tissue in each group was measured, type Ⅱalveolar epithelial cells were separated from lung tissue and labeled with Fluo?3AM, then concentration of Ca2+ was detected by Laser scanning confocal microscope. The mRNA levels of PI3K, AKT and mTOR in the typeⅡalveolar epithelial cells were tested by RT?PCR. Results Compared with control groupⅠ/Ⅱ at each time point, hydroxyproline content of lung tissue, Ca2+ concentration and expression of PI3K, AKT and mTOR in typeⅡalveolar epithelial cells in model groupⅠ/Ⅱand medication groupⅠ/Ⅱwere sig?nificantly raised( P<0.05 or P<0.01) , but these were significantly reduced in medication groupⅠ/Ⅱcompared with model groupⅠ/Ⅱ( P<0.05 or P<0.01) . Correlation analysis showed that there is significant positive correlation between Ca2+ concentration and mRNA expression levels of PI3K, AKT and mTOR in model groupⅠ/Ⅱ(r=0.5988, r=0.6230, r=0.6603,P<0.01)and medication groupⅠ/Ⅱ( r=0.701 2, r=0.632 3,r=0.740 3,P<0.01) . Conclusion The PI3K/AKT/mTOR pathway plays an important role in devel?opment of IPF. 1,25?( OH) 2 D3 is able to reduce Ca2+concentration in typeⅡalveolar epithelial cells and inhibit the PI3K/AKT/mTOR pathway, and then inhibit the development of IPF.
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Marrow stromal cells ( MSCs) , component of the mar-row stroma, play an important role in the growth of bone and metabolic balance. This paper mainly summarizes and reviews the molecular regulaory effects of vitamin D and its active form 1, 25 dihydroxy vitamin D3[1,25(OH)2D3] on the osteogenet-ic differentiation of MSCs by Wnt signaling pathway, Wnt5a/ROR2 axis, BMP/TGF-β/Samd signaling pathway and ROS/ERK signaling pathway, so as to clarify the molecular signaling pathway through which vitamin D regulates MSCs metabolically.
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Objective To investigate the effects of Dermatophagoides pteronyssinus ( Der p) on the expression of TLR4 and IL-4 in P815 mast cells and to further analyze the immunoregulatory effects of 1,25-(OH)2D3 on Der p treated P815 mast cells.Methods Different concentrations of Der p and 1,25-( OH) 2 D3 were used alone or in combination to stimulate P815 mast cells.The supernatants of the stimulated cell culture were analyzed by enzyme-linked immunosorbent assay ( ELISA) for the detection of IL-4.The stimulated cells were collected and analyzed by real-time PCR and Western blot assays for the detection of TLR4atmRNAandproteinlevels,respectively.Results (1)TLR4expressionwasdetectedinP815 cells.The expression of TLR4 was enhanced in P815 cells treated with various concentrations of Der p.A significant dose-dependent up-regulation of TLR4 was observed in P815 cells after incubation with Der p for 36 h.(2) Der p promoted the release of IL-4 in P815 cells (P0.05).(4) 10-8 mol/L of 1,25(OH)2D3 promoted the Der p-induced expression of TLR4 in P815 cells (P<0.01).However, 1,25(OH)2D3 inhibited the release of IL-4 in a dose-dependent manner(P<0.05orP<0.01).Conclusion (1)Derpcouldpromotetheinflammationandallergicreac-tion through up-regulating TLR4 and IL-4 in mast cells.(2) The possible mechanism for the inhibitory of 1, 25(OH)2D3 on Der p-induced immune responses was due to the suppression of Th2-type immune responses through inhibiting the synthesis and secretion of IL-4 in mast cells.
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Objective to investigate the curative effect of activated vitamin D3 combined with IFN-α on CCl4-induced hepatic fibrosis in BALB/c mouse model. Methods the experimental mice received 100 μL of 10% CCl4 intraperitoneally to induce a model of hepatic fibrosis,and the nor-mal control group were administrated with same volume of 0.9% saline(n = 6). the experimental mice then were randomly divided into four treat-ment groups and treated for six weeks:saline therapy group(0.9% saline,100 μL/mouse,n = 6),IFN-α therapy group(IFN-α 800 U/mouse,n =6),1,25-(OH)2D3 therapy group(50 μg/kg,by gavaged,n = 6)and combined treatment therapy group(combined treatment,n=6). Hepatic inju-ry and hepatic pathology were examined by HE staining and Masson staining. Interleukin-6(IL-6),interleukin-10(IL-10),tumor necrosis factor-α(tNF-α),transforming growth factor-β(tGF-β)and Smad3 were determined by real-time PCR and ELISA. Results Inflammatory cell infiltra-tion and periportal collagen deposition were significantly reduced in all the therapeutic group expect saline therapy group assessed by HE and Masson staining. the IFN-α therapy group,1,25-(OH)2D3 therapy group and combined treatment therapy group displayed significant decrease in Col1α1, IL-6,tNF-α,tGF-β and Smad3 expression and increase in IL-10 expression in liver tissue(P < 0.05). Conclusion the effect of 1,25-(OH)2D3 combined with IFN-α therapy is similar to separated treatment.
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Objective To study the expressions of Ki67 and mTOR in Thy-1 nephritis model of rat who were given 1,25-dihydroxyvitamin D3[1,25(OH)2D3] and to explore its mechanism. Methods Healthy male SD rats (n=90) were random?ly divided into three groups: control group, model group, 1,25(OH)2D3 treatment group (n=30 in each group). Model group and 1,25(OH)2D3 treatment group were intravenously injected with anti-Thy1 monoclonal antibody once via tail vein while the control group were administrated with same volume of normal saline through the same route. 1,25(OH)2D3 were adminis?trated at 0.5μg per day intra-gastrically for consecutive 21 days in 1,25(OH)2D3 treatment group while equal volume of pea?nut oil were given in control group and model group. Six rats were randomly selected from each group and sacrificed at the 1st , 3rd , 7th , 14th and 21st after drug intervention. Twenty four hour urine sample were collected in each rat just before it was culled to detect 24-hour urinary protein excretion. Renal tissue samples were harvested and stained with hematoxylin&eo?sin (H&E) and PAS to determine the renal pathological variation and the expressions of mTOR and Ki67 were assessed by immunohistochemistry. Results Urine protein begin to be detected at the first day after model was established, peaked at the 3rd days then started dropping until the 14th day when urine sample turned to normal. Urine protein levels were lower in 1, 25(OH)2D3 treatment group at the 1st,3rd,7th day after model establishment than those in model group(P<0.05). Compared with model group, the pathological damage of renal tissue in 1,25(OH)2D3 treatment group were alleviated at the 3rd and 7th day after model establishment (P < 0.05). Expressions of Ki67 and mTOR in 1, 25(OH)2D3 treatment group were reduced compared with those in model group (P<0.05). Twenty four hour urinary protein and expressions of Ki67 and mTOR as well as renal pathological damage were all positively correlated with each other. Conclusion 1,25(OH)2D3 can inhibit the proliferation of glomerular mesangial cells in Thy-1 nephritis model of rat. And its therapeutic mechanism may be associated with down reg? ulating expressions of Ki67 and mTOR.
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Objective To investigate the effects of 1,25-dihydroxyvitamin D3[1,25 (OH)2D3] on cell proliferation in hu?man glomerular mesangial cells and it′s effects on the regulation of mTOR/p70s6K signaling pathway in this cell line. Meth?ods The cultured human mesangial cells at passage 3-7 were divided into four groups:control group,VD group (addition of 10-8 mol/L of 1,25-dihydroxyvitamin D3 ),R group (addition of 5 mg/L of rapamycin) and R+VD group(addition of 5 mg/L ra?pamycin combined with 10-8 mol/L of 1,25-dihydroxyvitamin D3). Drug incubation last 48 h. The effect of mesangial cell pro?liferation was measured by CCK-8 colorimetric assay. The cell cycles were measured by flow cytometry. The expression of mTOR and p70s6K were detected by immunofluorescence. Results (1) The absorbance of A450 was higher in control group than that in VD group than that in R group than that in R+VD group. But the inhibition rate (IR) was lower in control group than that in VD group than that in R group than that in R+VD group. All comparisons were of statistic significance. ( 2) Cells in G1 phase were higher while cells in G2/M and S phases as well as proliferation rate (PI) were lower in control group than those in VD group than those in R group than those in R+VD group. All comparisons were of statistic significance except in?dexes between group R and group VD. (3) mTOR and p30s6K expressions in mesangial cells were higher in control group than those in VD group than those in R group than those in R+VD group. All comparisons were of statistic significance ex?cept indexes between group R and group VD. Conclusion 1,25-dihydroxyvitamin D3 might inhibit mesangial cell prolifera?tion significantly through mTOR/p70s6K signaling pathways.
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Objective To investigate the effects of 1,25-dihydroxyvitamin D3 [1,25 (OH)2D3] on PCNA expression and cell proliferation in human glomerular mesangial cells. Methods The cultured human mesangial cells, which was subcul?tured 3-8 generations, were randomly divided into four groups: normal control group (plus the DMEM medium containing 5%fetal bovine serum), proliferation in the control group (EGF group, plus 10μg/L of EGF), general intervention group [VD group, plus 10-8 mol/L of 1,25(OH)2D3], proliferation in the intervention group [EGF+VD group, plus 10μg/L EGF and 10-8 mol/L 1,25(OH)2D3] for treatment of 48 h. The cell cycle was detected by flow cytometry,and the expression of PCNA was detected by Western blot assay in four groups. Results (1) Compared with normal control group, G1 phase cells were signifi?cantly reduced, S, G2/M phase cells were increased and PI index was higher in EGF group. And G1 phase cells were signifi?cantly increased, S and G2/M phase cells were significantly decreased, and PI index was lower in VD group. Compared with the EGF group, G1 phase cells were significantly increased in VD group and EGF+VD group, and S, G2/M phase cells de?creased, PI index was lower. (2) Compared with normal control group, the expression of PCNA was higher in EGF group, and lower in VD group. Compared with EGF group, the expression of PCNA was lower in VD group and EGF+VD group. Conclu?sion 1,25 (OH) 2D3 inhibits the proliferation of human glomerular mesangial cells by arresting cell cycle and inhibiting the expression of PCNA protein.
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Objective To investigate the effects of different doses of 1 ,25(OH)2D3 on rat Thy‐1 nephritis mesangial cell ap‐optosis .Methods 120 clean SD rats were divided into the blank control group(group A) and the experimental group .The experi‐mental group was re‐divided into the nephritis group(group B) ,0 .25μg 1 ,25(OH)2D3 intervention group(group C) and 0 .50μg 1 , 25(OH)2D3 group(group D) ,30 cases in each group .The group C and D were performed the medication intervention after success‐fully constructing the model .6 rats were randomly killed in each group on 1 ,3 ,7 ,14 ,21 d after intervention .The renal tissues were taken for determining the renal pathological injury classification after hematoxylin and eosin staining and PAS staining .The expres‐sion of Caspase‐3 in the renal tissues was detected by the immunohistochemistry method ,and the glomerular cell apoptosis was de‐tected by TUNEL .Results The immunohistochemistry results showed that compared with the group A ,the Caspase‐3 expression in the group B was increased (P 0 .05);the Caspase‐3 expression in the group D was highest ,but the difference was not statistically significant compared with the group C .The TUNEL results showed that compared with the group A ,the apoptotic glomerular cells in the group B were increase obviously (P0 .05) .Conclusion 1 ,25(OH)2D3 has the effect for inducing glomerular mesangial cell apoptosis in rat , which participates in the regulation of Caspase‐3 ,induces glomerular mesangial cell apoptosis ,promotes the recovery of nephritis and could delay the progression of renal disease .
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Objective To study the functional roles of 1,25(OH)2D3 in osteogenic differentiation of the dental papilla stem cells. Methods The dental papilla stem cells were isolated and cultured in medium supplemented with different con-centrations of 1,25(OH)2D3 (1, 10 and 100 nmol/L). MTT assay was used to detect the cell growth, and flow cytometry was used to detect the cell cycle. Western blot assay was used to detect protein expression levels of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG) and vitamin D receptor (VDR). After siRNA silencing VDR expression, protein levels of RANKL and OPG were detected. Results MTT and flow cytometry results showed that there were no sig-nificant differences in the cell proliferation between different concentrations of 1,25(OH)2D3 (1, 10 and 100 nmol/L) and con-trol groups (P>0.05). Western blot results showed that there were protein expressions of VDR, RANKL and OPG in control group. The protein expressions of VDR, RANKL and OPG were increased after adding 1,25(OH)2D3, in which the upward trend was the most significant in VDR. After VDR expression was silenced by siRNA, the protein expression levels of VDR, RANKL and OPG were decreased. Conclusion 1,25(OH) 2D3 affects the osteoblast differentiation process of the dental pa-pilla stem cells by adjusting the VDR expression.
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Objective To investigate the protective effect of 1,25-(OH) 2D3 on radiation-induced bone marrow microenvironment injury and to explore the related molecular mechanism.Methods Sixty 7-week old male BALB/c mice were randomly divided into control group without any treatment; radiation group exposed to 6.0 Gy 60Co γ-rays with DMSO,and 1,25-(OH)2 D3 + radiation group treated with 1,25-(OH)2D32.5 μg/kg dissolved in DMSO each day and 6 Gy of γ-rays.The body weight and peripheral white blood cells,femur bone marrow histology,and the proportion of adipocyte area were measured.The expression of peroxisome proliferator-activated receptor-gamma (PPARγ) was detected immunohistochemistrically at 8 d after irradiation.Results After irradiation,the number of white blood cells and the body weight decreased obviously,and the percentage of adipocyte area was increased significantly.Compared with radiation group,1,25-(OH)2D3 reduced the decrease rate of body weight (t =-2.23,-2.34,P ＜ 0.05),partly recovered the number of white blood cells at 4 or 8 d after irradiation(t =-4.99,-4.46,P ＜ 0.05),and reduced the proportion of adipocyte area (t =-3.75,-2.10,P ＜ 0.05).With immunohistochemistrical assay,it was found that 1,25-(OH) 2D3 inhibited adipogenesis by reducing the expression of PPARγ.Conclusions 1,25-(OH) 2 D3 decreases radiationinduced adipogenesis and hence protects the bone marrow microenvironment from radiation damage.
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Objective To investigate the expression patterns of fibroblast growth factor 23 (FGF23) in osteoblast and its responses to calcium, phosphate, exogenous PTH and 1,25(OH)_2D~3. Methods The primary rat calvarial osteoblasts were cultured in MEM medium which containing 10% FBS, then were harvested when cells were in half-confluence, confluence, osteoid deposition and osteoid mineralization stages respectively. The procedure was monitored under microscopy. Total RNA was extracted from cells according to the Trizol procedure. FGF23 mRNA levels were determined by Real-time PCR. Further, the confluent osteoblasts were treated with 3.2 mmol/L CaCl_2, 4.4 mmol/L β-glycerophosphate, 10~(-9) mol/L rhPTH(1-34) and 10~(-8) mol/L 1,25(OH)_2D_3 respectively for 3 days, and same volume of the medium was added as the control. The gene expressions were determined by Real-time PCR. Results FGF23 expression was transiently up-regulated at cell confluent stage and down-regulated after that. The FGF23 mRNA levels were 7.5-fold higher in confluent cells compared with that in half-confluent cells (P<0.001). The markedlly stimulating effect (about 16 times) on FGF23 expression was stimulated by exogenous 1,25(OH)_2D_3 treatment while no significant effect was found on FGF23 mRNA levels by CaCl_2,β-glycerophosphate, and rhPTH(1-34) treatments when compared with the control. Conclusions The FGF23 expression in osteoblast is developmental stage-related and its powerful stimulator is 1,25(OH)_2D.
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OBJECTIVE: Several important roles of 1,25(OH)2D3 have been recognized in the immune system. The availability of 1,25(OH)2D3 at the cellular level is significantly influenced by the relative abundance of enzymes to synthesize (CYP27B1) and catabolize (CYP24) 1,25(OH)2D3. In this study, we examined the effect of 1,25(OH)2D3 on the expression of the CYP24 gene and the role of MAPK for the induction of CYP24 by 1,25(OH)2D3 in activated human macrophages. METHODS: For obtaining human activated macrophages, we treated U937 cells with PMA and we cultured these cells for 24 hours to adhere. After 24 hours treatment with PMA, the differentiated cells were washed with phosphate buffered saline (PBS), and then they were used for examining the effect of 1,25(OH)2D3 on the expression of the CYP24 gene. The mRNA expressions of the vitamin-D3 inducible genes were measured by real-time PCR, and the change of the protein expression by 1,25(OH)2D3 was measured by immunoblotting. RESULTS: 1,25(OH)2D3 significantly induced the expression of CYP24 in the U937 cells and the 1,25(OH)2D3-induced expression of CYP24 was strongly augmented in the PMA-differentiated U937 cells. The 1,25(OH)2D3-induced expression of CYP24 was mediated by Erk1/2 and p38 MAPKs. Parallel to the induced expression of CYP24, 1,25(OH)2D3 induced the expression and phosphorylation of the CCAAT enhancer-binding protein (C/EBPbeta). CONCLUSION: In this study, we found that 1,25(OH)2D3 inducedthe expression of CYP24 via activation of MAPKs. These results suggest that MAPK inhibitors may be useful for the treatment of inflammatory conditions, in which active vitamin D3 can be used as the therapeutic molecule, by increasing the availability of 1,25(OH)2D3.
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Humanos , Colecalciferol , Sistema Inmunológico , Immunoblotting , Macrófagos , Proteínas Quinasas p38 Activadas por Mitógenos , Fosforilación , Reacción en Cadena en Tiempo Real de la Polimerasa , ARN Mensajero , Esteroide Hidroxilasas , Células U937RESUMEN
"Rickets" is the term applied to impaired mineralization at epiphyseal growth plate, resulting in deformity and impaired linear growth of long bones. Rickets may arise as a result of vitamin D deficiency or abnormality in metabolism. Vitamin D-dependent rickets (VDDR) is rare autosomal recessive disorder in which affected individuals have clinical features of vitamin D deficiency. In 1961, Prader first described this disorder including severe clinical features of rickets, such as hypophosphatemia, hypocalcemia, muscle weakness and seizure. Two distinctive hereditary defects, type I VDDR and type II VDDR have been recognized in vitamin D metabolism. Type I VDDR may be due to congenital defects of renal 1 alpha-hydroxylase, the enzyme responsible for conversion of 25 (OH) D3. These patients have low to detectable 1,25(OH)2D3 in presence of normal to raised 25 (OH) D3. In type II VDDR, renal production of 1,25(OH)2D3 is intact but 1,25(OH)2D3 is not used effectively and target organ resistant to 1,25(OH)2D3 is respectively derived from the abnormality in the vitamin D receptor. We report a case of a 25 month-old girl with typical clinical features of VDDR type I rickets, hypocalcemia, increased alkaline phosphatase and secondary hyperparathyroidism.