Objective To investigate the effect of 1, 25-(OH)₂-VitD3 (VitD3) on renal tubuleinterstitial fibrosis in diabetic kidney disease. Methods NRK-52E renal tubular epithelial cells were divided into control group (5.5 mmol/L glucose medium treatment), high glucose group (25 mmol/L glucose medium treatment) and high glucose with added VitD3 group (25 mmol/L glucose medium combined with 10^-8 mmol/L VitD3). The mRNA and protein expression of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively. The expression and localization of Snail1, SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining. The binding of Snail1 with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor (CAR) was detected by chromatin immunoprecipitation. The interaction among Snail1, SMAD3/SMAD4 and E-cadherin were detected by luciferase assay. Small interfering RNA (siRNA) was used to inhibit the expression of Snail1 and SMAD4, and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR. SD rats were randomly divided into control group, DKD group and VitD3-treated group. DKD model was established by injection of streptozotocin (STZ) in DKD group and VitD3-treated group. After DKD modeling, VitD3-treated group was given VitD3 (60 ng/kg) intragastric administration. Control group and DKD group were given normal saline intragastric administration. In the DKD group and VitD3-treated group, insulin (1-2 U/kg) was injected subcutaneously to control blood glucose for 8 weeks. The mRNA and protein levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively. Immunohistochemistry was used to detect the expression and localization of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissue. Results Compared with the control group, the mRNA and protein expressions of Snail1, SMAD3, SMAD4 and α-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated, while E-cadherin expression was down-regulated. After the intervention of VitD3, the expression levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in the DKD model improved to be close to those in the control group. Chromatin immunoprecipitation showed that Snail1 and SMAD3/SMAD4 bound to CAR promoter IV, while VitD3 prevented Snail1 and SMAD3/SMAD4 from binding to CAR promoter IV. Luciferase assay confirmed the interaction among Snail1, SMAD3/SMAD4 and E-cadherin. After the mRNA of Snail1 and SMAD4 was inhibited by siRNA, the expression of E-cadherin induced by high glucose was up-regulated. Conclusion VitD3 could inhibit the formation of Snail1-SMAD3/SMAD4 complex and alleviate the renal tubulointerstitial fibrosis in DKD.
Animals ; Rats ; Cadherins/genetics* ; Diabetes Mellitus/pathology* ; Diabetic Nephropathies/pathology* ; Epithelial-Mesenchymal Transition ; Fibrosis/pathology* ; Glucose/pharmacology* ; Kidney/pathology* ; Rats, Sprague-Dawley ; RNA, Messenger ; RNA, Small Interfering ; Transforming Growth Factor beta1/metabolism* ; Vitamin D/pharmacology*

OBJECTIVES: To investigate the level of 25 hydroxyvitamin D [25(OH)D] in late preterm infants and the effect of vitamin D₃ supplementation on the neurobehavioral development of infants and young children. METHODS: In this prospective study, 161 late preterm infants who were admitted from June 2017 to June 2020 were enrolled. According to the level of 25(OH)D in umbilical cord blood, they were divided into three groups: sufficiency group (n=52), insufficiency group (n=53), and deficiency group (n=56). Each group was further divided into subgroup A (vitamin D₃ 800 IU/d) and subgroup B (individualized vitamin D₃ supplementation) using a random number table. The levels of 25(OH)D were measured at 3 months after birth and at the corrected ages of 10 months and 18 months. The neurobehavioral development levels were determined by the Gesell Developmental Scale at the corrected ages of 10 months and 18 months. RESULTS: Within 24 hours and 3 months after birth, the insufficiency group and the deficiency group had a significantly lower level of 25(OH)D than the sufficiency group (P<0.05), and the insufficiency group had a significantly higher level of 25(OH)D than the deficiency group (P<0.05). In the deficiency group, subgroup B had a significantly higher level of 25(OH)D than subgroup A (P<0.05) at 3 months after birth. At the corrected ages of 10 months and 18 months, the insufficiency and deficiency groups had significantly lower scores of five functional areas of the Gesell Development Scale than the sufficiency group (P<0.05). Compared with the insufficiency group, the deficiency group had a significantly lower score of language at the corrected age of 10 months and a significantly lower score of gross motor at the corrected age of 18 months (P<0.05). Compared with subgroup A of the deficiency group, subgroup B had a significantly higher score of adaptive ability at the corrected age of 10 months and significantly higher scores of adaptive ability and response ability at the corrected age of 18 months (P<0.05). CONCLUSIONS There is a significant difference in the level of 25(OH)D in umbilical cord blood in late preterm infants. Individualized vitamin D supplementation appears to be more effective for the treatment of vitamin D deficiency. Vitamin D level at birth and in early infancy has certain influence on neurobehavioral development.
Infant ; Child ; Infant, Newborn ; Humans ; Child, Preschool ; Cholecalciferol/pharmacology* ; Prospective Studies ; Fetal Blood ; Infant, Premature ; Dietary Supplements ; Vitamin D

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

OBJECTIVE: To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25-VitD3) supplementation on cerebral injury after ischemia/reperfusion (I/R) in mice with middle cerebral artery occlusion (MCAO). METHODS: Male C57BL6 mice were randomly divided into Sham group, Vehicle group and 1,25-VitD3 group, with 10 mice in each group. Vehicle group and 1,25-VitD3 group were given MCAO for 1 hour, and then killed after reperfusion for 24 hours. Mice in 1,25-VitD3 group were treated with 1,25-VitD3 at the dose of 100 ng/(kg·d) by injected intraperitoneally for 5 days before MCAO operation. Cerebral ischemic penumbra areas of each group were collected for TTC staining, RT-PCR, TTC staining and immunohistochemistry assay. The function defect of mice was evaluated by using neurological function score. RESULTS: Compared with the sham group, the volume of cerebral infarction in Vehicle group was increased significantly, and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues were increased significantly (P＜0.05); compared with Vehicle group, supplementation of 1,25-VitD3 reduced the volume of cerebral infarction by about 50% in I/R mice (P＜0.05), and the expressions of IL-6, IL-1beta and Gp91phox in brain tissues of 1,25-VitD3 group were decreased significantly (P＜0.05). The expression of Foxp3, a T-regulatory cell marker, was significantly increased in the brain of mice (P＜0.05), while the expression of Rorc, a transcription factor, was significantly decreased (P＜0.05), suggesting that Th17/gamma Delta T-cell response was reduced and the number of neutrophils in the brain injury site of mice was significantly reduced (P＜0.05). CONCLUSION Vitamin D could alleviate the development of cerebral infarction after arterial occlusion (MCAO) reperfusion, and its mechanism may be through regulating the inflammatory response in mouse brain I/R.
Animals ; Brain ; Cytokines ; metabolism ; Infarction, Middle Cerebral Artery ; drug therapy ; Inflammation ; Male ; Mice ; Mice, Inbred C57BL ; NADPH Oxidase 2 ; metabolism ; Protective Agents ; pharmacology ; Random Allocation ; Rats, Sprague-Dawley ; Reperfusion Injury ; drug therapy ; T-Lymphocytes ; Th17 Cells ; Vitamin D ; pharmacology

Vitamin D deficiency is a common health issue around the world. We therefore evaluated the associations of semen quality with both serum and seminal plasma vitamin D levels and studied the mechanisms underlying these by incubating spermatozoa with 1,25(OH)₂D In vitro. Two hundred and twenty-two men were included in our study. Vitamin D was detected using an electrochemiluminescence method. Spermatozoa used for In vitro experiments were isolated by density gradient centrifugation. Positive relationships of serum 25(OH)D with semen volume and seminal plasma fructose were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it was inversely associated with sperm concentration and positively correlated with semen volume and sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with 1,25(OH)₂D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol l^-1. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotide (NADH) were both enhanced, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of ATP production. Therefore, seminal plasma vitamin D may be involved in regulating sperm motility, and 1,25(OH)₂D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions.
Adenosine Triphosphate/metabolism* ; Adult ; Calcium/metabolism* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Humans ; Male ; Semen/metabolism* ; Semen Analysis ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Vitamin D/pharmacology* ; Vitamin D Deficiency/blood* ; Wit and Humor as Topic ; Young Adult

Serum sclerostin is positively associated with serum 25 hydroxyvitamin D concentration. Our preliminary studies confirmed that Qing'e formula (QEF) could effectively increase serum 25 hydroxyvitamin D concentration in patients with postmenopausal osteoporosis (PMOP), but the effect of supplementation with QEF on serum sclerostin is unknown. This study investigated the effects of supplementation of QEF on serum sclerostin levels in patients with PMOP. Totally 120 outpatients and inpatients with PMOP treated in our hospital between January and October 2012 were randomly divided into QEF+calcium group, alfacalcidol+calcium group, and placebo+calcium group (n=40 each), with a follow-up period of 2 years. The serum levels of sclerostin, 25 hydroxyvitamin D, and bone turnover markers (β-CTX, N-MID and T-PINP) at baseline and at the 6th month, 1st year, 1.5th year, and 2nd year after treatment were measured. The results showed that the levels of circulating sclerostin were increased significantly at the 6th month after treatment in QEF+calcium group and alfacalcidol+calcium group as compared with placebo+calcium group (P<0.05), but there was no significant difference between the former two groups (P>0.05). The levels of β-CTX, N-MID and T-PINP in serum were decreased in both QEF+calcium group and alfacalcidol+calcium group at the 6th month after treatment, without significant difference between the two groups (P>0.05). But the levels were significantly lower than that in placebo+calcium group (P<0.05). These results suggest that the mechanism by which QEF modulates bone metabolism in patients with PMOP might be related with the effect of QEF in increasing sclerostin expression. Our findings provide a scientific rationale for using QEF as an effective drug to prevent bone loss in PMOP.
Biomarkers ; blood ; Bone Density Conservation Agents ; administration & dosage ; pharmacology ; Calcium, Dietary ; administration & dosage ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Hydroxycholecalciferols ; administration & dosage ; Middle Aged ; Osteoporosis, Postmenopausal ; blood ; drug therapy ; Proteins ; drug effects ; metabolism ; Random Allocation ; Vitamin D ; analogs & derivatives ; blood

Cardiovascular Diseases ; physiopathology ; Humans ; Inflammatory Bowel Diseases ; metabolism ; Kidney Diseases ; metabolism ; Neoplasms ; Vitamin D ; pharmacology ; physiology

OBJECTIVETo investigate the effect of 1,25-dihydroxyvitamin D(3) and 5-fluorouracil, either alone or in combination, on the expression of IGFBP-3 in human esophageal carcinoma 109 cell xenograft in nude mice.

METHODSIn vitro cultured esophageal carcinoma Eca-109 cells were inoculated subcutaneously in BALB/c mice. The tumor-bearing mice were randomly divided into control group (A), 1,25-dihydroxyvitamin D(3) group (B), 5-fluorouracil group (C), and 1,25-dihydroxyvitamin D(3) plus 5-fluorouracil group (D). 1,25-dihydroxyvitamin D(3) and 5-fluorouracil were administered at the doses of 2.5 ug/kg and 25 mg/kg via intraperitoneal injections, respectively, and the mice in the control group received saline injection only. The tumor growth was observed and the expression of IGFBP-3 in the tumor xenograft was detected using immunohistochemistry. An automatic biochemistry analyzer was used to determine serum calcium levels, and Von Kossa staining was utilized for observation of calcium deposition in the kidneys.

RESULTSCompared with that in group A, the xenograft in groups B, C, and D all showed a lowered growth rate with a smaller tumor volume, and presented with stronger IGFBP-3 positivity and significantly higher levels of IGFBP-3 protein expression (P<0.05). In group D, the protein expression of IGFBP-3 was significantly increased compared with that in groups B and C (P<0.05). Compared with that in group A, serum calcium level was slightly increased in groups B, C, and D, , but no obvious calcium deposition was found in the kidney tissue sections.

CONCLUSIONBoth 1,25-dihydroxyvitamin D(3) and 5-fluorouracil can inhibit the growth of the tumor xenograft in nude mice, and their combination is more effective. This effect is probably associated with increased protein expression of IGFBP-3 in the xenograft tumor. No calcium deposition occurs in the kidney tissue of the tumor-bearing mice.

Animals ; Cell Line, Tumor ; Fluorouracil ; pharmacology ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Vitamin D ; analogs & derivatives ; pharmacology ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the effect of vitamin D on the expression of chemokine regulated on activation, normal T cells expressed and secreted (RANTES) in the lung tissue of asthmatic rats, and the role of vitamin D in the control of asthmatic airway inflammation and the synergistic action of hormones.

METHODSForty female Wistar rats were randomly and equally divided into normal control, asthma, vitamin D intervention, budesonide intervention, and budesonide+vitamin D intervention groups. Hematoxylin and eosin staining was used to observe pathological changes in the lung tissue. Immunohistochemistry was used to measure the protein expression of RANTES in lung tissue. Enzyme-linked immunosorbent assay was used to measure the level of RANTES in bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was used to measure the mRNA expression of RANTES.

RESULTSThe asthma group showed the most significant pathological changes in the lung tissue, including inflammatory cell infiltration, bronchial stenosis and distortion and smooth muscle rupture, while the intervention groups showed fewer pathological changes. Of the intervention groups, the budesonide intervention group showed fewer pathological changes than the vitamin D intervention group, and the budesonide+vitamin D intervention group showed the mildest pathological changes, which were similar to those observed in the normal control group. Protein expression of RANTES in the lung tissue and BALF was significantly higher in the asthma group than in the normal control group (P<0.05), while it was lower in the intervention groups than in the asthma group, exhibiting significant differences between each intervention group and the asthma group (P<0.05) (except the difference in protein expression of RANTES in BALF between the vitamin D intervention and asthma groups). The budesonide+vitamin D intervention group showed less protein expression of RANTES in the lung tissue and BALF than both the budesonide intervention and vitamin D intervention groups (P<0.05). The mRNA expression of RANTES was significantly higher in the asthma group than in the normal control group (P<0.05), while it was significantly lower in three intervention groups than in the asthma group (P<0.05), however no significant difference was found between the intervention groups in this regard. The budesonide+vitamin D intervention group showed the lowest level of RANTES mRNA, with no significant difference from the normal control group.

CONCLUSIONSThe mRNA and protein expression of RANTES in BALF and lung tissue increases significantly in asthmatic rats. Vitamin D intervention can decrease the expression of RANTES, suggesting that vitamin D can reduce airway inflammation by regulating the expression of RANTES. Vitamin D can be used together with budesonide to further decrease the mRNA and protein expression of RANTES.

Animals ; Asthma ; drug therapy ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Budesonide ; therapeutic use ; Chemokine CCL5 ; analysis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Female ; Immunohistochemistry ; Lung ; metabolism ; pathology ; Rats ; Rats, Wistar ; Vitamin D ; pharmacology

OBJECTIVETo explore the effect of different doses of 1,25-(OH)(2)VitD(3) early supplementation on airway inflammation and lung inflammatory factors in baby rats with asthma.

METHODSForty male weaned Wistar rats were divided into normal group, model group, low 1,25-(OH)(2)VitD(3) group, middle 1,25-(OH)(2)VitD(3) group, high 1,25-(OH)(2)VitD(3) group using random number table (8 rats each group). The rats in low, middle and high 1,25-(OH)(2)VitD(3) groups were given 1, 4, 10 µg/kg of 1,25-(OH)(2)VitD(3) every other day by intraperitoneal injection respectively for 25 days. Except normal group, the rats in other groups were challenged with ovalbumin to establish the asthma model. The pathologic changes of lung tissue, the total white blood cell and classified cell counts in bronchoalveolar lavage fluid (BALF) were measured. The concentrations of IL-4, IL-5 and IFN-γ in serum and BALF were measured by ELISA method.

RESULTSThe level of total white blood cell counts in BALF were (5.98 ± 1.67)×10(5)/ml, (25.34 ± 4.28)×10(5)/ml, (17.24 ± 3.3)×10(5)/ml, (9.31 ± 3.37)×10(5)/ml, (45.1 ± 15.75)×10(5)/ml, respectively (F = 33.453, P < 0.01). The percent ratio of EOS in BALF were (1.44 ± 0.78)%, (17.81 ± 6.88)%, (15.00 ± 5.70)%, (8.89 ± 3.66)%, (25.88 ± 5.57)%, respectively (F = 27.299, P < 0.01). The level of IL-4 in serum of normal, model, low, middle and high-1,25-(OH)(2)VitD(3) groups were (0.62 ± 0.54), (7.57 ± 1.04), (3.58 ± 0.56), (2.70 ± 0.78) and (5.27 ± 0.30) pg/ml, respectively (F = 116.287, P < 0.01); IL-5 in resume were (32.20 ± 4.23), (67.14 ± 18.14), (37.51 ± 0.47), (40.69 ± 2.47) and (124.60 ± 36.19) pg/ml, respectively (F = 23.902, P < 0.01); IFN-γ in serum were (79.71 ± 10.08), (49.06 ± 4.46), (59.15 ± 2.51), (59.27 ± 2.33) and (53.85 ± 1.97) pg/ml, respectively (F = 39.954, P < 0.01). Also in BLAF, the IL-4 of all groups were (0.51 ± 0.30), (102.92 ± 54.61), (8.64 ± 4.07), (3.10 ± 1.28) and (33.67 ± 8.1) pg/ml, respectively (F = 24.062, P < 0.01); the IFN-γ were (247.37 ± 189.18), (43.82 ± 13.76), (81.32 ± 17.07), (86.50 ± 14.26) and (59.89 ± 34.17) pg/ml, respectively (F = 7.157, P < 0.01); the IL-5 in BALF were (38.81 ± 0.60), (80.48 ± 17.90), (45.11 ± 1.33), (43.39 ± 1.11) and (149.60 ± 45.87) pg/ml, respectively (F = 35.978, P < 0.01). Pathologic changes in lung of asthma rat groups were obvious. The lung pathologic changes in low and middle dose groups showed a significant improvement compared to the asthma group and high dosage group showed more serious pathologic changes compared to the low and middle dose groups.

CONCLUSIONIntervention with appropriate dose of 1,25-(OH)(2)VitD(3) in the early life could improve lung pathologic changes and reduce the effect of inflammatory factors in air way of baby rat asthma model. However, overdose might play detrimental effect.

Animals ; Asthma ; metabolism ; pathology ; Bronchoalveolar Lavage Fluid ; Disease Models, Animal ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-5 ; metabolism ; Lung ; metabolism ; pathology ; Male ; Pneumonia ; metabolism ; pathology ; Rats ; Rats, Wistar ; Vitamin D ; administration & dosage ; pharmacology