Absence of claudin-3 does not alter intestinal absorption of phosphate in mice.

Intestinal absorption of phosphate is bimodal, consisting of a transcellular pathway and a poorly characterized paracellular mode, even though the latter one contributes to the bulk of absorption under normal dietary conditions. Claudin-3 (Cldn3), a tight junction protein present along the whole intestine in mice, has been proposed to tighten the paracellular pathway for phosphate. The aim of this work was to characterize the phosphate-related phenotype of Cldn3-deficient mice. Cldn3-deficient mice and wildtype littermates were fed standard diet or challenged for 3 days with high dietary phosphate. Feces, urine, blood, intestinal segments and kidneys were collected. Measurements included fecal, urinary, and plasma concentrations of phosphate and calcium, plasma levels of phosphate-regulating hormones, evaluation of trans- and paracellular phosphate transport across jejunum and ileum, and analysis of intestinal phosphate and calcium permeabilities. Fecal and urinary excretion of phosphate as well as its plasma concentration was similar in both genotypes, under standard and high-phosphate diet. However, Cldn3-deficient mice challenged with high dietary phosphate had a reduced urinary calcium excretion and increased plasma levels of calcitriol. Intact FGF23 concentration was also similar in both groups, regardless of the dietary conditions. We found no differences either in intestinal phosphate transport (trans- or paracellular) and phosphate and calcium permeabilities between genotypes. The intestinal expression of claudin-7 remained unaltered in Cldn3-deficient mice. Our data do not provide evidence for a decisive role of Cldn3 for intestinal phosphate absorption and phosphate homeostasis. In addition, our data suggest a novel role of Cldn3 in regulating calcitriol levels.

Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

Microbial bile acid metabolite ameliorates mycophenolate mofetil-induced gastrointestinal toxicity through vitamin D3 receptor.

Mycophenolate mofetil (MMF) is one of the most used immunosuppressive drugs in organ transplantation, but frequent gastrointestinal (GI) side effects through unknown mechanisms limit its clinical use. Gut microbiota and its metabolites were recently reported to play a vital role in MMF-induced GI toxicity, but the specific mechanism of how they interact with the human body is still unclear. Here, we found that secondary bile acids (BAs), as bacterial metabolites, were significantly reduced by MMF administration in the gut of mice. Microbiome data and fecal microbiota transfer model supported a microbiota-dependent effect on the reduction of secondary BAs. Supplementation of the secondary BA lithocholic acid alleviated MMF-induced weight loss, colonic inflammation, and oxidative phosphorylation damage. Genetic deletion of the vitamin D3 receptor (VDR), which serves as a primary colonic BA receptor, in colonic epithelial cells (VDRΔIEC) abolished the therapeutic effect of lithocholic acid on MMF-induced GI toxicity. Impressively, we discovered that paricalcitol, a Food and Drug Administration-approved VDR agonist that has been used in clinics for years, could effectively alleviate MMF-induced GI toxicity. Our study reveals a previously unrecognized mechanism of gut microbiota, BAs, and VDR signaling in MMF-induced GI side effects, offering potential therapeutic strategies for clinics.

Nutraceutically-enhanced oral delivery of vitamin D3 via Bio-SNEDDS: Demonstrating in vivo superiority over pediatric formulations.

BACKGROUND: Vitamin D3 (VD3) deficiency among children in Saudi Arabia remains a pressing concern due to its poor bioavailability and the limitations of current pediatric formulations. To address this challenge, we developed a groundbreaking pediatric self-nanoemulsifying drug delivery system (Bio-SNEDDS) for VD3, fortified with black seed oil and moringa seed oil for dual therapeutic benefits. Through meticulous formulation optimization using ternary phase diagrams and comprehensive testing, our Bio-SNEDDS demonstrated exceptional performance. METHODS: Bio-SNEDDS were manufactured by incorporating Black seed oil and moringa seed oil as bioactive nutraceutical excipients along with various cosurfactant and surfactants. Bio-SNEDDS were systematically optimized through ternary phase diagrams, visual tests, droplet size analysis, drug solubilization studies, dispersion assessments, and pharmacokinetic testing in rats compared to Vi-De 3®. RESULTS: Pseudoternary phase diagrams identified oil blends producing large nanoemulsion regions optimal for SNEDDS formation. The optimized F1 Bio-SNEDDS showed a mean droplet diameter of 33.7 nm, solubilized 154.46 mg/g VD3 with no metabolite formation, and maintained >88% VD3 in solution during 24 h dispersion testing. Notably, in vivo pharmacokinetic evaluation at a high VD3 dose demonstrated an approximately two-fold greater relative bioavailability over Vi-De 3®, validating the superb oral delivery performance of Bio-SNEDDS even under challenging high-dose conditions. CONCLUSIONS: The Bio-SNEDDS provides an effective VD3 delivery strategy with established in vivo superiority over marketed products, along with offering additional health benefits from the natural oils.

Vitamin D3 promotes fish oocyte development by directly regulating gonadal steroid hormone synthesis†.

Vitamin D receptors and vitamin D3-metabolizing enzymes have been found to be highly expressed in the ovaries and spermatophores of fish. However, the role of vitamin D3 on fish gonadal development has rarely been reported. In this study, 2-month-old female zebrafish were fed with different concentrations of vitamin D3 diets (0, 700, 1400, and 11 200 IU/kg) to investigate the effects of vitamin D3 on ovarian development. The diet with 0 IU/kg vitamin D3 resulted in elevated interstitial spaces, follicular atresia, and reproductive toxicity in zebrafish ovaries. Supplementation with 700 and 1400 IU/kg of vitamin D3 significantly increased the oocyte maturation rate; upregulated ovarian gonadal steroid hormone synthesis capacity; and elevated plasma estradiol, testosterone, and ovarian vitellogenin levels. Furthermore, the current study identified a vitamin D response element in the cyp19a1a promoter and demonstrated that 1.25(OH)2D3-vitamin D response directly activated cyp19a1a production through activating the vitamin D response element. In conclusion, this study shows that an appropriate concentration of vitamin D3 can promote zebrafish ovarian development and affect vitellogenin synthesis through the vdr/cyp19a1a/er/vtg gene axis.

Daily versus fortnightly oral vitamin D3 in treatment of symptomatic vitamin D deficiency in children aged 1-10 years: An open labelled randomized controlled trial.

OBJECTIVE: Compare the efficacy and safety of daily versus fortnightly oral vitamin D3 in treating symptomatic vitamin D deficiency in children aged 1-10 years. DESIGN: Open labelled randomized controlled trial. PATIENTS: Eighty children with symptomatic vitamin D deficiency were randomized into group daily (D) and group bolus (B) [40 in each group] to receive oral vitamin D3, 4000 IU daily or 60,000 IU fortnightly for 12 weeks respectively. Both groups received daily oral calcium of 500 mg/day. MEASUREMENTS: Serum calcium (Ca), phosphate (P), alkaline phosphatase (ALP), 25-hydroxy cholecalciferol (25(OH)D), parathyroid hormone (PTH) levels, urine calcium: creatinine ratio and radiological score were assessed at baseline, 4 weeks and 12 weeks. At the end of 12 weeks, 74 children were available for evaluation of the efficacy and safety of both regimens. RESULTS: Both regimens led to a significant increase in Ca and P levels and a fall in ALP and PTH levels from baseline to 4 and 12 weeks of therapy, with no intergroup difference. At 4- and 12-week assessments, all children in both treatment arms achieved 25(OH)D level in sufficiency range, with no significant difference in their geometric mean. Both regimens were associated with asymptomatic transient hypercalcemia [group D-51.4% vs. group B-34.3%; p -0.14] and hypercalciuria (5.7%) in group D that resolved spontaneously on follow-up. CONCLUSIONS: Daily and fortnightly oral vitamin D3 in similar cumulative doses are efficacious for treating symptomatic vitamin D deficiency in children (1-10 years). Treated children should be monitored for serum 25(OH)D, Ca and urinary calcium creatinine ratio.

The Effect of Vitamin D3 and Valproic Acid on the Maturation of Human-Induced Pluripotent Stem Cell-Derived Enterocyte-Like Cells.

Cytochrome P450 3A4 (CYP3A4) is involved in first-pass metabolism in the small intestine and is heavily implicated in oral drug bioavailability and pharmacokinetics. We previously reported that vitamin D3 (VD3), a known CYP enzyme inducer, induces functional maturation of iPSC-derived enterocyte-like cells (iPSC-ent). Here, we identified a Notch activator and CYP modulator valproic acid (VPA), as a promotor for the maturation of iPSC-ent. We performed bulk RNA sequencing to investigate the changes in gene expression during the differentiation and maturation periods of these cells. VPA potentiated gene expression of key enterocyte markers ALPI, FABP2, and transporters such as SULT1B1. RNA-sequencing analysis further elucidated several function-related pathways involved in fatty acid metabolism, significantly upregulated by VPA when combined with VD3. Particularly, VPA treatment in tandem with VD3 significantly upregulated key regulators of enterohepatic circulation, such as FGF19, apical bile acid transporter SLCO1A2 and basolateral bile acid transporters SLC51A and SLC51B. To sum up, we could ascertain the genetic profile of our iPSC-ent cells to be specialized toward fatty acid absorption and metabolism instead of transporting other nutrients, such as amino acids, with the addition of VD3 and VPA in tandem. Together, these results suggest the possible application of VPA-treated iPSC-ent for modelling enterohepatic circulation.

Selective Oxidation of Vitamin D3 Enhanced by Long-Range Effects of a Substrate Channel Mutation in Cytochrome P450BM3 (CYP102A1).

Vitamin D deficiency affects nearly half the population, with many requiring or opting for supplements with vitamin D3 (VD3), the precursor of vitamin D (1α,25-dihydroxyVD3). 25-HydroxyVD3, the circulating form of vitamin D, is a more effective supplement than VD3 but its synthesis is complex. We report here the engineering of cytochrome P450BM3 (CYP102A1) for the selective oxidation of VD3 to 25-hydroxyVD3. Long-range effects of the substrate-channel mutation Glu435Ile promoted binding of the VD3 side chain close to the heme, enhancing VD3 oxidation activity that reached 6.62 g of 25-hydroxyVD3 isolated from a 1-litre scale reaction (69.1 % yield; space-time-yield 331 mg/L/h).

Dual effect of vitamin D3 on breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Despite the well-known in vitro antitumoral effect of vitamin D3 (VD3), its impact on breast CAFs is almost unknown. In this study, we analyzed the ex vivo effects of calcitriol on CAFs isolated from breast cancer tissues. METHODS: CAFs were cultured with 1 and 10 nM calcitriol and their phenotype; gene expression, protein expression, and secretion were assessed. Calcitriol-treated CAFs-conditioned media (CM) were used to analyze the effect of CAFs on the migration and protein expression of MCF-7 and MDA-MB-231 cells. RESULTS: Tumor tissues from VD3-deficient patients exhibited lower levels of ß-catenin and TGFß1, along with higher levels of CYP24A1 compared to VD3-normal patients. In VD3-deficient patients, CAF infiltration was inversely associated with CYP24A1 levels and positively correlated with OPN levels. Calcitriol diminished CAFs' viability, but this effect was weaker in premenopausal and VD3-normal patients. Calcitriol reduced mRNA expression of CCL2, MMP9, TNC, and increased PDPN, SPP1, and TIMP1. It also decreased the secretion of CCL2, TNC, and the activity of MMP-2, while increasing cellular levels of TIMP1 in CAFs from all patient groups. In nonmetastatic and postmenopausal patients, PDPN surface expression increased, and CAFs CM from these groups decreased MCF-7 cell migration after ex vivo calcitriol treatment. In premenopausal and VD3-deficient patients, calcitriol reduced IDO1 expression in CAFs. Calcitriol-treated CAFs CM from these patients decreased OPN expression in MCF-7 and/or MDA-MB-231 cells. However, in premenopausal patients, calcitriol-treated CAFs CM also decreased E-cadherin expression in both cell lines. CONCLUSION: The effects of calcitriol on breast CAFs, both at the gene and protein levels, are complex, reflecting the immunosuppressive or procancer properties of CAFs. The anticancer polarization of CAFs following ex vivo calcitriol treatment may result from decreased CCL2, TNC (gene and protein), MMP9, and MMP-2, while the opposite effect may result from increased PDPN, TIMP1 (gene and protein), and SPP1. Despite these multifaceted effects of calcitriol on molecule expression, CAFs' CMs from nonmetastatic and postmenopausal patients treated ex vivo with calcitriol decreased the migration of MCF-7 cells.

Maternal Vitamin D3 Supplementation in an Oxidized-Oil Diet Protects Fetus from Developmental Impairment and Ameliorates Oxidative Stress in Mouse Placenta and Fetus.

BACKGROUND: Fried food has increased in popularity worldwide. However, deep frying can increase the production of peroxidative toxins in food, which might be harmful to fetal development. The antioxidative effect of vitamin D3 (VD3) has been reported previously. OBJECTIVES: This study aimed to explore how maternal VD3 supplementation in an oxidized-oil diet during gestation affects fetal antioxidative ability and development. METHODS: Pregnant mice were randomly assigned into 3 groups: Control group (diet with fresh soybean oil), OSO group [diet with oxidized soybean oil (OSO)], and OSOV group (diet with OSO and 10,000 IU/Kg VD3). Mice were fed with the corresponding diet during gestation. On day 16.5 of gestation, the placenta and fetus were harvested to analyze antioxidative status. RESULTS: Maternal oxidized-oil diet during gestation significantly reduced placental vessel abundance, labyrinth zone area, and fetal body weight. However, dietary VD3 supplementation prevented these negative effects of oxidized-oil diet. Maternal intake of oxidized-oil diet increased serum concentrations of malondialdehyde, total-nitric oxide synthase, and inducible nitric oxide synthase, whereas VD3 supplementation showed a protection effect on it. Additionally, maternal VD3 supplementation increased the levels of antioxidative enzymes and the nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), thereby protecting placenta and fetus from apoptosis and oxidative stress caused by an oxidized-oil diet. The gene expression and protein levels of a fatty acid transporter solute carrier family 27 member 1 in the fetal liver were increased by maternal VD3 supplementation under oxidized-oil diet. Notably, NRF2 could be co-immunoprecipitated with the VD receptor in the placenta. CONCLUSIONS: Maternal VD3 supplementation could protect fetus from oxidized-oil diet induced developmental impairment by alleviating oxidative stress in the placenta and fetus through the VD receptor/NRF2 pathway, at least partially. Thus, ensuring adequate levels of VD3 through supplementation is often critical during pregnancy.

Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling.

BACKGROUND: The global prevalence of vitamin D (VitD) deficiency associated with numerous acute and chronic diseases has led to strategies to improve the VitD status through dietary intake of VitD-fortified foods and VitD supplementation. In this context, the circulating form of VitD3 (cholecalciferol) in the human body, 25-hydroxy-VitD3 (calcifediol, 25OHVitD3), has a much higher efficacy in improving the VitD status, which has motivated researchers to develop methods for its effective and sustainable synthesis. Conventional monooxygenase-/peroxygenase-based biocatalytic platforms for the conversion of VitD3 to value-added 25OHVitD3 are generally limited by a low selectivity and yield, costly reliance on cyclodextrins and electron donor systems, or by the use of toxic co-substrates. RESULTS: In this study, we used a whole-cell approach for biocatalytic 25OHVitD3 synthesis, in which a molybdenum-dependent steroid C25 dehydrogenase was produced in the denitrifying bacterium Thauera aromatica under semi-aerobic conditions, where the activity of the enzyme remained stable. This enzyme uses water as a highly selective VitD3 hydroxylating agent and is independent of an electron donor system. High density suspensions of resting cells producing steroid C25 dehydrogenase catalysed the conversion of VitD3 to 25OHVitD3 using either O2 via the endogenous respiratory chain or externally added ferricyanide as low cost electron acceptor. The maximum 25OHVitD3 titer achieved was 1.85 g L-1 within 50 h with a yield of 99%, which is 2.2 times higher than the highest reported value obtained with previous biocatalytic systems. In addition, we developed a simple method for the recycling of the costly VitD3 solubiliser cyclodextrin, which could be reused for 10 reaction cycles without a significant loss of quality or quantity. CONCLUSIONS: The established steroid C25 dehydrogenase-based whole-cell system for the value-adding conversion of VitD3 to 25OHVitD3 offers a number of advantages in comparison to conventional oxygenase-/peroxygenase-based systems including its high selectivity, independence from an electron donor system, and the higher product titer and yield. Together with the established cyclodextrin recycling procedure, the established system provides an attractive platform for large-scale 25OHVitD3 synthesis.

The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis.

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

Selective dual sensing strategy for free and vitamin D3 micelles in food samples based on S,N-GQDs photoinduced electron transfer.

A reliable nanotechnological sensing strategy, based on an S,N-co-doped graphene quantum dot (GQD) platform, has been developed to distinctly detect two key variants of vitamin D3, specifically the free (VD3) and the nanoencapsulated form (VD3Ms). For this purpose, food-grade vitamin D3 micelles were self-assembled using a low-energy procedure (droplet size: 49.6 nm, polydispersity index: 0.34, ζ-potential: -33 mV, encapsulation efficiency: 90 %) with an innovative surfactant mixture (Tween 60 and quillaja saponin). Herein, four fluorescent nanoprobes were also synthesized and thoroughly characterized: S,N-co-doped GQDs, α-cyclodextrin-GQDs, ß-cyclodextrin-GQDs, and γ-cyclodextrin-GQDs. The goal was to achieve a selective dual sensing strategy for free VD3 and VD3Ms by exploiting their distinctive quenching behaviors. Thus, the four nanosensors allowed the individual sensing of both targets to be performed (except α-CD-GQD for VD3Ms), but S,N-GQDs were finally selected due to selectivity and sensitivity (quantum yield, QY= 0.76) criteria. This choice led to a photoinduced electron transfer (PET) mechanism associated with static quenching, where differentiation was evidenced through a displayed 13-nm hypsochromic (blue) shift when interacting with VD3Ms. The reliability of this dual approach was demonstrated through an extensive evaluation of analytical performance characteristics. The feasibility and accuracy were proven in commercial food preparations and nutritional supplements containing declared nanoencapsulated and raw VD3, whose results were validated by a paired Student's t-test comparison with a UV-Vis method. To the best of our knowledge, this represents the first non-destructive analytical approach addressing the groundbreaking foodomic trend to distinctly detect different bioactive forms of vitamin D3, while also preserving their native nanostructures as a chemical challenge, thus providing reliable information about their final stability and bioavailability.

The effect of vitamin D3 and omega-3 combination, taken orally, on triglycerides, lining of intestine, and the biodiversity of gut microbiota in healthy rats.

AIM: The gut microbiota plays a key role in host health. An intake of omega-3 and vitamin D3 in a separate manner is vital for maintaining good health of gut microbiota and controlling some illness manifestations. The aim of this study is to investigate the potential change in biodiversity of the gut microbiome in healthy rats supplemented with vitamin D3, omega-3 alone and their combination and to reflect onto the triglyceride levels in serum and fecal samples. METHODS AND RESULTS: Using the 16S rRNA gene Miseq Illumina NGS, and monitoring triglyceride levels in serum and fecal samples coupled with several clinical parameters, we examined the effect of orally taken combination of omega-3 and vitamin D3 alongside the separate intake of supplements on gut microbiota in 24 healthy white Wistar rats for six weeks. The study findings showed that combination treatment encouraged the growth of opportunistic Clostridia class during day 21 and 42 of treatment by 7.7 and 7.4 folds, respectively, exhibited incomplete absorption levels for both supplements when used concomitantly, demonstrated a damaging effect on the gut intestinal lining wall thickness (126 µm) when compared to control group (158 µm), increasing lumen diameter (400 µm), and showed higher triglyceride level in fecal samples. CONCLUSIONS: These findings indicate that omega-3 and vitamin D3 supplements as combination intake reveal unfavorable effects, thus, it is advised to conduct further in-depth studies to clarify the presence or absence of any chemical interaction between both supplements' molecules and to investigate based on human model to attain a superior perspective.

A study on the effects of vitamin D supplementation on hematological parameters and serum 25-hydroxy vitamin D in healthy dogs.

BACKGROUND: Limited studies are available on vitamin D supplementation in dogs. This study evaluates the effect of a commercial vitamin D3 supplement on serum 25-hydroxy vitamin D as well as selected biochemical and hematological parameters in healthy dogs. Eight intact male adult dogs with a mean body weight of 20 kg from mixed breeds were included in the study. After adaptation period, dogs received vitamin D3 supplement at the dose of 50 IU/kg body weight per day. Blood samples were collected on days 0, 14, 28 and 42 of supplementation. Food was used for analysis of vitamin D3 content. RESULTS: Significant increase in serum level of 25-hydroxy vitamin D3 was detected since day 14 of supplementation. Changes in serum 25-hydroxy vitamin D3 concentration during time showed an upward significance (p < 0.05). Vitamin D3 content of the food was 2900 IU/kg dry matter. Changes in serum phosphorus levels were upward significant. No dog showed calcium or phosphorus levels above the highest reference level. Liver and kidney parameters remained in the reference range during the experiment. A gradual significant increase was observed in hemoglobin and hematocrit which was started from day 14. Vitamin D3 supplementation had no significant effect on neutrophils, monocytes and lymphocytes percent during the study. CONCLUSIONS: Vitamin D3 supplementation at 50 IU/kg BW daily, increases serum levels of 25-hydroxy vitamin D in healthy dogs fed with a diet containing proper amount of this vitamin. It also increases hemoglobin and hematocrit levels in a time dependent manner without inducing adverse effects.

Combined effects of vitamin D3 and dioxopiperidinamide derivative on lipid homeostasis, inflammatory pathways, and redox imbalance in non-alcoholic fatty liver disease in vivo zebrafish model.

Liver damage and metabolic dysfunctions, the defining features of non-alcoholic fatty liver disease (NAFLD), are marked by inflammation, oxidative stress, and excessive hepatic fat accumulation. The current therapeutic approaches for NAFLD are limited, necessitating exploring novel treatment strategies. Dioxopiperidinamide derivatives, particularly DOPA-33, have shown effective anti-inflammatory and antioxidant properties, potentially offering therapeutic benefits against NAFLD. This study investigated the combined potential of vitamin D3 (Vit D3) and DOPA-33 in treating NAFLD. The network pharmacology analysis identified key NAFLD targets modulated by Vit D3 and DOPA-33, emphasizing their potential mechanisms of action. In NAFLD-induced zebrafish models, Vit D3 and DOPA-33 significantly reduced hepatic lipid accumulation, oxidative stress, and apoptosis, demonstrating superior efficacy over individual treatments. The treatment also lowered reactive oxygen species (ROS) levels, decreased liver damage, and enhanced antioxidant defense mechanisms. Moreover, behavioral analyses showed improved locomotion and reduced weight gain in treated zebrafish. Biochemical analyses revealed lower triglycerides (TG) and glucose levels with improved oxidative markers. Furthermore, histological analyses indicated reduced hepatic steatosis and inflammation, with decreased expression of lipogenesis-related genes and inflammatory mediators. Finally, high-performance liquid chromatography (HPLC) confirmed a significant reduction in hepatic cholesterol levels, indicating the effectiveness of the combination therapy in addressing key NAFLD-related dyslipidemias. These findings suggest that Vit D3 + DOPA-33 targets pathways involved in lipid metabolism, inflammation, and oxidative stress by offering a promising therapeutic approach for NAFLD.

Aortic calcification accelerates cardiac dysfunction via inducing apoptosis of cardiomyocytes.

Vascular calcification (VC) is a known predictor of cardiovascular events in patients with atherosclerosis and chronic renal disease. However, the exact relationship between VC and cardiovascular mortality remains unclear. Herein, we investigated the underlying mechanisms between VC progression, arterial stiffness, and cardiac dysfunction. C57BL/6 mice were administered intraperitoneally vitamin D3 (VD3) at a dosage of 35×104 IU/day for 14 days. At day 42, VC extent, artery elasticity, carotid artery blood flow, aorta pulse propagation velocity, cardiac function, and pathological changes were evaluated. Heart apoptosis was detected using TUNEL and immunohistochemistry staining. In vitro, rat cardiomyocytes H9C2 were exposed to media from calcified rat vascular smooth muscle cells (VSMCs) cultured in calcification medium, and then H9C2 apoptosis and gene expression related to cardiac function were assessed. VD3-treated mice displayed a significant aortic calcification, increased pulse propagation velocity of aortae, and reduced cardiac function. Aortae showed increased calcification and elastolysis, with increased heart apoptosis. Hearts demonstrated higher levels of ANP, BNP, MMP2, and lower levels of bcl2/bax. Moreover, calcified rat VSMC media induced H9C2 apoptosis and upregulated genes expression linked to cardiac dysfunction. Our data provide evidence that VC accelerates cardiac dysfunction, partially by inducing cardiomyocytes apoptosis.

Vitamin D3 improved hypoxia-induced lung injury by inhibiting the complement and coagulation cascade and autophagy pathway.

BACKGROUND: Pulmonary metabolic dysfunction can cause lung tissue injury. There is still no ideal drug to protect against hypoxia-induced lung injury, therefore, the development of new drugs to prevent and treat hypoxia-induced lung injury is urgently needed. We aimed to explore the ameliorative effects and molecular mechanisms of vitamin D3 (VD3) on hypoxia-induced lung tissue injury. METHODS: Sprague-Dawley (SD) rats were randomly divided into three groups: normoxia, hypoxia, and hypoxia + VD3. The rat model of hypoxia was established by placing the rats in a hypobaric chamber. The degree of lung injury was determined using hematoxylin and eosin (H&E) staining, lung water content, and lung permeability index. Transcriptome data were subjected to differential gene expression and pathway analyses. In vitro, type II alveolar epithelial cells were co-cultured with hepatocytes and then exposed to hypoxic conditions for 24 h. For VD3 treatment, the cells were treated with low and high concentrations of VD3. RESULTS: Transcriptome and KEGG analyses revealed that VD3 affects the complement and coagulation cascade pathways in hypoxia-induced rats, and the genes enriched in this pathway were Fgb/Fga/LOC100910418. Hypoxia can cause increases in lung edema, inflammation, and lung permeability disruption, which are attenuated by VD3 treatment. VD3 weakened the complement and coagulation cascade in the lung and liver of hypoxia-induced rats, characterized by lower expression of fibrinogen alpha chain (Fga), fibrinogen beta chain (Fgb), protease-activated receptor 1 (PAR1), protease-activated receptor 3 (PAR3), protease-activated receptor 4 (PAR4), complement (C) 3, C3a, and C5. In addition, VD3 improved hypoxic-induced type II alveolar epithelial cell damage and inflammation by inhibiting the complement and coagulation cascades. Furthermore, VD3 inhibited hypoxia-induced autophagy in vivo and in vitro, which was abolished by the mitophagy inducer, carbonyl cyanide-m-chlorophenylhydrazone (CCCP). CONCLUSION: VD3 alleviated hypoxia-induced pulmonary edema by inhibiting the complement and coagulation cascades and autophagy pathways.

Intralesional vitamin D3 versus intralesional triamcinolone acetonoid in patchy alopecia areata: a comparative clinical and dermoscopic study.

BACKGROUND: Alopecia areata (AA) is a common autoimmune T-cell mediated non-scarring, form of hair loss. It affects people of all ages and sexes. AIM: To compare the efficacy of intralesional vitamin D3 injection versus that of intralesional triamcinolone acetonide in the treatment of patchy alopecia areata. PATIENTS AND METHODS: This clinical study was carried on 40 adult patients with patchy alopecia areata, the patients were categorized into two groups. Group I involved 20 patients who received 1 ml of intralesional injection of vitamin D3 (cholecalciferol aqueous preparation 200 000 IU/2 ml) every 4 weeks for a maximum of three sessions. Group II involved 20 patients who received 1 ml of intralesional injection of triamcinolone acetonide 40 mg/mL every 4 weeks for a maximum of three sessions. Clinical and trichoscopic evaluations were done at the baseline, each session and for 3 months after the last session. RESULTS: There was no statistically significant difference between the two studied groups regarding the degree of clinical improvement (p = .8). A statistically significant reduction in AA specific trichoscopic signs was detected at the end of the sessions and after 3 months of follow-up in both groups, without any statistically significant difference between them. Also a statistically significant difference was found between both groups regarding the reported adverse effects with a significant better patient satisfaction encountered toward the intralesional vitamin D3 injection. CONCLUSION: Intralesional vitamin D3 is a promising effective, simple, safe, and inexpensive, therapeutic modality for patchy AA.

Vitamin D3 formation in milk by UV treatment - Novel insights into a rediscovered process.

Vitamin D3 is essential for several functions in the human body and the demand is usually covered by natural reactions in skin with UV radiation delivered by the sun. But living beyond a latitude of 35° can lead to a lack of sufficient exposition to the deciding wavelength. Here, many countries fortify their milk prophylactically with artificial vitamin D3. However, the precursor molecule of vitamin D3 (7-deydrocholesterol) is already naturally located in the milk fat globule membrane. Thus, this study deals with the transformation of the naturally occurring 7-dehydrocholesterol into vitamin D3 through UV treatment of the milk - a mechanism that was observed a century ago only indirectly. Different parameters such as temperature (10 - 50°C), fluid flow regimen (turbulent vs. laminar thin film, i.e., 0.6 mm) and wavelength (254, 280 and 313 nm) were investigated in this study for their efficiencies. The UV dose of each experiment was measured with chemical actinometry delivering the actually applied dose reaching the milk. Thus, the connection between applied UV dose and generated vitamin D3 content in the milk measured quantitively with LC-MS/MS was evaluated here that both were not possible a hundred years ago. The experimental results revealed that temperature generally promotes the vitamin D3 formation at 254 nm. Further, a turbulent flow is not as efficiently treated as a laminar thin film flow that is as narrow as 0.6 mm. As expected from absorbance spectra of the precursor molecule 7-dehydrocholesterol, 280 nm turned out to be the most efficient wavelength, followed by intermediate success through irradiation with 254 nm and almost no effect by 313 nm. Generally, it was shown that vitamin D3 concentration of milk was easily increased by UV treatment with today's technologies and that adjustment of certain physical parameters have a significant effect on the efficiency.