Effect of vitamin D3 on production of progesterone in porcine granulosa cells by regulation of steroidogenic enzymes

1,25-dihydroxyvitamin D3 （VD3）, an active form of Vitamin D, is photosynthesized in the skin of vertebrates in response to solar ultraviolet B radiation （UV-B）. VD3 deficiency can cause health problems such as immune disease, metabolic disease, and bone disorders. It has also been demonstrated that VD3 is involved in reproductive functions. Female sex hormones such as estrogen and progesterone are biosynthesized mainly in ovarian granulosa cells as the ovarian follicle develops. The functions of sex hormones include regulation of the estrus cycle and puberty as well as maintenance of pregnancy in females. In this study, we isolated granulosa cells from porcine ovaries and cultured them for experiments. To examine the effects of VD3 on ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by Real-time PCR and Western blotting assay. Production of progesterone from granulosa cells was also measured by ELISA assay. As a result, transcriptional and translational regulation of progesterone biosynthesis-related genes in granulosa cells was significantly altered by VD3. Furthermore, progesterone concen- trations in porcine granulosa cell-cultured media decreased in response to VD3. These results show that VD3 was a strong regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

Vitamin D3 supplementation influences ovarian histomorphometry and follicular development in prepubertal albino rats

Objective:To evaluate the development of ovarian follicles in female albino rats following vitamin D3 supplementation.Methods:Eighteen prepubertal female albino rats,aged 3-4 weeks,weighing(70.25±9.16)g,were assigned to three groups(n=6 in each group).Group A was treated with 5.00 mL/kg of distilled water and served as the control group,group B was treated with 0.025 mg/kg of vitamin D3 dissolved in distilled water,and group C was treated with 0.125 mg/kg of vitamin D3 dissolved in distilled water.All treatments were administered orally,twice weekly for 28 days.Blood and ovaries were harvested under anaesthesia.Serum vitamin D3 levels were determined by using spectrophotometric method.Ovaries were processed for histology and every10th hematoxylin and eosin stained-section was selected for histomorphometry.The number of follicles at each developmental stage was estimated.Results:Both 0.025 mg/kg and 0.125 mg/kg of vitamin D3 significantly increased serum concentrations of vitamin D3 and calcium(P<0.05),but did not alter inorganic phosphorus concentration(P>0.05).The control group had fewer growing follicles(primary,secondary and antral follicles)and more non-growing follicles(primordial and atretic follicles)when compared with the vitamin D3-supplemented groups(P<0.05).Vitamin D3 at 0.025 mg/kg significantly increased antral follicles and corpora lutea counts(P<0.05).Vitamin D3 at 0.125 mg/kg significantly increased total,primordial and atretic follicles counts(P<0.05),but significantly decreased primary,secondary,antral follicles count,ovarian weight,relative ovarian weight,and ovarian surface area when compared with the control group and rats treated with 0.025 mg/kg of vitamin D3(P<0.05).Conclusions:Vitamin D3 supplementation at 0.025 mg/kg can enhance optimal ovarian follicle recruitment and development in female rats.

A Syndrome of Vitamin D3 Deficiency/Fibromyalgia/Hyperparathyroidism Mimicking Rheumatoid Arthritis, a Clinical Prospective Study

Objectives: Rheumatoid arthritis is sometimes misdiagnosed for other diseases, like psoriatic arthritis, erosive OA, viral arthritis, reactive arthritis, IBD arthritis, Lyme’s disease, and palindromic rheumatism. Secondary hyperparathyroidism was not included in the differential diagnosis of RA, though it sometimes presents with joint pains and tenderness, and even arthritis. Fibromyalgia is a psychosomatic disorder characterized by chronic widespread pain and tender areas. Mimicry of some manifestations of these diseases sometimes results in misdiagnosis as RA. Methods: Two hundred patients previously diagnosed as RA from outpatient clinics in Al-Azhar Faculty of Medicine, attended as not responding to medical treatment of RA. All patients were subjected to a re-evaluation of disease activity including HAQ, and DAS 28, CDAI, and SDAI. Also, we measured serum vitamin D3, PTH, total and ionized Calcium, Phosphorus, Uric acid, ACPA, and other routine lab. All patients were exposed to various radiological imaging modalities. Results: Cases not responding to RA treatment were reevaluated and were found to have a syndrome of fibromyalgia associated with vitamin D3 deficiency and secondary hyperparathyroidism. Conclusions: Fibromyalgia/Hyperparathyroidism syndrome is an underdiagnosed disease, which results from chronic vitamin D3 deficiency. SHPT can cause bone erosions, which are mostly shaggy in appearance and distributed in the radiocarpal, radioulnar, metacarpophalangeal and distal interphalangeal joints, in contrast to that which predominate proximal IP joints of rheumatoid arthritis. Radiology of FM/HPT syndrome patients revealed a sign of spur-like excrescences in terminal finger tufts unilaterally or bilaterally, which we think is pathognomonic.

The Role of Vitamin D3 Therapy in Pediatric Bronchiectasis Severity (CF versus non-CF Patients)

Objective: To determine and compare the effect of vitamin D3 supplementation on modifying the disease severity in cystic fibrosis (CF) and non-CF bronchiectasis pediatric patients. Methods: A randomized clinical trial evaluating the role of oral vitamin D3 supplementation for six months, was performed in forty patients with CF and non-CF bronchiectasis under the age of 18 years with vitamin D deficiency or insufficiency. The primary outcome was to reach the sufficient Vitamin D level, the secondary outcome was to reevaluate bronchiectasis severity by following up the frequency, severity of pulmonary exacerbations and lung function after vitamin D3 supplementation. Results: Forty patients completed the trial. The percentage of improvement of vitamin D level after vitamin D3 supplementation for six months was significantly higher in CF (88.3%) than non-CF bronchiectasis patients (59.82%) (P = 0.03). Additionally, moderate to severe pulmonary exacerbations significantly decreased by more than 60%, 45% (P = 0.001, 0.005) and frequent exacerbations decreased by 15%, 10% (P = 0.327, 0.490), while the forced expiratory volume in 1 (FEV1) significantly increased by 17% and 15% in non CF bronchiectasis and CF patients respectively (p < 0.001). Conclusions: Vitamin D3 therapy was effective in decreasing the frequency and severity of pulmonary exacerbations and preserving lung function. Thereby, improving the disease severity even more in non-CF bronchiectasis than CF patients.

Efficient Synthesis of 2-Ethyl-A-ring Analogues of 19-Nor-1α,25-dihydroxy Vitamin D_3

The novel 19 nor l α ,25 dihydroxy vitamin D 3 analogues possessing an ethyl at the 2 position(4 and 5), were synthesized by coupling 25 hydroxy Windaus Grundmann ketone derivative 20 with A ring synthons(15 and 19) respectively. The enantioselective synthesis of substituted bicyclic hexanes structure A ring synthons, started from all cis 3,5 dihydroxy 4 ethyl 1 (methoxycarbonyl)cyclohexane via lipase catalyzd asymmetrization, was demonstrated.

重组枯草芽孢杆菌全细胞催化合成钙二醇的初步研究

钙二醇(25-hydroxy vitamin D_(3),25-OH V_(D3))是一种具有广泛生理活性及药用价值的物质。该文旨在通过分子生物学手段构建1株异源表达V_(D3)羟化酶(V_(D3)hydroxylase,Vdh)的重组枯草芽孢杆菌Bacillus subtilis WB600/pMA5-vdh,使其可以利用细胞催化系统合成25-OH V_(D3)。该重组菌株经诱导表达后,首先利用CO差光谱法评价Vdh的体外活性,并将其用作全细胞催化剂合成25-OH V_(D3),随后通过优化温度、时间、转速及pH等全细胞转化条件,提高其转化合成25-OH V_(D3)的产量。结果表明,37℃下培养24 h后,重组枯草芽孢杆菌B.subtilis WB600/pMA5-vdh的菌体裂解物酶活力达0.56 nmol/g;并且在底物质量浓度为0.1 g/L、外加18 g/L葡萄糖和22.5 g/L的2-羟丙基-β-环糊精的条件下,控制初始pH为7、反应温度30℃、转速200 r/min时进行全细胞转化反应48 h,得到25-OH V_(D3)的转化率为10.36%,以上研究结果为制备和生产食品级25-OH V_(D3)提供了新的思路。

Synthesis of Silica-anchored Sensitizer and its Application in the Preparation of Previtamin D_3 from Tachysterol

A silica gel-anchored photosensitizer was synthesized with anthracene as sensitizer moiety. The photoisomerization of tachysterol to previtamin D3 was carried out with this heterogeneous photosensitizer in ethanol. The experiment results demonstrate that this solid phase photosensitizer is efficient for the photoisomerization and can be simply separated from the reaction mixture by filtration.

Vitamin D_(3) attenuates anxiety-like behavior in long-term ovariectomized rats with unpredictable mild stress

The impact of various vitamin D3(VD3)doses(1.0,2.5,or 5 mg/kg,s.c.)in mitigating the negative consequences of chronic unpredictable mild stress(CUMS)was investigated.Adult female rats with long-term estrogen deficiency were assessed using the sucrose preference test(SPT),the elevated plus-maze(EPM),the light/dark test(LDT),and the open-field test(OFT)to measure anhedonia-like and anxiety-like behavior.The corticosterone(CS)and adrenocorticotrophic hormone(ACTH)concentrations in blood serum and the brain-derived neurotrophic factor(BDNF)expression in the hippocampus of long-term ovariectomized(OVX)rats were measured by ELISA kits and/or western blotting.Treatment with VD3(5.0 mg/kg),similarly to fluoxetine(10.0 mg/kg),significantly reduced the anhedonia profile in the SPT and anxiety-like behavior in the EPM and LDT,and CS and ACTH levels in blood serum.It also elevated BDNF levels in the hippocampus of long-term OVX/CUMS compared to OVX/CUMS/solvent rats.Thus,these findings suggest that VD3(5.0 mg/kg)administration might attenuate the anxiety-like profile in long-term OVX adult rats subjected to the CUMS.This might occur via activation of the BDNF signaling pathway in the hippocampus and via restoration of CS and ACTH levels in blood serum.

Stereochemistry Control in Direct 1α-Hydroxylation of 5,6-trans-Vitamin D_(3) by Solid Support

A solid-phase synthesis of 1α-hydroxylation of 5,6-trans-vitamin D3 8 is described.The solid phase resin acts as a special protecting group, which gives a higher stereoselectivity in oxidation step than classical protecting groups. The stereochemistry control is favored by using high crosslinkage polymer support in a poor solvent. This work may be of benefit to the synthesis of vitamin D system.

Effect of Reaction Media on Photoreaction of Provitamin D_3

The photoisomerization of provitamin D-3 is carrried out in silica gel-hexane matrix, ethanol and hexane. The results show that in silica gel-hexane matrix and ethanol, the desired product of provitamin D-3 and vitamin D-3 can be obtained in high yield, while the undesirable byproducts are greatly limited.

Efficacy and safety of vitamin D3 in patients with diabetic nephropathy： a meta-analysis of randomized controlled trials

Background Several studies found that vitamin D3 might alter glucose metabolism,protect kidney from injury and even proposed the mechanisms.But results from previous studies have been conflicting.The aim of this study was to evaluate the efficacy and safety of vitamin D3 in patients with diabetic nephropathy.The underlying mechanism of vitamin D3 decreasing proteinuria is also discussed.Methods We conducted a search of English and Chinese articles using database of Pubmed,Embase,Sinomed,CNKI,Wanfang and clinical trial register centers,for randomized controlled trials of vitamin D3 in diabetic nephropathy patients.Two reviewers performed independently.Meta-analysis was used when studies were homogeneous enough.Results Twenty studies,including 1 497 patients with diabetic nephropathy,were involved in this systemic review.Vitamin D3-treated patients with diabetic nephropathy had a statistically significant reduction in 24-hour proteinuria （weighted mean difference-0.44,95% CI-0.54 to-0.34,Z=8.80,P 〈0.000 01） and urine albumin/creatine ratio （standardized mean difference-0.29,95% CI-0.48 to-0.10,Z=2.96,P=0.003）.But vitamin D3 supplementation did not significantly reduce blood pressure and hemoglobin A1c compared with control group.The potential mechanisms about the renal protection of vitamin D3,including the inhibition of rennin-angiotensin system,the protection of kidney from inflammation,fibrosis and the structure change of kidney are discussed.In addition,vitamin D3 did not significantly increase the incidence of adverse effects,including total adverse effects,gastrointestinal adverse effects and fluctuation of blood pressure.Conclusions Vitamin D3 can ameliorate proteinuria and protect kidney from injury in patients with diabetic nephropathy.This renoprotective effect is independent of blood pressure and glucose reduction.And it does not increase any adverse effects than control,even in combination therapy with angiotensin converting enzyme inhibitors/angiotensin receptor blockers.But due to the limited randomized controlled trials of high quality,more clinical researches should be taken in the future.

1,25-Dihydroxyvitamin D3 protects obese rats from metabolic syndrome via promoting regulatory T cell-mediated resolution of inflammation

Vitamin D_3 has been found to produce therapeutic effects on obesity-associated insulin resistance and dyslipidemia through its potent anti-inflammatory activity, but the precise immunomodulatory mechanism remains poorly understood. In the present study we found that 1,25-dihydroxyvitamin D_3[1,25(OH)_2D_3], the biologically active form of vitamin D_3, significantly attenuated monosodium glutamate(MSG)-induced obesity and insulin resistance as indicated by body weight reduction, oral glucose tolerance improvement, and a glucose infusion rate increase as detected with hyperinsulinemiceuglycemic clamp. Moreover, 1,25(OH)_2D_3 not only restored pancreatic islet functions but also improved lipid metabolism in insulin-targeted tissues. The protective effects of 1,25(OH)_2D_3 on glycolipid metabolism were attributed to its ability to inhibit an obesity-activated inflammatory response in insulin secretory and targeted tissues, as indicated by reduced infiltration of macrophages in pancreas islets and adipose tissue while enhancing the expression of Tgf-β1 in liver tissue, which was accompanied byincreased infiltration of Treg cells in immune organs such as spleen and lymph node as well as in insulintargeted tissues such as liver, adipose, and muscle. Together, our findings suggest that 1,25(OH)_2D_3 serves as a beneficial immunomodulator for the prevention and treatment of obesity or metabolic syndrome through its anti-inflammatory effects.

1,25-Dihydroxyvitamin D3 pretreatment enhances the efficacy of allergen immunotherapy in a mouse allergic asthma model

Background Allergen-specific immunotherapy can induce immune tolerance to specific allergens by regulating immune status of individuals. However, its clinical application is limited due to individual differences in efficacy among patients and un-confirmed safety. 1,25 Dihydroxyvitamin D3 （1,25（OH）2D3） has been shown to be involved in a variety of physiological processes, including immune response regulation. In the present study we explored the role of 1,25（OH）2D3 pretreatment for immunotherapy.Methods Seventy-five BALB/c mice were randomly divided into five groups （15 mice per group）. The mouse allergic asthma model was established by intra-peritoneal injection of ovalbumin （OVA, 10 μg） and aluminium hydroxide （2 mg）as an adjuvant. Intra-peritoneal injection of 50 ng of 1,25（OH）2D3 served as a pretreatment, subcutaneous injection of OVA （100 μg） as an immunotherapy, and 1% OVA inhalation as a challenge. Histopathological analysis was performed on four mice per group. The number of cells and their classification in bronchoalvolar lavage （BAL） fluid were assayed.Levels of serum OVA-specific immunoglobulin E （slgE） and IFN-Y, IL-4, IL-5 and IL-10 in BAL fluid were measured by ELISA.Results After 1,25（OH）2D3 pretreatment, immunotherapy could significantly inhibit the infiltration of inflammatory cells into lung tissues and BAL fluid of mice with allergic asthma when compared with un-treated animals （eosinophils：（7.46±1.34）×104/ml vs. （13.41±1.67）×104/ml, P ＜0.05）. In addition, levels of IL-4 （（36.g1±7.87） pg/ml vs. （43.70±6.42）pg/ml, P ＞0.05） and IL-5 （（41.97±7.93） pg/ml vs. （60.14±8.35） pg/ml, P ＜0.05） in BAL fluid and serum slgE （（0.42±0.05）vs. （0.75±0.06） OD units, P ＜0.05） were profoundly reduced. However, the IL-10 level in BAL fluid was significantly increased （（67.74±6.57） pg/ml vs. （44.62±8.81） pg/ml, P ＜0.05）.Conclusions These results indicated that 1,25（OH）2D3 pretreatment enhanced the inhibitory effects of immunotherapy on allergic airway inflammation. In the treatment of allergic diseases, 1,25（OH）2D3 pretreatment may be beneficial for improving the efficacy of immunotherapy.

SMURF2 regulates bone homeostasis by disrupting SMAD3 interaction with vitamin D receptor in osteoblasts

Subject Code:H06 With the support by the National Natural Science Foundation of China,a study by the research group led by Prof.Zou Weiguo(邹卫国)from the Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,CAS demonstrates that E3ligase SMURF2maintains bone

Molecular mechanism mediating enteric bacterial translocation after severe burn:the role of cystic fibrosis transmembrane conductance regulator

Background:Gut ischemia and hypoxia post severe burn leads to breakdown of intestinal epithelial barrier and enteric bacterial translocation(EBT),resulting in serious complications,such as systemic inflammatory response syndrome,sepsis and multiple organ failure.Cystic fibrosis transmembrane conductance regulator(CFTR)is known to be downregulated by hypoxia and modulate junctional complexes,which are crucial structures maintaining the intestinal barrier.This study aimed to investigate whether CFTR plays a role in both regulating the intestinal barrier and mediating EBT post severe burn,as well as the signaling pathways involved in these processes.Methods:An in vitro Caco-2 cell model subjected to hypoxic injury and an in vivo mouse model with a 30%total body surface area full-thickness dermal burn were established.DF 508 mice(mice with F508del CFTR gene mutation)were used as an in vivo model to further demonstrate the role of CFTR in maintaining normal intestinal barrier function.QRT-PCR,western blot,ELISA,TER assay and immunofluorescence staining were used to detect the expression and localization of CFTR and tight junction proteins,as well as the function of tight junctions.Results:Our data indicated that,in Caco-2 cells,the hypoxia condition significantly reduced CFTR expression;activated extracellular signal-regulated kinase and nuclear factor-κB signaling;elevated secretion of inflammatory factors(tumor necrosis factor-α,interleukin-1βand interleukin-8);downregulated zonula occludens-1,occludin and E-cadherin expression;decreased transepithelial electrical resistance values;and led to a cellular mislocation of ZO-1.More importantly,knockdown of CFTR caused similar alterations.The upregulation of inflammatory factors and downregulation of tight junction proteins(ZO-1 and occludin)induced by knockdown of CFTR could be reversed by specific extracellular signal-regulated kinase or nuclear factor-κB inhibition.In support of the in vitro data,exuberant secretion of pro-inflammatory mediators and EBT was observed in the intestine of severely burnt mice in vivo.EBT occurred in DF508 mice(mice with the F508del CFTR gene mutation),accompanied by augmented tumor necrosis factor-α,interleukin-1βand interleukin-8 levels in the ileum compared to wildtype mice.In addition,vitamin D3 was shown to protect the intestinal epithelial barrier from hypoxic injury.Conclusions:Collectively,the present study illustrated that CFTR and downstream signaling were critical in modulating the intestinal epithelial junction and EBT post severe burn.

The relationships of vitamin D, vitamin D receptor gene polymorphisms, and vitamin D supplementation with Parkinson's disease

In recent years,many studies have investigated the correlations between Parkinson's disease(PD)and vitamin D status,but the conclusion remains elusive.The present review focuses on the associations between PD and serum vitamin D levels by reviewing studies on the associations of PD with serum vitamin D levels and vitamin D receptor(VDR)gene polymorphisms from PubMed,Web of Science,Cochrane Library,and Embase databases.We found that PD patients have lower vitamin D levels than healthy controls and that the vitamin D concentrations are negatively correlated with PD risk and severity.Furthermore,higher vitamin D concentrations are linked to better cognitive function and mood in PD patients.Findings on the relationship between VDR gene polymorphisms and the risk of PD are inconsistent,but the Fokl(C/M)polymorphism is significantly linked with PD.The occurrence of Fokl(CT)gene polymorphism may influence the risk,severity,and cognitive ability of PD patients,while also possibly influencing the effect of Vitamin D3 supplementation in PD patients.In view of the neuroprotective effects of vitamin D and the close association between vitamin D and dopaminergic neurotransmission,interventional prospective studies on vitamin D supplementation in PD patients should be conducted in the future.

Improved and efficient synthesis of Maxacalcitol

Maxacalcitol,the 22-oxa-derivative of 1a,25-dihydroxyvitamin D3,has been used as an antihyperparathyroidism and antipsoriatic drug.In this paper,an alternative synthetic route has been developed using commercially available vitamin D2as the starting material.In addition,as a key intermediate,the asymmetric synthesis of 20（S）-alcohol 14 with the chiral auxiliary（R-CBS） was described for the first time.This new synthetic route afforded Maxacalcitol with improved overall yield（13.9%） in eleven steps.

Synthesis of Polymer-bound Sensitizers and their Applications in the Preparation of Previtamin D_3 from Tachysterol

Two kinds of polymer-bound photosensitizers were synthesized, in which anthracene acts as sensitizer moiety, polystyrene (PS) and poly(methyl methylacrylate) (PMMA) serve as solid phase supports, respectively. The photoisomerization of tachysterol to previtamin D3 was carried out with these polymer-bound photosensitizers in ethanol and toluene solutions. The experiment results demonstrate that these solid phase photosensitizers are effi-cient for the photoisomerization with good recycle property and can be simply separated from the reaction mixtures by filtration.