Increase in the Tight Junction Protein Claudin-1 in Intestinal Inflammation

Background and Aims

Studies have shown a decrease in key tight junction (TJ) proteins such as ZO-1 and occludin in both inflammatory bowel disease (IBD) and experimental models of inflammation. Our group has also shown an increase in claudin-1 in experimental colitis.

Methods

IEC-18 cells were treated with increasing doses of tumor necrosis factor alpha (TNF??). The TJ was assessed by transepithelial resistance (TER), permeability, Western blot, PCR, and immunofluorescence. Mucosal samples from patients with ulcerative colitis (UC), Crohn??s disease (CD), and without IBD (normal) were assayed for TJ proteins occludin and claudin-1 by Western blot and a ratio of claudin-1 to occludin (C:O) was calculated.

Results

IEC-18 cells had increased permeability, decreased TER and an increase in claudin-1 with TNF?? treatment. In human specimens, there was a decrease in occludin and an increase in claudin-1 leading to a significant increase in the C:O ratio in diseased UC colon compared to non-diseased UC colon (P < 0.001) and normal colon (P < 0.01). In CD, the C:O ratio was similar in all CD tissue irrespective of disease status.

Conclusions

Treatment of IEC-18 cells with TNF??, a key inflammatory cytokine in IBD, led to a significant increase in claudin-1 expression. There was a significant increase in the C:O ratio in diseased colon in UC compared to the healthy appearing UC colon and normal controls. The C:O ratio was unchanged in CD despite presence or abscence of gross disease. This suggests that there may be an underlying difference in the TJ between UC and CD.     

Vitamin D and S-Farnesylthiosalicylic Acid Have a Synergistic Effect on Hepatic Stellate Cells Proliferation

Background

Hepatic stellate cells (HSCs) have a key role in the formation of hepatic fibrosis. The active form of vitamin D, 1,25(OH)₂D₃, has been found to have antiproliferative and antifibrotic effects in various tissues including liver. Farnesylthiosalicylic acid (FTS), a novel Ras antagonist, was also found to inhibit hepatic fibrosis.

Aims

The purpose of this study was to examine the antiproliferative and antifibrotic effects of the combined treatment of 1,25(OH)₂D₃ and FTS on primary cultured HSCs.

Methods

Primary HSCs, isolated from rat’s livers, were treated with 1,25(OH)₂D₃, FTS or a combination of both. Proliferation was assessed by bromodeoxyuridine. Expression of p-ERK, ERK, Ras-GTP, total-Ras, CyclinD1 and fibrotic markers was measured by western blotting analysis and real-time PCR. Cytotoxicity was assessed by lactate dehydrogenase method.

Results

The combined treatment inhibited HSCs proliferation by threefold. The effect was synergistic and non-cytotoxic. In concordance, the combined treatment suppressed CyclinD1 expression by ~2-fold, whereas 1,25(OH)₂D₃ or FTS alone showed a significantly lower inhibitory effect. The effect of the combined treatment on CyclinD1 expression was mediated via Ras-GTP and p-ERK signal transduction pathway. The effect on fibrotic markers showed that 1,25(OH)₂D₃ decreased collagen Iα1 expression by ~40 %, FTS by ~50 % and the combined treatment by ~60 %. 1,25(OH)₂D₃ inhibited tissue inhibitor of metalloproteinases-1 (TIMP-1) expression by 20 %. FTS alone or 1,25(OH)₂D₃ + FTS inhibited TIMP-1 expression by 60 %. FTS inhibited transforming growth factor-β (TGF-β) expression by 25 %, while 1,25(OH)₂D₃ had no effect.

Conclusion

Although the combination of 1,25(OH)₂D₃ and FTS did not demonstrate an additive antifibrotic effect, it showed a synergistic antiproliferative effect on primary HSCs. Therefore, the combined treatment may have a potential therapeutic value in the initiation of fibrotic process.     

Role of vitamin D derivatives in intestinal tissue of patients with inflammatory bowel diseases

Background and aimThe adhesion molecule expression and matrix metalloproteinases (MMPs) are proposed to be major factors for intestinal injury mediated by T cells in (IBD) and are up-regulated in intestinal mucosa of IBD patients. To investigate the effect of vitamin D derivatives on adhesion molecules and MMPs in colonic biopsies of IBD patients.MethodsBiopsies from inflamed and non-inflamed tract of terminal ileum and colon and PBMC from the same IBD patients were cultured with or without vitamin D derivatives. MMP activity and adhesion molecule levels were determined.Results1,25(OH)₂D₃ and ZK 191784 significantly decrease ICAM-1 protein levels in the biopsies obtained only from the inflamed region of intestine of UC patients, while MAdCAM-1 levels decrease in the presence of 1,25(OH)₂D₃ in the non-inflamed region, and, in the presence of ZK, in the inflamed one. In CD patients 1,25(OH)₂D₃ and ZK decrease ICAM-1 and MAdCAM-1 in the biopsies obtained from the non-inflamed and inflamed regions, with the exception of ICAM-1 in the inflamed region in the presence of 1,25(OH)₂D₃. The expression of MMP-9, MMP-2, and MMP-3 decreases in the presence of vitamin D derivatives in UC and CD with the exception of 1,25(OH)₂D₃ that does not affect the levels of MMP-9 and MMP-2 in CD. Vitamin D derivatives always affect MMP-9, MMP-2 and ICAM-1 in PBMC of UC and CD patients.ConclusionsBased on the increased expression of ICAM-1, MAdCAM-1 and MMP-2,-9,-3 in IBD, our study suggests that vitamin D derivatives may be effective in the management of these diseases.     

Cardiac metabolism,inflammation, and peroxisome proliferator-activated receptors modulated by 1,25-dihydroxyvitamin D3 in diabetic rats

Background

High free fatty acid with reduced glucose utilization in diabetes mellitus (DM) impairs cardiac function. Peroxisome proliferator-activated receptors (PPARs) modulate myocardial lipid and glucose homeostasis. The active 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) regulates oxidative stress and inflammation, which may play a key role in the modulation of PPARs. The aim of this study was to investigate whether 1,25(OH)₂D₃ can modulate the cardiac PPARs and fatty acid metabolism.

Methods

Electrocardiogram, echocardiogram, and Western blot analysis were used to evaluate cardiac fatty acid metabolism, inflammation, and PPAR isoform expression in Wistar-Kyoto (WKY) rats, DM rats, and DM rats treated with 1,25(OH)₂D₃.

Results

Compared to healthy rats, DM and 1,25(OH)₂D₃-treated DM rats had lower body weight. DM rats had larger left ventricular end-diastolic diameter, and longer QT interval than healthy or 1,25(OH)₂D₃-treated DM rats. Moreover, compared to healthy or 1,25(OH)₂D₃-treated DM rats, DM rats had fewer cardiac PPAR-α and PPAR-δ protein expressions, but had increased cardiac PPAR-γ protein levels, tumor necrosis factor-α, interleukin-6, 5′ adenosine monophosphate-activated protein kinaseα2, phosphorylated acetyl CoA carboxylase, carnitine palmitoyltransferase 1, PPAR-γ coactivator 1-α, cluster of differentiation 36, and diacylglycerol acyltransferase 2 protein expressions.

Conclusions

1,25(OH)₂D₃ significantly changed the cardiac function and fatty acid regulations in DM hearts, which may be caused by its regulations on cardiac PPARs and proinflammatory cytokines.     

Vitamin D regulates the tight-junction protein expression in active ulcerative colitis

Objective: Epithelial barrier function is primarily regulated by the tight-junction proteins. Ulcerative colitis (UC) is characterized by Th2 immune response with inflammation and epithelial barrier dysfunction, including an elevation of claudin-2 protein function. Recent studies support an important role of vitamin D in the pathogenesis as well as potential therapy of IBD. Vitamin D deficiency is in fact common in patients with IBD. The aim of the study was to determine whether vitamin D could affect IL-13 and IL-6 levels, and regulate the activity of tight-junction proteins. Claudin-1, -2, -4, and -7 in the inflamed and non-inflamed colonic mucosa of UC patients.

Material and methods: Biopsies from inflamed and non-inflamed tract of colon and rectum from the same active UC patients were cultured with1,25(OH)₂D₃. IL-13, IL-6 and the tight-junction proteins level were determined.

Results: Claudin-1 and claudin-2 proteins were up-regulated in active UC. The treatment with 1,25(OH)₂D₃ decreases the claudin-1 and claudin-2 protein levels in both inflamed and non-inflamed tract. Claudin-4 and claudin-7 proteins were down-regulated and their levels increase after incubation with the 1,25(OH)₂D₃. When the biopsies were incubated with 1,25(OH)₂D₃, a decrease in IL-13 and IL-6 levels was registered.

Conclusions: Our results, indicating the inhibition of cytokine levels and the regulation of claudin-2, claudin-4, and claudin-7 by 1,25(OH)₂D₃, suggest that vitamin D may represent a potential therapeutic agent for the treatment of active UC.     

Claudin-2 Regulates Colorectal Inflammation via Myosin Light Chain Kinase-Dependent Signaling

Background

Claudins have been demonstrated to be associated with inflammatory bowel disease (IBD), but the specific role of claudin-2 in colorectal inflammation remains undefined.

Aims

We aimed to determine the role of claudin-2 in TNFα-induced colorectal inflammation.

Methods

We used claudin-2 (?/?) mice to assess the role of claudin-2 in colon. The mice were intraperitoneally injected with 3 μg of recombinant murine TNFα, and the NF-κB signaling and mRNA expression levels of proinflammatory cytokines and myosin light chain kinase (MLCK) were evaluated. Moreover, in claudin-2 (?/?) mice, colitis was induced by the administration of dextran sodium sulfate (DSS). The involvement of claudin-2 in colorectal inflammation was also investigated using the Caco-2 human colon adenocarcinoma cell line, and the expression of claudin-2 was downregulated using claudin-2 siRNA.

Results

TNFα-induced colorectal inflammation via NF-κB signaling activation was enhanced in claudin-2 (?/?) mice compared with that in claudin-2 (+/+) mice. MLCK expression level in the colon tissue of claudin-2 (?/?) mice treated with TNFα was enhanced in comparison to that of the claudin-2 (+/+) mice. DSS-induced colitis was more severe in the claudin-2 (?/?) mice than in the claudin-2 (+/?) mice. In in vitro experiments, the decreased expression of claudin-2 enhanced the expressions of IL-6, IL-1β and MLCK.

Conclusions

Our findings concerning the role of claudin-2 in epithelial inflammatory responses enrich our collective understanding of mucosal homeostasis and intestinal diseases such as IBD. Furthermore, the results of this study indicate that claudin-2 and MLCK are potential therapeutic targets for treatments against intestinal disease.     

1,25‐dihydroxyvitamin D3 modulates Th17 polarization and interleukin‐22 expression by memory T cells from patients with early rheumatoid arthritis

Objective

To examine the immunologic mechanism by which 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃) may prevent corticosteroid‐induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology.

Methods

Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO− (non‐memory) T cells separated by fluorescence‐activated cell sorting (FACS) from treatment‐naive patients with early RA were stimulated with anti‐CD3/anti‐CD28 in the absence or presence of various concentrations of 1,25(OH)₂D₃, dexamethasone (DEX), and 1,25(OH)₂D₃ and DEX combined. Levels of T cell cytokines were determined by enzyme‐linked immunosorbent assay and flow cytometry.

Results

The presence of 1,25(OH)₂D₃ reduced interleukin‐17A (IL‐17A) and interferon‐γ levels and increased IL‐4 levels in stimulated PBMCs from treatment‐naive patients with early RA. In addition, 1,25(OH)₂D₃ had favorable effects on tumor necrosis factor α (TNFα):IL‐4 and IL‐17A:IL‐4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL‐17A– and IL‐22–expressing CD4+ T cells and IL‐17A–expressing memory T cells were observed in PBMCs from treatment‐naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO− cells between these 2 groups. Interestingly, 1,25(OH)₂D₃, in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL‐17A, IL‐17F, TNFα, and IL‐22 production by memory T cells sorted by FACS from patients with early RA.

Conclusion

These data indicate that 1,25(OH)₂D₃ may contribute its bone‐sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL‐4.

     

Vitamin D treatment of senescence accelerated mice (SAM-P/6) induces several regulators of stromal cell plasticity

In an attempt to understand the regulation of bone marrow multipotential cells plasticity in vivo, we treated 4-month-old SAM-P/6 mice with a constant infusion of either 18 pmol/24 h of 1,25(OH)₂D₃ or vehicle alone for 6 weeks. In vehicle treated animals 78% ± 4 adipose volume vs. total volume was stained positive with oil red O as compared to only 32 ± 3% in 1,25(OH)₂D₃treated animals (P < 0.001). Furthermore, we aimed to identify the changes in gene expression induced by 1,25(OH)₂D₃in bone marrow cells by analyzing a set of 5440 genes in the NIA 15K Mouse cDNA microarray. Overall, a coordinated regulation of genes which both stimulate osteoblastogenesis and inhibit adipogenesis was observed in 1,25(OH)₂D₃-treated mice when compared to vehicle treated mice. In summary, this study illustrates the anti-adipogenic effect of 1,25(OH)₂D₃in bone cells and identifies some of the possible key signals involved in bone cell plasticity.     

The effect of cholecalciferol supplementation on vitamin D levels and insulin sensitivity is dose related in vitamin D-deficient HIV-1-infected patients

Objective

The aim of this study was to explore the effects of cholecalciferol supplementation on vitamin D levels, bone mineral density (BMD), body fat distribution and insulin sensitivity in vitamin D‐deficient HIV‐1‐infected patients.

Methods

Twenty vitamin D‐deficient HIV‐1‐infected patients were prospectively treated with 2000 IU cholecalciferol/day for 14 weeks, whereafter treatment was continued with half this dosage until 48 weeks. BMD, body fat distribution, 1,25‐dihydroxy vitamin D₃ (1,25(OH)₂D₃), fasting glucose, insulin, adiponectin, leptin, interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α were measured at baseline, and at 24 and 48 weeks. Parathyroid hormone (PTH), 25‐hydroxy vitamin D₃ [25(OH)D₃], cholesterol and triglycerides were measured at baseline, and at 12, 24 and 48 weeks.

Results

After 24 weeks, cholecalciferol supplementation significantly increased 25(OH)D₃ and 1,25(OH)₂D₃ levels and decreased PTH and insulin sensitivity. After 48 weeks, however, only 25(OH)D₃ levels remained significantly different from baseline, while the other parameter levels returned to baseline, suggesting a dose–response effect. Cholecalciferol had no effect on BMD, adipokines and triglycerides.

Conclusions

The effect of cholecalciferol treatment in this cohort appears to be dose dependent. Cholecalciferol dosages of ≥2000 IU are necessary to achieve 1,25(OH)₂D₃ levels that significantly decrease PTH, but also negatively affect insulin sensitivity. The results of this hypothesis‐driven explorative study need to be confirmed in larger clinical trials.     

Effects of analogs of 1,25(OH)2 Vitamin D3 on the proliferation and differentiation of the human chronic myelogenous leukemia cell line,RWLeu-4

Summary We evaluated the proliferative and differentiative effects of analogs of 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] on a chronic myelogenous leukemia cell line, RWLeu-4, which is growth-inhibited and differentiates in response to 1,25(OH)₂D₃ (ED₅₀-3-10 nM). Side-chain-fluorinated analogs were more potent (ED₅₀=0.7–2 nM) while most of those with altered saturation of the D ring or side-chain carbon-carbon bonds were equally or less effective than 1,25(OH)₂D₃. However, the two analogs with either two additional double bonds or an extra double and triple bond in the D ring had greater antiproliferatiive activity [1,25(OH)₂-16,23-diene D₃ (ED₅₀=2.7 nM) and 1,25(OH)₂-16-ene-23-yne D₃ (ED₅₀=0.7 nM)]. Since the latter of these has been reported to be less potent at mobilizing calcium than 1,25(OH)₂D₃, it (or a similar compound) may be a candidate for clinical use as an antineoplastic agent.Abbreviations 1,25(OH)₂D₃ 1,25-(OH)₂ vitamin D₃ - CML chronic myelogenous leukemia - NBT nitroblue tetrazolium - ED₅₀ 50% effective dose - IC₅₀ 50% inhibitory concentration     

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1alpha,25-dihydroxyvitamin D3

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK₁ kidney cells. To induce 24-hydroxylase expression, 1,25(OH)₂D₃ (10⁻⁷ M) and dexamethasone were added simultaneously to the medium of LLC-PK₁ cells, and 24 h before dexamethasone treatment, 1,25(OH)₂D₃ was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells. Maximal stimulation was observed with 10⁻⁶ M dexamethasone in both cell lines. The addition of 10⁻⁶ M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10⁻⁶ M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)₂D₃-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)₂D₃-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)₂D₃ enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.     

Roseburia intestinalis inhibits oncostatin M and maintains tight junction integrity in a murine model of acute experimental colitis

Objective: Levels of oncostatin M (OSM) and the composition of gut microbiota predict responses to anti-TNF agents used for IBD therapy. Here, the aim was to investigate the effects of Roseburia intestinalis, a gut microbiota, on OSM and on intestinal barrier in colitis.

Methods: In the murine model of 3% dextran sulfate sodium (DSS)-induced colitis, we tested disease activity index (DAI), colon length, histological score and expression of tight junction (TJ) proteins (ZO-1, occludin and claudin-1), OSM, TNF-α and TLR5. In addition, a cellular model was used to examine the role of R. intestinalis during secretion of OSM by lipopolysaccharide (LPS)-induced bone marrow-derived macrophages (BMDMs) isolated from wild-type (WT) and TLR5 knockout (TLR5 KO) mice. Furthermore, we evaluated the impact of OSM on expressions of TJ proteins by Caco-2 cells.

Results: R. intestinalis in DSS-induced colitis decreased DAI score (p?<?.001), colon length shortening (6.46?±?0.36?cm vs 5.65 ± 0.47 cm, p?=?.022), histological score (2.667?±?1.15 vs 5.33 ± 1.14, p?=?.018) and increased expression of TJ proteins (p?<?.05). In addition, R. intestinalis reduced expression of OSM (p?<?.05) and TNF-α (p?<?.05), while increasing expression of TLR5 (p?<?.05). Furthermore, R. intestinalis reduced secretion of OSM (p?<?.05) by LPS-induced BMDMs isolated from WT and TLR5 KO mice. Moreover, OSM downregulated expression of TJ proteins (p?<?.05) by Caco-2 cells in a concentration-dependent manner.

Conclusions: These results indicate that R. intestinalis attenuates inflammation in IBD by decreasing secretion of OSM and by promoting intestinal barrier function. Taken together, the data provide insight into the role of the gut microbiota in patients with IBD who are resistant to anti-TNF therapy.     

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10⁻⁷ M, 1,25(OH)₂D₃ for 24 h resulted in a 20–30% decrease in PKI mRNA. 1,25(OH)₂D₃ treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)₂D₃ directly and tissue-specifically downregulates PKI mRNA in the chick kidney.     

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10⁻¹⁰–10⁻⁸ M 1,25-(OH)₂D₃ or 10⁻⁹–10⁻⁷ M 24,25-(OH₂)D₃, and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [³⁵S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)₂D₃ caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)₂D₃ responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)₂D₃; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)₂D₃. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.     

STW 5 is effective in dextran sulfate sodium-induced colitis in rats

Purpose

An herbal preparation, STW 5, used clinically in functional dyspepsia and irritable bowel syndrome, has been shown to possess properties that may render it useful in inflammatory bowel disease (IBD). The present work was conducted to study its effectiveness in a rat model of IBD.

Methods

An experimental model reflecting ulcerative colitis in man was adopted, whereby colitis was induced in Wistar rats by feeding them 5?% dextran sulfate sodium (DSS) in drinking water for one week. STW 5 and sulfasalazine (as a reference standard) were administered orally daily for 1?week before colitis induction and continued during DSS feeding. The animals were then sacrificed, and the severity of colitis was evaluated macroscopically and microscopically. Colon samples were homogenized for determination of reduced glutathione, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-3 as well as myeloperoxidase, glutathione peroxidase, and superoxide dismutase. In addition, colon segments were suspended in an organ bath to test their reactivity towards carbachol, KCl, and trypsin.

Results

STW 5 and sulfasalazine were both effective in preventing the shortening of colon length and the increase in both colon mass index and total histology score as well as the changes in biochemical parameters measured except changes in dismutase activity. DSS-induced colitis led to marked depression in colonic responsiveness to the agents tested ex vivo, an effect which was normalized by both drugs.

Conclusions

The findings point to a potential usefulness of STW 5 in the clinical setting of ulcerative colitis.     

Lactulose Mediates Suppression of Dextran Sodium Sulfate-Induced Colon Inflammation by Increasing Hydrogen Production

Background

Molecular hydrogen (H₂) is a potent antioxidant and able to protect organs from oxidative stress injuries. Orally administered lactulose, a potent H₂ inducer, is digested by colon microflora and significantly increases H₂ production, indicating its potential anti-inflammatory action.

Objective

To evaluate the anti-inflammatory effects of lactulose on dextran sodium sulfate (DSS)-induced colitis in mice.

Methods

Mice were randomly assigned into seven groups, receiving regular distilled water, H₂-rich saline (peritoneal injection), DSS, oral lactulose (0.1, 0.15, 0.2 ml/10 g, respectively), and lactulose (0.2 ml/10 g) + oral antibiotics. The mouse model of human ulcerative colitis was established by supplying mice with water containing DSS. The H₂ breath test was used to determine the exhaled H₂ concentration. Body weight, colitis score, colon length, pathological features and tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), maleic dialdehyde (MDA) and marrow peroxidase (MPO) levels in colon lesions were evaluated.

Results

After 7 days, DSS-induced loss of body weight, increase of colitis score, shortening of colon length, pathological changes and elevated levels of TNF-α, IL-1β, MDA, and MPO in colon lesions, were significantly suppressed by oral lactulose administration and intraperitoneally injected H₂-rich saline. Ingestion of antibiotics significantly compromised the anti-inflammatory effects of lactulose. The H₂ breath test showed that lactulose administration significantly induced hydrogen production and that antibiotics administration could inhibit H₂ production.

Conclusion

Lactulose can prevent the development of DSS-induced colitis and alleviate oxidative stress in the colon, as measured by MDA and MPO, probably by increasing endogenous H₂ production.     

The cecum is the site with the highest calcium absorption in rat intestine

In the absence of electrochemical gradients, mucosa-to-serosa Ca transport across the rat cecum is about six times higher than the serosa-to-mucosa flux, resulting in a marked Ca absorption, which is considerably higher when compared with Ca absorption reported for other intestinal segments. The voltage-clamp experiments reveal that 45% of the total mucosa-to-serosa Ca transport measured across the short-circuited tissue is cellular whereas 55% is paracellular. The serosa-to-mucosa Ca flux, however, is purely paracellular. Dexamethasone or 1,25(OH)₂D₃ has no effect on the Ca transport across the cecum. Diphosphonate, known to inhibit 1,25(OH)₂D₃ synthesis, abolishes cellular mucosa-to-serosa Ca transport but this effect can be restored by simultaneous exogenous 1,25(OH)₂D₃ application. It is concluded that the cecum is the site with the highest calcium absorption in the rat intestine. The cellular mucosa-to-serosa Ca transport is dependent on 1,25(OH)₂D₃ but cannot be increased by exogenous, 1,25(OH)₂D₃ application, suggesting that the Ca carrier in this segment already under normal nutritional conditions works at the maximal level. Ca absorption across the proximal colon therefore seems to be of physiological importance in the regulation of intestinal Ca homeostasis.Supported by Deutsche Forschungsgemeinschaft (Ka 639/13).