Trinitrobenzene Sulfonic Acid-induced Colitis Research Articles

Hookworm-Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key Inflammatory Cytokines in Primary Human Leukocytes.

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts.

Human Mesenchymal Stem Cell-Treated Regulatory CD23+CD43+ B Cells Alleviate Intestinal Inflammation.

Rationale: Mesenchymal stem cells (MSCs) have been demonstrated to ameliorate inflammatory bowel disease by their actions on multiple immune cells, especially on regulatory B cells (Breg cells). However, the phenotypes and functions of human MSCs (hMSCs)-treated Breg cell subsets are not yet clear.Methods: Purified B cells were cocultured with MSCs and the phenotypes and immunomodulatory functions of the B cells were analyzed by FACS and proliferation assays in vitro. Also, a trinitrobenzenesulfonic acid-induced mouse colitis model was employed to detect the function of MSC-treated Breg cells in vivo.Results: We demonstrated that coculturing with hMSCs significantly enhanced the immunomodulatory activity of B cells by up-regulating IL-10 expression. We then identified that a novel regulatory B cell population characterized by CD23 and CD43 phenotypic markers could be induced by hMSCs. The CD23+CD43+ Breg cells substantially inhibited the inflammatory cytokine secretion and proliferation of T cells through an IL-10-dependent pathway. More significantly, intraperitoneal injection of hMSCs ameliorated the clinical and histopathological severity in the mouse experimental colitis model, accompanied by an increase in the number of CD23+CD43+ Breg cells. The adoptive transfer of CD23+CD43+ B cells effectively alleviated murine colitis, as compared with the CD23-CD43- B cells. Treatment with CD23+CD43+ B cells, and not hMSCs, substantially improved the symptoms of colitis in B cell-depleted mice.Conclusion: the novel CD23+CD43+ Breg cell subset appears to be involved in the immunomodulatory function of hMSCs and sheds new light on elucidating the therapeutic mechanism of hMSCs for the treatment of inflammation-related diseases.

Effect of hesperetin on inflammatory and oxidative status in trinitrobenzene sulfonic acid-induced experimental colitis model

In our study, the effect of hesperetin on inflammatory and oxidative status in trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis model was investigated through different methods. Eighteen Wistar albino male rats were divided in to three groups: Group I (Control, n = 8; 1 ml physiological saline), Group II (Colitis, n = 8; 1 ml TNBS), Group III (Hesperetin, n = 8; 1 ml TNBS and 100 mg/kg hesperetin). Macroscopic and microscopic scores were calculated to determine the damage to the colon at the end of the experiment. Serum tumor necrosis factor-α (TNF-α) and tissue interleukin-6 (IL-6) levels were determined using the ELISA method. Myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) levels were investigated spectrophotometrically. The TUNEL method was used for the detection of apoptotic cells in the colon tissue. Inducible nitric oxide synthase (iNOS) and nuclear factor-kappa-B (NF-ĸβ) expression in the colon were determined immunohistochemically. Hesperetin administration has shown to significantly reduce levels of MPO, MDA, and proinflammatory agents (TNF-α, IL-6, and NF-ĸβ). It has also been proven to inhibit mucosal apoptosis. This study indicates that hesperetin is protective against TNBS-induced colitis model via antiinflammatory, antioxidant and antiapoptotic effects.

Anti-Inflammatory Local Effect of Hydroxytyrosol Combined with Pectin-Alginate and Olive Oil on Trinitrobenzene Sulfonic Acid-Induced Colitis in Wistar Rats

ABSTRACTPurpose: Evaluate the efficacy of hydroxytyrosol in the local treatment of inflammatory colitis. Currently, the existing treatments for inflammatory bowel diseases does not cure the disease and it is associated with high rates of side effects and complications. Hydroxytyrosol is a phenyl-ethyl-alcohol derived from the hydrolysis of oleuropein and present in olive oil, previous studies have demonstrated the anti-inflammatory effect of dietary hydroxytyrosol supplement, with no toxicity. Materials & Methods: Colitis has been induced by using Trinitrobenzene Sulfonic Acid at 40 rats. They were divided into four groups randomly: 10 rats without treatment; 10 rats with pectin/alginate mixture; 10 rats treated with pectin/alginate + olive oil; 10 rats treated with pectin/alginate + olive oil + hydroxytyrosol. Animals were sacrificed 10 days after induction of trinitrobenzene sulfonic acid, receiving 5 days of continuous treatment. Samples of the rectal area were studied and observed under a microscope to determine the damage by Hunter scoring modified, assessing inflammatory infiltration, number of intestinal walls involved, damage to the mucosal architecture, and edema. Results: When the rectum was analyzed in a global way, nonsignificant differences were observed; however, when performing an individualized analysis, statistically significant differences in the inflammatory infiltrate are present in the samples, which were evaluated using the ANOVA and Student-T statistics. Conclusions: Local treatment with the natural antioxidant hydroxytyrosol combined with pectin/alginate and olive oil of inflammatory bowel disease has been shown to be effective against inflammatory infiltration of TNBS-induced colitis.

Elevation of arachidonoylethanolamide levels by activation of the endocannabinoid system protects against colitis and ameliorates remote organ lesions in mice.

The endocannabinoid system (ECS) is a potential pharmaceutical target for the treatment of inflammatory bowel diseases (IBDs). The aim of this study was to explore the effects of activation of the ECS on IBD and the associated neural inflammation-induced disruption of the blood-brain barrier (BBB). In a mouse model of trinitrobenzene sulfonic acid-induced colitis, the inhibition of fatty acid amide hydrolase with URB597 elevated the arachidonoylethanolamide concentration of the colon. Macroscopic alterations of the colons were evaluated, and the 7-day survival rate of mice was analyzed. BBB integrity was assessed using a dye tracer method, and the cognitive function of mice was examined using a fear-conditioning test. URB597 treatment significantly reduced macroscopic alterations of the colon, decreased the mortality rate, and protected the integrity of the BBB in the mice (P<0.05). No significant changes were observed in the cognitive functions of the mice (P>0.05); therefore, the neuroprotective effect of ECS in this colitis model requires further investigation. Activation of the ECS was efficient in ameliorating colitis and increasing the survival rate of the mice, and reducing remote organ changes induced by colitis. The results suggest that modulation of the ECS is a potential therapeutic approach for IBDs and the associated remote organ lesions.

The Probiotic VSL#3 Modulates Colonic Macrophages, Inflammation, and Microflora in Acute Trinitrobenzene Sulfonic Acid Colitis.

The probiotic mixture VSL#3 attenuates colitis in patients with Inflammatory Bowel Disease (IBD) and in animal models of this condition, but the mechanisms involved are incompletely understood. VSL#3 alters macrophage morphology and secretory profile in vitro in a polarization-dependent manner. We examined the effect of VSL#3 on macrophages in acute trinitrobenzene sulfonic acid-induced colitis. Rats were randomized to normal, colitis, or colitis+VSL#3 groups. After sacrifice, the colons were evaluated for macroscopic and microscopic damage. Serum cytokine levels were measured, and microbiome analysis undertaken. Total and M1 colonic macrophages, and total and proliferating hepatic macrophages were assessed by double immunofluorescence staining. Colitis+VSL#3 rats had lower macroscopic damage, with less microscopic damage in the proximal colon, compared with colitis alone. Colitis significantly increased colonic macrophage infiltration, which was significantly reduced by VSL#3 treatment. VSL#3 did not decrease the colitis-induced surge of colonic M1 macrophages or hepatic macrophages. VSL#3 reduced colitis-induced serum cytokine levels, and induced restoration of colonic transcript levels for pro-inflammatory, anti-inflammatory, and barrier proteins to, or past, normal levels. Fecal bacteria distribution changed between groups. In summary, the probiotic VSL#3 reduces colitis severity, colonic macrophage infiltration, and serum cytokine levels, but does not dampen the pro-inflammatory phenotype of M1 macrophages.

Indigo Naturalis ameliorates murine dextran sodium sulfate-induced colitis via aryl hydrocarbon receptor activation

Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3-RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Intestinal anti-inflammatory effects of RGD-functionalized silk fibroin nanoparticles in trinitrobenzenesulfonic acid-induced experimental colitis in rats.

BackgroundCurrent treatment of inflammatory bowel disease is based on the use of immunosuppressants or anti-inflammatory drugs, which are characterized by important side effects that can limit their use. Previous research has been performed by administering these drugs as nanoparticles that target the ulcerated intestinal regions and increase their bioavailability. It has been reported that silk fibroin can act as a drug carrier and shows anti-inflammatory properties.PurposeThis study was designed to enhance the interaction of the silk fibroin nanoparticles (SFNs) with the injured intestinal tissue by functionalizing them with the peptide motif RGD (arginine–glycine–aspartic acid) and to evaluate the intestinal anti-inflammatory properties of these RGD-functionalized silk fibroin nanoparticles (RGD-SFNs) in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis.Materials and methodsSFNs were prepared by nanoprecipitation in methanol, and the linear RGD peptide was linked to SFNs using glutaraldehyde as the crosslinker. The SFNs (1 mg/rat) and RGD-SFNs (1 mg/rat) were administered intrarectally to TNBS-induced colitic rats for 7 days.ResultsThe SFN treatments ameliorated the colonic damage, reduced neutrophil infiltration, and improved the compromised oxidative status of the colon. However, only the rats treated with RGD-SFNs showed a significant reduction in the expression of different pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, and IL-12) and inducible nitric oxide synthase in comparison with the TNBS control group. Moreover, the expression of both cytokine-induced neutrophil chemoattractant-1 and monocyte chemotactic protein-1 was significantly diminished by the RGD-SFN treatment. However, both treatments improved the intestinal wall integrity by increasing the gene expression of some of its markers (trefoil factor-3 and mucins).ConclusionSFNs displayed intestinal anti-inflammatory properties in the TNBS model of colitis in rats, which were improved by functionalization with the RGD peptide.

The effect of memantine on trinitrobenzene sulfonic acid-induced ulcerative colitis in mice

Previous reports suggest a significant role for N-Methyl-D-aspartate (NMDA) activation in inflammatory processes. So, this study was conducted to investigate the effect of memantine, a commonly used NMDA receptor antagonist, on inflammatory changes in mice model of colitis. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS) (40mg/kg). Animals received memantine (12.5, 25 and 50mg/kg, i.p.), glutamate (2g/kg, p.o.) or dexamethasone (1mg/kg, i.p.) 24h before TNBS instillation and daily thereafter for 4 days. The colonic injury was measured by clinical, macroscopic, microscopic and biochemical analysis. Memantine significantly attenuated the body weight loss, colon weight, the plasma levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and colon level of tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO); as well as macroscopic and microscopic signs of colitis. Oral administration of glutamate had no significant effect on investigated parameters. Memantine as a NMDA antagonist may provide a novel venue for the development of strategies for the treatment of ulcerative colitis.

Role of transient receptor potential melastatin 2 (TRPM2) channels in visceral nociception and hypersensitivity

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca2+-permeable cation channel. TRPM2 contributes to the pathogenesis of inflammatory bowel disease, and inflammatory and neuropathic pain. We hypothesized that TRPM2 is important for visceral nociception and the development of visceral hypersensitivity. Therefore, we investigated the expression of TRPM2 channels and their involvement in visceral nociception in normal physiology and under pathological conditions that cause visceral hypersensitivity in rats. TRPM2 immunoreactivities were detected in the mucosa and muscle layer of the rat gastrointestinal tract. TRPM2 immunopositive cell bodies were almost completely co-localized with calretinin- and NeuN-positive cells in the myenteric plexus. We found that the majority of the TRPM2-immunoreactive cells were double-labeled with the retrograde marker fluorogold in lumbar 6/sacral 1 dorsal root ganglia (DRG), indicating that TRPM2 is expressed in spinal primary afferents innervating the distal colon. Subtypes of TRPM2-immunopositive DRG neurons were labeled by the A-fiber marker NF200, the C-fiber marker IB4, substance P, calcitonin gene-related peptide, or P2X3 receptor. We found that oral administration of the TRPM2 inhibitor econazole (30mg/kg) reduced the visceromotor response (VMR) to noxious colorectal distention (CRD) at 80mmHg in control rats. Expression of TRPM2 in the mucosa of the distal colon was increased in a trinitrobenzene sulfonic acid-induced colitis model. The VMR to CRD significantly increased in colitis model rats compared with control rats at 40, 60, and 80mmHg. Econazole restored visceral hypersensitivity to the control level. Furthermore, TRPM2-deficient mice showed significantly attenuated trinitrobenzene sulfonic acid induced visceral hypersensitivity compared with wild-type mice. In conclusion, TRPM2 channels contribute to visceral nociception in response to noxious stimuli under normal conditions and visceral hypersensitivity in pathological conditions.

Transplantation of a bacterial consortium ameliorates trinitrobenzenesulfonic acid-induced colitis and intestinal dysbiosis in rats.

To investigate the effects of a defined bacterial consortium on trinitrobenzenesulfonic acid (TNBS)-induced colitis and intestinal dysbiosis in rats. Rats with TNBS-induced colitis were treated with ceftriaxone and/or a mixture of ten bacterial strains isolated from mouse feces for continuous 24 days. Macroscopic and histopathological parameters in colonic tissue were compared, as were myeloperoxidase enzyme activity and cytokine levels. Patterns of intestinal microbiota were assessed by PCR-denaturing gradient gel electrophoresis, the abundance of selected microbial groups was evaluated by qPCR. Transplantation of the bacterial consortium showed anti-inflammatory activity in the intestines of rats with TNBS-induced colitis and contributed to the rapid re-establishment of intestinal microbial equilibrium. A defined bacterial consortium may be a viable therapeutic option for the treatment inflammatory bowel disease.

VBP15, a novel dissociative steroid compound, reduces NFκB-induced expression of inflammatory cytokines in vitro and symptoms of murine trinitrobenzene sulfonic acid-induced colitis.

The goal of this study was to assess the capacity of VBP15, a dissociative steroidal compound, to reduce pro-inflammatory cytokine expression in vitro, to reduce symptoms of colitis in the trinitrobenzene sulfonic acid-induced murine model, and to assess the effect of VBP15 on growth stunting in juvenile mice. In vitro studies were performed in primary human intestinal epithelial cells. Colitis was induced in mice by administering trinitrobenzene sulfonic acid. Growth stunting studies were performed in wild type outbred mice. Cells were treated with VBP15 or prednisolone (10μM) for 24h. Mice were subjected to 3days of VBP15 (30mg/kg) or prednisolone (30mg/kg) in the colitis study. In the growth stunting study, mice were subjected to VBP15 (10, 30, 45mg/kg) or prednisolone (10mg/kg) for 5weeks. Cytokines were measured by PCR and via Luminex. Colitis symptoms were evaluated by assessing weight loss, intestinal blood, and stool consistency. Growth stunting was assessed using an electronic caliper. VBP15 significantly reduced the in vitro production of CCL5 (p<0.001) IL-6 (p<0.001), IL-8 (p<0.05) and reduced colitis symptoms (p<0.05). VBP15 caused less growth stunting than prednisolone (p<0.001) in juvenile mice. VBP15 may reduce symptoms of IBD, while decreasing or avoiding detrimental side effects.

Azadirachta indica Attenuates Colonic Mucosal Damage in Experimental Colitis Induced by Trinitrobenzene Sulfonic Acid.

Azadirachta indica leaves indicated the presence of active principles with proven antioxidants, antiinflammatory, immunomodulatory, free radical scavenging and healing properties. In the present study we evaluated the healing effects of 50% ethanol extract of dried leaves of Azadirachta indica on trinitrobenzene sulfonic acid-induced colitis in rats. Azadirachta indica extract (500 mg/kg) was administered orally, once daily for 14 days and studied for its effects on diarrhoea, food and water intake, body weight changes, colonic damage and inflammation, histology, antibacterial activity and free radicals (nitric oxide and lipid peroxidation), antioxidants (superoxide dismutase, catalase and reduced glutathione) and myeloperoxidase activities in colonic tissue. Intracolonic trinitrobenzene sulfonic acid increased colonic mucosal damage and inflammation, diarrhea, but decreased body weight which were reversed by Azadirachta indica extract and sulfasalazine (positive control) treatments. Azadirachta indica extract showed antibacterial activity. Azadirachta indica extract and sulfasalazine enhanced the antioxidants but decreased free radicals and myeloperoxidase activities affected in trinitrobenzene sulfonic acid-induced colitis. Azadirachta indica extract, thus seemed to be effective in healing trinitrobenzene sulfonic acid-induced colitis in rats.

Allograft inflammatory factor-1 in the pathogenesis of bleomycin-induced acute lung injury.

Allograft inflammatory factor-1 (AIF-1) is a protein expressed by macrophages infiltrating the area around the coronary arteries of rats with an ectopic cardiac allograft. Some studies have shown that expression of AIF-1 increased in a mouse model of trinitrobenzene sulfonic acid-induced acute colitis and in acute cellular rejection of human cardiac allografts. These results suggest that AIF-1 is related to acute inflammation. The current study used bleomycin-induced acute lung injury to analyze the expression of AIF-1 and to examine its function in acute lung injury. Results showed that AIF-1 was significantly expressed in lung macrophages and increased in bronchoalveolar lavage fluid from mice with bleomycin-induced acute lung injury in comparison to control mice. Recombinant AIF-1 increased the production of IL-6 and TNF-α from RAW264.7 (a mouse macrophage cell line) and primary lung fibroblasts, and it also increased the production of KC (CXCL1) from lung fibroblasts. These results suggest that AIF-1 plays an important role in the mechanism underlying acute lung injury.

Pharmaceutical Activation or Genetic Absence of ClC-2 Alters Tight Junctions During Experimental Colitis.

We have previously reported that the ClC-2 chloride channel has an important role in regulation of tight junction barrier function during experimental colitis, and the pharmaceutical ClC-2 activator lubiprostone initiates intestinal barrier repair in ischemic-injured intestine. Thus, we hypothesized that pharmaceutical ClC-2 activation would have a protective and therapeutic effect in murine models of colitis, which would be absent in ClC-2 mice. We administered lubiprostone to wild-type or ClC-2 mice with dextran sulfate sodium (DSS) or 2, 4, 5-trinitrobenzene sulfonic acid-induced colitis. We determined the severity of colitis and assessed intestinal permeability. Selected tight junction proteins were analyzed by Western blotting and immunofluorescence/confocal microscopy, whereas proliferative and differentiated cells were examined with special staining and immunohistochemistry. Oral preventive or therapeutic administration of lubiprostone significantly reduced the severity of colitis and reduced intestinal permeability in both DSS and trinitrobenzene sulfonic acid-induced colitis. Preventive treatment with lubiprostone induced significant recovery of the expression and distribution of selected sealing tight junction proteins in mice with DSS-induced colitis. In addition, lubiprostone reduced crypt proliferation and increased the number of differentiated epithelial cells. Alternatively, when lubiprostone was administered to ClC-2 mice, the protective effect against DSS colitis was limited. This study suggests a central role for ClC-2 in restoration of barrier function and tight junction architecture in experimental murine colitis, which can be therapeutically targeted with lubiprostone.

Probiotic yeast Kluyveromyces marxianus CIDCA 8154 shows anti-inflammatory and anti-oxidative stress properties in in vivo models.

Inflammatory bowel diseases (IBDs) are complex affections with increasing incidence worldwide. Multiple factors are involved in the development and maintenance of the symptoms including enhanced oxidative stress in intestinal mucosa. The conventional therapeutic approaches for IBDs are based on the use anti-inflammatory drugs with important collateral effects and partial efficacy. In the present work we tested the anti-inflammatory capacity of Kluyveromyces marxianus CIDCA 8154 in different models. In vitro, we showed that the pretreatment of epithelial cells with the yeast reduce the levels of intracellular reactive oxygen species. Furthermore, in a murine model of trinitro benzene sulfonic acid-induced colitis, yeast-treated animals showed a reduced histopathological score (P<0.05) and lower levels of circulating interleukin 6 (P<0.05). The capacity to modulate oxidative stress in vivo was assessed using a Caenorhabditis elegans model. The yeast was able to protect the nematodes from oxidative stress by modulating the SKN-1 transcription factor trough the DAF-2 pathway. These results indicate that K. marxianus CIDCA 8154 could control the intestinal inflammation and cellular oxidative stress. Deciphering the mechanisms of action of different probiotics might be useful for the rational formulation of polymicrobial products containing microorganisms targeting different anti-inflammatory pathways.

1,25-Dihydroxyvitamin D Protects Intestinal Epithelial Barrier by Regulating the Myosin Light Chain Kinase Signaling Pathway.

The myosin light chain kinase (MLCK) pathway controls intestinal epithelial barrier permeability by regulating the tight junction. 1,25-dihydroxyvitamin D (1,25(OH)2D3)-vitamin D receptor (VDR) signaling protects the epithelial barrier, but the molecular mechanism is incompletely understood. MLCK activation and barrier permeability were studied using monolayers of HCT116, Caco-2, and SW480 cells treated with tissue necrosis factor α with or without 1,25(OH)2D3. The MLCK pathway was analyzed in normal and inflamed colonic biopsies from patients with ulcerative colitis. Colonic mucosal barrier permeability and MLCK activation were also investigated using trinitrobenzene sulfonic acid-induced colitis models in vitamin D analog paricalcitol-treated wild-type mice and mice carrying VDR deletion in colonic epithelial cells. Tissue necrosis factor α increased cell monolayer permeability and induced long isoform of MLCK expression and myosin II regulatory light chain (MLC) phosphorylation, and 1,25(OH)2D3 blocked tissue necrosis factor α-induced increases in monolayer permeability and MLCK-MLC pathway activation by a VDR-dependent fashion. 1,25(OH)2D3 directly suppressed long MLCK expression by attenuating NF-κB activation, and chromatin immunoprecipitation assays confirmed that 1,25(OH)2D3 disrupted p65 binding to 3 κB sites in long MLCK gene promoter. In human ulcerative colitis biopsies, VDR reduction was associated with increases in long MLCK expression and MLC phosphorylation. In trinitrobenzene sulfonic acid colitis models, paricalcitol ameliorated colitis, attenuated the increase in mucosal barrier permeability, and inhibited long MLCK induction and MLC phosphorylation. In contrast, mice with colonic epithelial VDR deletion exhibited more robust increases in mucosal barrier permeability and MLCK activation compared with wild-type mice. These data demonstrate that 1,25(OH)2D3-VDR signaling preserves the mucosal barrier integrity by abrogating MLCK-dependent tight junction dysregulation during colonic inflammation.

Methacrylic acid-co-butylmethacrylate copolymers: design, characterization and evaluation as encapsulating material for colon targeted formulations

Hydrophilic monomer methacrylic acid was copolymerized with hydrophobic monomer butylmethacrylate in different molar ratio, using AIBNfree radical initiator. Successful polymerization was confirmed by FTIR and 1H NMR, while DSC and XRD revealed the amorphous nature of the polymers. Copolymers were highly hemocompatible. The polymers showed pH-dependent swelling and erosion which increased with increase in pH. As the amount of methacrylic acid increased, the swelling and erosion also increased. Polymers were used for the microencapsulation of a model anti-inflammatory drug, aceclofenac by an oil-in-oil solvent evaporation method. Microparticles were characterized using FTIR, XRD, DSC, and SEM analysis. The negatively charged particles were almost spherical in shape. The drug release was best explained by zero-order kinetics with anomalous release mechanism. Less than 1% of drug was released at pH 1.2 while the bulk of the drug load was released at pH 6.8 or above, confirming the colon-targeting nature. The microparticles showed significant lowering in myeloperoxidase activity in trinitrobenzene sulfonic acid-induced colitis rat model. There was a significant lowering in the severity of disease symptoms and colon-to-body weight ratio. The histopathological studies also confirmed successful treatment with microparticle formulation.

Evaluation of glycine-bearing celecoxib derivatives as a colon-specific mutual prodrug acting on nuclear factor-κB, an anti-inflammatory target.

In an inflammatory state where HOCl is generated, glycine readily reacts with HOCl to produce glycine chloramine, an anti-inflammatory oxidant. Colonic delivery of celecoxib elicits anticolitic effects in a trinitrobenzene sulfonic acid-induced rat colitis model. Glycine-bearing celecoxib derivatives were prepared and evaluated as a colon-specific mutual prodrug acting on nuclear factor-κB (NFκB), an anticolitic target. Glycylcelecoxib (GC), N-glycylaspart-1-ylcelecoxib (N-GA1C), and C-glycylaspart-1-ylcelecoxib (C-GA1C) were synthesized and their structures identified using infrared and proton nuclear magnetic resonance spectrometer. The celecoxib derivatives were chemically stable in pH 6.8 and 1.2 buffers. GC and C-GA1C were resistant to degradation in the small intestinal contents, while N-GA1C was substantially cleaved to release celecoxib. In contrast, all the celecoxib derivatives were degraded to liberate celecoxib in the cecal content. These results suggest that GC and C-GA1C could be delivered to and liberate celecoxib and glycine in the large intestine. In human colon carcinoma HCT116 and murine macrophage RAW264.7 cells, combined celecoxib–glycine chloramine treatment additively suppressed the production of proinflammatory NFκB target gene products. Collectively, our data suggest that C-GA1C is a potential colon-specific mutual prodrug acting against NFκB.

Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces.

Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, "Candidatus Saccharibacteria," Verrucomicrobia, Tenericutes, and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.