TNBS Research Articles

Melatonin ameliorates TNBS-induced colitis in rats through the melatonin receptors: involvement of TLR4/MyD88/NF-κB signalling pathway.

The aim of the present study is to investigate the anti-inflammatory effect of melatonin in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis through the inhibition of Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signalling pathway and activation of melatonin receptor. Colitis was induced in Wistar rats by administration of 100mg/kg TNBS dissolved in 0.25ml of 50% ethanol solution using a flexible plastic rubber catheter into the colon via the anus. This resulted in incidence of colitis on the first day, and all treatments were conducted for 10days after induction of colitis. Melatonin was administered intraperitoneally (i.p.) at doses of 1, 5, and 10mg/kg/day. Luzindole (non-selective MT1/MT2 receptor antagonist) was administered i.p. at dose of 5mg/kg/day 15min prior to melatonin injection. During the experiment, animals were monitored for the appearance of diarrhoea, body weight loss, and rectal bleeding. Myeloid peroxidase enzyme and tumour necrosis factor-α (TNF-α) activities were detected by immunohistochemistry. The protein expression level of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB p65, and inhibitor of kappa B (I-κB) were detected by western blotting analysis. Treatment with melatonin improved weight loss, mucosal, and histological damage compared with TNBS group. In addition, melatonin decreased TNBS-induced up-regulation of TLR4, MyD88, and NF-κB p65, and increased down-regulation of I-κB proteins. On the other hand, the administration of luzindole resulted in the inhibition of melatonin effects. It seems that the inhibition of TLR4/NF-κB signalling pathway may mediate the anti-inflammatory effects of melatonin in TNBS-induced rat colitis.

Preventive effects of bovine colostrum supplementation in TNBS-induced colitis in mice.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder for which the current medical therapy is not completely effective. Bovine colostrum (BC) is a biological fluid rich in bioactive molecules that may have beneficial effects on several gastrointestinal disorders. The objectives of this study were to assess the preventive effects of BC supplementation in a mouse model of 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced colitis using a multidisciplinary approach. Specifically, the following parameters were evaluated: (i) disease activity index (DAI), (ii) histological score, (iii) expression levels of TLR4, anti- and pro-inflammatory cytokines, and (iv) count of some bacterial species of the intestinal microbiota. Mice received a daily suspension of BC (BC group, n = 12) or saline solution (control, CN group, n = 12) for 21 days before the intrarectal inoculation with 1% of TNBS solution. BC was well tolerated and did not induce any histological damage or clinical symptoms. After TNBS treatment, BC group showed a reduction of body weight (BW) loss (P<0.01) and histological score (P<0.05) compared to CN. Moreover, the expression levels of TLR4 (P<0.05), IL-1β (P<0.001), IL-8 (P<0.001), and IL-10 (P<0.001) were lower in mice administered with BC, while the concentrations of TNF-α did not show any differences between groups. Finally, the supplementation with BC resulted in a differential response to TNBS treatment in the bacterial count. In CN group, E. coli and Enterococci increased (P<0.001), while Anaerobes (P<0.01), Lactobacilli, and Bifidobacteria (P<0.001) reduced. Conversely, no significant changes in bacterial load were found after the inoculation of TNBS in BC pre-treated mice. This study confirms that TNBS-induced colitis model in mice is useful for studying the mechanisms involved in IBD pathogenesis and shows that pre-treatment with BC reduces the intestinal damages and clinical signs of the colitis. Molecular mechanisms and intestinal microflora could be involved in the protective effect of colostrum.

Intake of Protein Hydrolysates and Phenolic Fractions Isolated from Flaxseed Ameliorates TNBS‐Induced Colitis

In the attempt to develop new therapeutic treatments for colitis, fractions containing phenolic compound isolate (Phi) and phenolic reduced-flaxseed protein hydrolysate (phr-FPH) from flaxseed are evaluated for their effects on the in vitro production of pro-inflammatory mediators and on the course of experimental colitis. The anti-inflammatory effects of Phi and phr-FPH from flaxseeds are studied in RAW264.7 cells and in trinitrobenzene sulphonic acid (TNBS) colitis model. It is observed that the incubation with Phi or phr-FPH result in lower levels of tumor necrosis factor α and nitric oxide in macrophages stimulated with bacterial lipopolysaccharide + interferon-γ. Prophylactic and therapeutic treatments with Phi and phr-FPH, respectively, greatly contribute to the prevention of weight loss and colon inflammation in colitic BALB/c mice. T cell proliferation, expansion of TH1 and TH17 cells, and pro-inflammatory cytokines are lower, whereas Treg cells are higher in spleen cell cultures from Phi-treated mice. In addition, therapeutic phr-FPH treatment is able to reduce the expansion of TH17 in splenic cell cultures. The consumption of phenolic and protein compounds extracted from flaxseeds has a protective effect on TNBS-induced colitis, and may be useful in the control of other inflammatory disorders.

Adenylyl cyclase 6 is involved in the hyposecretory status of experimental colitis.

One of the cardinal symptoms of intestinal inflammation is diarrhea. Acute intestinal inflammation is associated with inhibition of ion absorption and increased secretion, along with fluid leakage due to epithelial injury and changes in permeability. However, in the chronic situation, a downregulation of both absorptive and secretory transport has been reported. We investigated how experimental colitis reduces cAMP levels in intestinal epithelial cells through modulation of adenylyl cyclases (AC). Primary colonic epithelial cells obtained from rats with trinitrobenzenesulfonic acid colitis and non-colitic controls were analyzed for AC expression by RT-qPCR and Western blot, following a preliminary microarray analysis. AC6 and AC5 were found to be expressed in colonocytes, and downregulated by inflammation, with the former exhibiting considerably higher mRNA levels in both cases. To test the hypothesis that inflammatory cytokines may account for this effect, Caco 2 cells were treated with IL-1β, TNF-α, or IFN-γ. All three cytokines inhibited forskolin evoked short-circuit currents in Ussing chambers and lowered intracellular cAMP, but failed to alter AC6 mRNA levels. AC5/AC6 expression was however inhibited in mouse jejunal organoids treated with IFN-γ and TNF-α, but not IL-1β. Gene knockdown of AC6 resulted in a significant decrease of ion secretion in T84 cells. We conclude that the disturbances in ion secretion observed in rat TNBS colitis are associated with low intracellular levels of cAMP in the epithelium, which may be explained in part by the downregulation of AC5/AC6 expression by proinflammatory cytokines.

Analgesic effects of electroacupuncture at ST25 and CV12 in a rat model of postinflammatory irritable bowel syndrome visceral pain.

Treatment with electroacupuncture (EA) at ST25 and CV12 has a significant analgesic effect on postinflammatory irritable bowel syndrome (PI-IBS) visceral pain. Enterochromaffin (EC) cells and serotonin (5-hydroxytryptamine (5-HT)) are important in the development of visceral hyperalgesia. To investigate the analgesic effect and underlying mechanisms of EA at ST25 and CV12 on the treatment of trinitrobenzene sulfonic acid (TNBS)-induced PI-IBS visceral hyperalgesia in rats. After EA at ST25 and CV12, changes in abdominal withdrawal reflex (AWR), electromyography (EMG) recordings, colonic EC cell numbers, and expression of tryptophan hydroxylase (TPH), 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) of TNBS-induced PI-IBS visceral hyperalgesia in rats were examined. The results of AWR tests and EMG recordings indicated a significant analgesic effect of EA stimulation at ST25 and CV12on PI-IBS visceral hyperalgesia (p<0.05). In addition, the increased EC cell numbers and colonic expression of TPH and 5-HT in rats with TNBS-induced PI-IBS visceral hyperalgesia were significantly reduced by EA (p<0.05). EA stimulation at ST25 and CV12 can attenuate visceral hyperalgesia. This analgesic effect may be mediated via reduction of both colonic EC cell number and 5-HT concentration.

Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome.

Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS). Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates. VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αβ-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals. Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain.

Oral treatment with Lactobacillus rhamnosus 64 during the early postnatal period improves the health of adult rats with TNBS-induced colitis

The current study investigated a potential modulating effect of orally applied Lactobacillus rhamnosus 64 (LB64) during the early postnatal period (day of life: ∼3–30), during young adult period (day of life: 31–70) or throughout experiment, on parameters of trinitrobenzenesulfonic acid (TNBS)-induced colitis in adult rats. Treatment with LB64 during early postnatal, but not during young adult period reduced clinical damage score, neutrophil and macrophage infiltration into colon, the level of cytokine and myeloperoxidase (MPO) activity, but had no influence on other parameters of oxidative damage. Early postnatal treatment with LB64 also increased the diversity of fecal Bifidobacteria and Eubacteria, and improved maturation of ileal villi in 30-days old rats. When LB64 is applied during a critical period early in life, it affects immune system functioning of adults, probably by interactions with the mucosal immune system of the gastrointestinal tract that provides immune system maturation and shapes the overall immune response.

Effect of moxibustion on CRF and CRFR1 expressions in hypothalamus of TNBS-induced experimental colitis rats

To observe the effect of moxibustion on the protein and mRNA expressions of corticotropin-releasing factor (CRF) and corticotropin-releasing factor receptor 1 (CRFR1) in hypothalamus of trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis rats, and to explore the central mechanisms of moxibustion in improving visceral pain and the pain-related emotions in experimental colitis rats. Thirty-six Sprague-Dawley (SD) rats were randomly divided into a normal group (NG), a model group (MG), a herb-partitioned moxibustion group (HPMG) and a sham herb-partitioned moxibustion group (SHPMG). Except the NG, rats in the remaining three groups all received TNBS enema to establish experimental colitis models. The HPMG received herb-partitioned moxibustion (HPM) at bilateral Tianshu (ST 25) and Qihai (CV 6) for intervention; for the SHPMG, the herbal cakes and moxa cones were only placed on the acupoints but not ignited; rats in the MG and NG were only fixed in the same way as those in the HPMG but did not receive any treatment. At the end of the intervention, the abdominal withdrawal reflex (AWR) score, the open field test (OFT) score and the elevated plus maze (EPM) score were observed to measure the changes in visceral pain and pain-related emotions of the rats. The enzyme-linked immunosorbent assay (ELISA) was used to examine the expressions of CRF and CRFR1 proteins in hypothalamus; the fluorescence-based quantitative polymerase chain reaction (PCR) was used to detect the expressions of CRF and CRFR1 mRNAs in hypothalamus. Compared with the NG, the AWR score increased significantly and the OFT and EPM scores dropped significantly in the MG (all P 0.05). HPM can down-regulate the abnormally increased expressions of CRF and CRFR1 proteins and mRNAs in hypothalamus of the TNBS-induced experimental colitis rats, which is plausibly one of its action mechanisms in mitigating visceral pain and the pain-related emotions in the experimental colitis rats.

Colonic migrating motor complexes are inhibited in acute tri-nitro benzene sulphonic acid colitis.

BackgroundInflammatory Bowel Disease (IBD) is characterized by overt inflammation of the intestine and is typically accompanied by symptoms of bloody diarrhea, abdominal pain and cramping. The Colonic Migrating Motor Complex (CMMC) directs the movement of colonic luminal contents over long distances. The tri-nitrobenzene sulphonic acid (TNBS) model of colitis causes inflammatory damage to enteric nerves, however it remains to be determined whether these changes translate to functional outcomes in CMMC activity. We aimed to visualize innate immune cell infiltration into the colon using two-photon laser scanning intra-vital microscopy, and to determine whether CMMC activity is altered in the tri-nitro benzene sulphonic (TNBS) model of colitis.MethodsEpithelial barrier permeability was compared between TNBS treated and healthy control mice in-vitro and in-vivo. Innate immune activation was determined by ELISA, flow cytometry and by 2-photon intravital microscopy. The effects of TNBS treatment and IL-1β on CMMC function were determined using a specialized organ bath.ResultsTNBS colitis increased epithelial barrier permeability in-vitro and in-vivo. Colonic IL-1β concentrations, colonic and systemic CD11b+ cell infiltration, and the number of migrating CD11b+ cells on colonic blood vessels were all increased in TNBS treated mice relative to controls. CMMC frequency and amplitude were inhibited in the distal and mid colon of TNBS treated mice. CMMC activity was not altered by superfusion with IL-1β.ConclusionsTNBS colitis damages the epithelial barrier and increases innate immune cell activation in the colon and systemically. Innate cell migration into the colon is readily identifiable by two-photon intra-vital microscopy. CMMC are inhibited by inflammation, but this is not due to direct effects of IL-1β.

Kuijieling regulates the differentiation of Treg and Th17 cells to ameliorate experimental colitis in rats.

Regulatory T (Treg) cells and T helper 17 (Th17) cells play crucial roles in ulcerative colitis (UC). Kuijieling (KJL) is an effective Chinese medicine formula for treating UC in clinic. Kuijieling has shown remedy effect on the imbalance between Treg and Th17 cells. This study aimed to further reveal the exact underlying mechanism of how Kuijieling regulates the differentiation of Treg and Th17 cells in the treatment of UC. Colitis was induced by trinitrobenzene sulfonic acid in rats and treated by KJL. Pathological injury was evaluated by HE staining and pathological score. Transforming growth factor-β1 (TGF-β1), interleukin(IL)-2, IL-6, IL-10, IL-17, IL-23 and IL-21 in plasma were assayed by ELISA. Forkhead box P3 (Foxp3), signal transducer and activator of transcription (STAT) 5 expressed in colon mucosa were measured by western blot. Immunohistochemistry was employed for quantifying retinoic acid-related orphan receptor γt (RORγt) and STAT3 in colon. RT-PCR was used to analyze the expression of IL-2, IL-17, IL-23, IL-21 mRNA in colon. After the administration of KJL, pathological injury in colon mucosa was reduced and histological score was decreased, transforming growth factor-β1 (TGF-β1), interleukin(IL)-2, IL-10 in blood and Foxp3, STAT5, IL-2 in colon increased significantly, IL-6, IL-23, IL-17, IL-21 in blood and RORγt, STAT3, IL-23, IL-17, IL-21 in colon decreased. Our result showed that KJL regulates the related cytokines and transcription factors to promote Treg cells and suppress Th17 cells. KJL restores the balance between Treg and Th17 cells through regulating the differentiation of them, therefore contributes to the treatment of UC.

Portulacaoleraceal extract alleviates trinitrobenzene sulfonic acid-induced colitis in rats.

Portulacaoleraceal (POL) has been widely used as an edible plant and a folk medicine in many countries, due to its several health benefits. This study examined the effects of POL on trinitrobenzene sulfonic acid (TNBS)-induced colitis via enema administration. Sixty male Sprague-Dawley rats were randomly divided into five groups: untreated, TNBS, TNBS + POL 10 g/kg, TNBS + POL 5 g/kg and TNBS + POL 2.5 g/kg groups. Rats were subjected to enema treatment once a day for 10 consecutive days with POL extract or distilled water after induction of TNBS. The changes of body weight, histological parameters, myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide synthase activity (NOS), malondialdehyde (MDA) and nitric oxide (NO) levels in colon tissues were investigated. After POL extract treatment, body weights of rats significantly increased, macroscopic and microscopic damage scores reduced, MPO and NOS activity, as well as MDA and NO level significantly decreased, while SOD activity increased in a dose-dependent manner in the TNBS + POL groups compared with the TNBS group. Our results demonstrated that POL enema treatment attenuated pathologic changes of TNBS-induced colitis in rats through restoring colonic damage and reducing inflammatory response in the intestine. Thus, POL enema might be considered as a potential effective treatment for ulcerative colitis patients.

Exclusive enteral nutrition protects against inflammatory bowel disease by inhibiting NF‑κB activation through regulation of the p38/MSK1 pathway.

Although enteral nutrition therapy for inflammatory bowel disease has been confirmed to be an effective treatment method, the exact mechanism responsible for the effects of enteral nutrition remains unclear. The aim of the present study was to investigate the protective effect of exclusive enteral nutrition (EEN) against colitis, and to elucidate the potential mechanisms by inhibiting p65 activation via regulating the p38/mitogen- and stress-activated protein kinase-1 (MSK1) pathway. Experiments were performed by establishing dextran sulfate sodium (DSS)-mice colitis and picrylsulfonic acid solution (TNBS)-induced rat colitis, and the results demonstrated that EEN treatment attenuated body weight loss, colon length shortening and colonic pathological damage caused by colitis. EEN also inhibited inflammatory cells infiltration and decreased myeloperoxidase and inducible nitric oxide synthase activities. Furthermore, EEN significantly reduced the production of pro-inflammatory mediators in serum and the colon. Mechanically, EEN suppressed activation of p65 by inhibiting the p38/MSK1 pathway. In conclusion, the present study demonstrated that EEN attenuated DSS- and TNBS-induced colitis by inhibiting p65 activation via regulating the p38/MSK1 pathway, thus suggesting that EEN is effective in the treatment of colitis.

Treatment with Obestatin-A Ghrelin Gene-Encoded Peptide-Reduces the Severity of Experimental Colitis Evoked by Trinitrobenzene Sulfonic Acid.

Obestatin is a 23-amino acid peptide derived from proghrelin, a common prohormone for ghrelin and obestatin. Previous studies showed that obestatin exhibited some protective and therapeutic effects in the gut. The aim of our presented study was to examine the effect of treatment with obestatin on trinitrobenzene sulfonic acid (TNBS)-induced colitis. In rats anesthetized with ketamine, colitis was induced through intrarectal administration of 25 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Obestatin was administered intraperitoneally at doses of 4, 8, or 16 nmol/kg, twice per day for four consecutive days. The first dose of obestatin was given one day before the induction of colitis, and the last one was given two days after administration of TNBS. Fourteen days after the induction of colitis, rats were anesthetized again with ketamine, and the severity of colitis was determined. The administration of obestatin had no effect on the parameters tested in rats without the induction of colitis. In rats with colitis, administration of obestatin at doses of 8 or 16 nmol/kg reduced the area of colonic damage, and improved mucosal blood flow in the colon. These effects were accompanied by a reduction in the colitis-evoked increase in the level of blood leukocytes, and mucosal concentration of pro-inflammatory interleukin-1β. Moreover, obestatin administered at doses of 8 or 16 nmol/kg reduced histological signs of colonic damage. The administration of obestatin at a dose of 4 nmol/kg failed to significantly affect the parameters tested. Overall, treatment with obestatin reduced the severity of TNBS-induced colitis in rats. This effect was associated with an improvement in mucosal blood flow in the colon, and a decrease in local and systemic inflammatory processes.

Reversing Ongoing Chronic Intestinal Inflammation and Fibrosis by Sustained Block of IL-12 and IL-23 Using a Vaccine in Mice.

Interleukin (IL)-12 and IL-23 that share subunit p40 are important cytokines in the pathogenesis of inflammatory bowel disease. We reported that mouse p40 peptide-based vaccines ameliorated intestinal inflammation in the prevention of trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Here, we evaluated whether administration of the vaccine after establishment of colitis would be effective in modifying both TNBS-induced and dextran sulfate sodium (DSS)-induced chronic colitis and the underlying immune mechanisms. We further examined whether vaccination could exacerbate infections. Chronic colitis was developed by either intrarectally administrating TNBS or drinking 4% DSS water. Vaccination started after two TNBS administrations or 7 days of DSS treatment. Results showed that administrating p40 vaccine induced high tittered antibodies to IL-12 and IL-23, improved clinical scores, reduced intestinal inflammation and fibrosis, and down-regulated proinflammatory cytokine productions in colon tissue, compared with control mice. Furthermore, in lamina propria mononuclear cells and/or mesenteric lymph nodes, mice immunized with p40 peptide vaccine exhibited high ratios of Treg/Th1 and Treg/Th17 cells and increased IL-10 expression in CD11c+IL-10+cells. In mice infected with lung chlamydia, in which the protective role of Th1/Th17 is well documented, vaccine immunization did not increase lung bacterial burden. We conclude that p40 vaccine may provide a potential and safe approach for treatment of IBD. 10.1093/ibd/izy142_videoizy142.video5785979965001.

Cytokine IL9 Triggers the Pathogenesis of Inflammatory Bowel Disease Through the miR21-CLDN8 Pathway.

Cytokine interleukin-9 (IL9) plays an essential role in the pathogenesis of inflammatory bowel disease. However, the molecular mechanism underlying the IL9 pathway remains unknown. Here, we initiate a series of studies to characterize the essential components of this pathway. The expression of IL9 in colon biopsies from Crohn's disease (CD) and controls were examined by quantitative polymerase chain reaction, immunoblot, and immunohistochemistry. The trinitrobenzene sulfonic acid (TNBS)-induced colitis model was used to verify the therapeutic efficiency of anti-IL9 mAb. Bioinformatics analysis was performed to predict putative candidate microRNAs that mediate the crosstalk between the IL9 proinflammatory signal and the downstream target gene in intestinal barrier function. Caco-2, NCM460, and SW480 cells were used to assess the specific pathway in vitro. We demonstrated the proinflammatory role of IL9 in the colonic mucosa of patients with CD. The junction complex protein Claudin 8 (CLDN8) was identified as a critical downstream component of the IL9 inflammatory cascade. Anti-IL9 treatment alleviated TNBS-induced colitis by restoring CLDN8 levels in the colonic mucosa. Notably, we characterized miR21 as a critical player that mediates the crosstalk between the proinflammatory IL9 and the downstream CLDN8 in both in vitro and in vivo models. Our results, for the first time, uncover a critical role of miR21 and CLDN8 in the complex network that IL9 regulates the intestinal epithelium barrier in the pathogenesis of CD. Interventional blockade of the IL9-miR21-CLDN8 pathway could be a novel therapeutic approach for the management of CD.

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.

Oral treatment with plecanatide or dolcanatide attenuates visceral hypersensitivity via activation of guanylate cyclase-C in rat models.

AIMTo investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity.METHODSTransport of fluorescein isothiocyanate (FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1 (ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid (TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress (PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests.RESULTSTreatment of T84 and Caco-2 monolayers with lipopolysaccharide (LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model.CONCLUSIONOur results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent anti-nociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.

Luteolin Ameliorates Experimental Chronic Pancreatitis Induced by Trinitrobenzenesulfonic Acid in Rats.

The purpose of this study is to assess the effect and possible mechanism of luteolin on chronic pancreatitis (CP). Trinitrobenzenesulfonic acid-induced CP was used as CP models in vivo. After the intervention of luteolin for 28 days, chronic pancreatic injury was assessed by serum hydroxyproline and pancreatic histology. α-Smooth muscle actin (α-SMA) expression was performed to detect the activation of pancreatic stellate cells (PSCs). Pancreatic stellate cells were also isolated and cultured in vitro, and the effect of luteolin on PSCs was evaluated. Transforming growth factor β (TGF-β1) signaling and its regulated mRNA expression was tested by Western blot and quantitative real-time polymerase chain reaction, respectively. The protective role of luteolin on CP was confirmed by increased pancreas/body weight ratio, decreased pancreas hydroxyproline level, and reduced fibrosis. α-SMA expressions in PSCs were significantly decreased both in vitro and in vivo after the management of luteolin. Pancreas TGF-β1 expression was significantly decreased by luteolin. Luteolin inhibited the proliferation and activation of PSCs in a dose-dependent manner. Luteolin played a protective role in CP in many aspects, partly by regulating release of inflammatory cytokines through TGF-β1 signaling pathway.

IL-6 Drives Neutrophil-Mediated Pulmonary Inflammation Associated with Bacteremia in Murine Models of Colitis

Inflammatory bowel disease (IBD) is associated with several immune-mediated extraintestinal manifestations. More than half of all IBD patients have some form of respiratory pathology, most commonly neutrophil-mediated diseases, such as bronchiectasis and chronic bronchitis. Using murine models of colitis, we aimed to identify the immune mechanisms driving pulmonary manifestations of IBD. We found increased neutrophil numbers in lung tissue associated with the pulmonary vasculature in both trinitrobenzenesulfonic acid- and dextran sulfate sodium-induced models of colitis. Analysis of systemic inflammation identified that neutrophilia was associated with bacteremia and pyrexia in animal models of colitis. We further identified IL-6 as a systemic mediator of neutrophil recruitment from the bone marrow of dextran sulfate sodium animals. Functional inhibition of IL-6 led to reduced systemic and pulmonary neutrophilia, but it did not attenuate established colitis pathology. These data suggest that systemic bacteremia and pyrexia drive IL-6 secretion, which is a critical driver for pulmonary manifestation of IBD. Targeting IL-6 may reduce neutrophil-associated extraintestinal manifestations in IBD patients.

Role of miR-22 in intestinal mucosa tissues and peripheral blood CD4+ T cells of inflammatory bowel disease

ObjectivemiR-22 is known to be involved in the pathogenesis of several autoimmune diseases, but it remains unclear whether miR-22 is associated with inflammatory intestinal disease (IBD). MethodsThe patients with ulcerative colitis (UC) and Crohn’s disease (CD) were enrolled in this study. After the CD4+ T cells from healthy controls and active IBD patients were isolated and then transfected with miR-22 mimics/inhibitors, Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure expressions of miR-22, HDAC4, specific transcription factors in intestinal mucosa tissue and CD4+ T cells, while enzyme-linked immuno sorbent assay (ELISA) to detect expressions of inflammatory cytokines in PB. Antisense miR-22 was administered into mice during trinitrobenzene sulphoni cacid (TNBS)-induced colitis to determine its role in IBD. ResultsA significant elevation of miR-22 but an evident decrease of HDAC4 was found in CD4+ T cells in PB and intestinal mucosa tissues from IBD patients. In addition, there was a great reduction in HDAC4 and a dramatic enhancement in Th17 cell specific transcription factor (RORC) and inflammatory cytokines (IL-17A, IL-6 and TNF-α) after overexpression miR-22, which was opposite to the effect of inhibition of miR-22. Furthermore, administration of antisense miR-22 in TNBS-induced mouse colitis model significantly decreased numbers of interleukin (IL)-17A+ CD4+ T cells and the expressions of IL-17A, RORC, IL-6 and TNF-α. ConclusionMiR-22 was up-regulated in CD4+ T cells in PB and intestinal mucosa tissues of IBD patients, which could promote Th17 cell differentiation via targeting HDAC4 to be involved in IBD progression.