黄芪多糖对三硝基苯磺酸诱导溃疡性结肠炎大鼠的保护作用

本研究探讨黄芪多糖(APS)对2,4,6-三硝基苯磺酸(TNBS)诱导溃疡性结肠炎(UC)大鼠的保护作用。将72只SD大鼠随机分为正常组、模型组、柳氮磺胺吡啶(SASP)阳性组、APS低、中、高剂量组(100、200、400mg·kg-1),每组12只,除正常组外,每组以150mg·kg-1 TNBS乙醇溶液灌肠制备大鼠结肠炎模型。造模24h后,SASP阳性组及黄芪多糖组分别给予相应的药物进行灌胃干预,每日1次,连续给药14d。研究过程中观察大鼠体重、评价其活动指数(DAI)及结肠黏膜损伤(CMDI),并于给药结束后检测大鼠血清和组织中的炎症因子及生化指标。结果表明,黄芪多糖可以显著改善UC大鼠活动指数,减轻大鼠黏膜损伤,且APS高剂量组大鼠组织中丙二醛(MDA)、髓过氧化物酶(MPO)水平相比模型对照组显著降低,超氧化物歧化酶(SOD)水平显著增加(p<0.05);同时血清中炎症因子TNF-α、IL-6含量相比模型对照组显著降低,IL-10含量显著增加(p<0.05)。因此,黄芪多糖对TNBS诱导的大鼠溃疡性结肠炎具有一定的保护作用。     

N-(3-(aminomethyl)benzyl)acetamidine, an inducible nitric oxide synthase inhibitor, decreases colonic inflammation induced by trinitrobenzene sulphonic acid in rats.

Gastrointestinal inflammation has been associated with an increased generation of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS). Using an experimental model of colitis induced by trinitrobenzene sulphonic acid (TNBS), we sought to determine whether the administration of N-(3-(Aminomethyl)benzyl)acetamidine (1400W), a specific inhibitor of iNOS, has a beneficial action on the colonic injury. 1400W (0.4 and 2 mg/kg/day) was administered intraperitoneally from day 5 to 10 after intrarectal instillation of TNBS. TNBS led to colonic ulceration and inflammation, an increase of colonic myeloperoxidase activity and the expression of the calcium-independent NOS from days 1 to 15. 1400W reduced the macroscopic damage and the histological changes induced by TNBS as well as the calcium-independent NOS activity and myeloperoxidase activity determined over 30 min after sacrifice. These findings indicate that the expression of iNOS accounts for most of the damage caused by TNBS and that the administration of 1400W after the onset of colitis has a beneficial action on the colonic injury.     

Central effects of selective NK1 and NK3 tachykinin receptor agonists on two models of experimentally-induced colitis in rats

Peripheral tachykinins (TKs) are believed to play a role in the pathogenesis of inflammatory bowel diseases (IBD). In this study we investigated changes induced by central administration of two natural TK receptor agonists, NK(1) (PG-SPI) and NK(3) (PG-KII), on trinitrobenzene sulphonic acid (TNBS)- and dextran sodium sulphate (DSS)-induced experimental colitis in rats. Colitis was induced by instilling a single intracolonic dose of TNBS 50 mgkg(-1) (0.5 ml in 50% ethanol) or by oral administration of 5% DSS for 7 days. Each group of rats was intracerebroventricularly injected daily with PG-SPI and PG-KII (0.5, 5, and 50 microgkg(-1)). On day 3, TNBS-treated animals were killed and the severity of gut inflammation was evaluated by measuring myeloperoxidase (MPO) activity, interleukin-1beta (IL-1beta) production and by scoring macroscopic and histologic colonic damage. DSS-treated animals were checked daily for the length of survival and for stool consistency and faecal blood. In the TNBS group, PG-SPI and PG-KII increased scores for the severity of colonic damage, stimulated the production of IL-1beta and increased granulocyte infiltration into the colon (MPO activity). In the DSS group, PG-SPI and PG-KII decreased the percentage of surviving animals, and increased the number of rats that developed loose stools and blood in the faeces and the MPO activity. These results indicate that centrally injected NK(1) and NK(3) tachykinin receptor agonists play a proinflammatory role in experimentally-induced colitis in rats.     

Etanercept attenuates TNBS-induced experimental colitis: role of TNF-α expression

Crohn′s disease (CD) is associated with gut barrier dysfunction. Tumour necrosis factor-α (TNF-α) plays an important role into the pathogenesis of several inflammatory diseases because its expression is increased in inflamed mucosa of CD patients. Anti-TNF therapy improves significantly mucosal inflammation. Thus, this study aimed to evaluate the effect of Etanercept (ETC), a tumour necrosis factor alpha (TNF-α) antagonist on the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis. A total of 18 Wistar rats were randomized into four groups, as follows: (1) Sham: sham induced-colitis; (2) TNBS: non-treated induced-colitis; (3) ETC control; (4) ETC-treated induced-colitis. Rats from group 4 presented significant improvement either of macroscopic or of histopathological damage in the distal colon. The gene expression of TNF-α mRNA, decreased significantly in this group compared to the TNBS non-treated group. The treatment with etanercept attenuated the colonic damages and reduced the inflammation caused by TNBS. Taken together, our results suggest that ETC attenuates intestinal colitis induced by TNBS in Wistar rats by TNF-α downregulation.     

Effects of proanthocyanidins from grape seed on treatment of recurrent ulcerative colitis in rats

The aim of the present study was to investigate the therapeutic effect and mechanism of proanthocyanidins from grape seed (GSPE) in the treatment of recurrent ulcerative colitis (UC) in rats. To induce recurrent colitis, rats were instilled with 2,4,6-trinitrobenzenesulfonic acid (TNBS) (80?mg/kg) into the colon through the cannula in the first induced phase, and then the rats were instilled a second time with TNBS (30?mg/kg) into the colon on the sixteenth day after the first induction UC. Rats were intragastrically administered GSPE (200?mg/kg) per day for 7?days after twice-induced colitis by TNBS. Sulfasalazine at 500?mg/kg was used as a positive control drug. Rats were killed 7?days after GSPE treatment. The colonic injury and inflammation were assessed by macroscopic and macroscopic damage scores, colon weight/length ratio (mg/cm), and myeloperoxidase activity. Then, superoxide dismutase, glutathione peroxidase, inducible nitric oxide synthase (iNOS) activities, and the levels of malonyldialdehyde, glutathione, and nitric oxide in serum and colonic tissues were measured. Compared with the recurrent UC group, GSPE treatment facilitated recovery of pathologic changes in the colon after induction of recurrent colitis, as demonstrated by reduced colonic weight/length ratio and macroscopic and microscopic damage scores. The myeloperoxidase and iNOS activities with malonyldialdehyde and nitric oxide levels in serum and colon tissues of colitis rats were significantly decreased in the GSPE group compared with those in the recurrent UC group. In addition, GSPE treatment was associated with notably increased superoxide dismutase, glutathione peroxidase activities, and glutathione levels of colon tissues and serum of rats. GSPE exerted a protective effect on recurrent colitis in rats by modifying the inflammatory response, inhibiting inflammatory cell infiltration and antioxidation damage, promoting damaged tissue repair to improve colonic oxidative stress, and inhibiting colonic iNOS activity to reduce the production of nitric oxide.     

Luminal antioxidants enhance the effects of mesalamine in the treatment of chemically induced colitis in rats

Previous experiments in rats with chemically induced colitis have shown that the antioxidant N-acetylcysteine plus mesalamine (5-ASA) exerted a significantly greater therapeutic effect in promoting mucosal healing when compared to either agent alone. The aims of the present study were to compare the effects of three antioxidants plus mesalamine vs. 5-ASA alone in treatment of colitis induced by trinitrobenzene sulfonic acid (TNBS) in rats. Methods: Three days following induction of TNBS colitis, rats received 8 days of rectal therapy with 5-ASA, or 5-ASA plus vitamin C (ascorbic acid), 5-ASA plus phenyl butylnitrone (PBN) and 5-ASA plus vitamin E (alpha-tocopherol). Distal colonic tissues were examined for microscopic colitis and myeloperoxidase (MPO) activity. Results: Global assessments of microscopic colitis induced by TNBS indicated that 5-ASA alone significantly changed colonic injury by -31%. Combination therapy with ascorbic acid plus 5-ASA or alpha-tocopherol plus 5-ASA caused further significant change in TNBS colitis by -65 and -82%, respectively. Each of these values was significantly below scores observed with 5-ASA as monotherapy. Reduction in colitis with PBN plus 5-ASA was not different from 5-ASA alone. MPO activity was decreased significantly in response to monotherapy with 5-ASA and each of the antioxidants plus 5-ASA when compared to TNBS. alpha-Tocopherol plus 5-ASA, however, was the only treatment strategy that reduced significantly MPO activity below that recorded for 5-ASA alone. In conclusion, our results indicate that antioxidants other than N-acetylcysteine significantly enhance the therapeutic effectiveness of 5-ASA in the treatment of TNBS colitis. alpha-Tocopherol plus 5-ASA exerted profound anti-inflammatory and reparative effects upon colitis induced by TNBS.     

Immunohistochemical examination of anti-inflammatory and anti-apoptotic effects of hesperetin on trinitrobenzene sulfonic acid induced colitis in rats

The trinitrobenzene sulfonic acid (TNBS) induced colitis model is used to investigate the pathogenesis of ulcerative colitis. Colon inflammation and apoptosis are associated with tissue damage in ulcerative colitis. Hesperetin is a natural flavonoid that exhibits antioxidative, anti-inflammatory and anti-apoptotic properties. We investigated the effects of hesperetin on tumor necrosis factor-alpha (TNF-α), protein tyrosine phosphatase, receptor type C (CD45), caspase-3 and Bax expressions in TNBS in induced colitis model in rats. Male rats were divided into three groups: control group treated with 1 ml physiological saline, colitis group, and colitis + hesperetin group treated with TNBS and hesperetin. Hesperetin treatment was applied for 10 days starting 3 days prior to colitis induction. At the end of the experiment, TNF-α, CD45, caspase-3 and Bax expressions in colon tissue were determined using indirect immunohistochemistry. Increased immunoreactivity of both inflammation markers, TNF-α, CD45, and apoptotic markers, caspase-3 and Bax, was detected in the colitis group. Hesperetin treatment effected significant reduction of all parameters. Hesperetin treatment prevents colon damage owing to its anti-inflammatory and anti-apoptotic effects.     

Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

A comparative study of the preventative effects exerted by three probiotics, Bifidobacterium lactis, Lactobacillus casei and Lactobacillus acidophilus, in the TNBS model of rat colitis

AIMS: The intestinal anti-inflammatory effects of three probiotics with immunomodulatory properties, Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium lactis, were evaluated and compared in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis. METHODS AND RESULTS: Colitis was induced in rats by intracolonic administration of 10 mg of TNBS dissolved in 0.25 ml of 50% ethanol. Each probiotic was administered orally (5x10(8) CFU suspended in 0.5 ml of skimmed milk) for 3 weeks, starting 2 weeks before the administration of TNBS. Colonic damage was evaluated histologically and biochemically 1 week after TNBS instillation. The results obtained revealed that all probiotics assayed showed intestinal anti-inflammatory effects, macroscopically evidenced by a significant reduction in the colonic weight/length ratio. Only B. lactis showed a lower incidence of diarrhoea in comparison with untreated rats. Biochemically, all probiotics restored colonic glutathione levels, depleted as a consequence of the oxidative stress of the inflammatory process. Bifidobacterium lactis treatment reduced colonic tumour necrosis factor (TNF)-alpha production, and inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) expression; L. acidophilus administration reduced colonic leukotriene B4 production and iNOS expression and L. casei intake was associated with a decrease in colonic COX-2 expression. CONCLUSION: The three probiotics assayed have shown intestinal anti-inflammatory activity in the TNBS model of rat colitis, although each probiotic shows its own anti-inflammatory profile. SIGNIFICANCE AND IMPACT OF THE STUDY: These probiotics could be considered as potential adjuvants in the treatment of inflammatory bowel disease, although more studies are required in order to demonstrate their efficacy in humans.     

Total Glucosides of Peony Attenuates 2,4,6-Trinitrobenzene Sulfonic Acid/Ethanol-Induced Colitis in Rats Through Adjustment of Th1/Th2 Cytokines Polarization

The present study is to investigate effects of total glucosides of peony (TGP) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of TGP for treatment of inflammatory bowel disease. Sixty Sprague–Dawley rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100 mg/kg/day), and low, medium, and high-dose TGP groups (25, 50, and 100 mg/kg/day, respectively). 24 h following colonic instillation of TNBS, TGP, and SASP were given by gastric gavage three times a day for 7 days. Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS), and myeloperoxidase (MPO) activity were evaluated. Levels of serum TNF-α, IL-1β, and IL-10 were measured by ELISA, and expression of TNF-α, IL-1β, and IL-10 mRNA and protein in colonic tissues was detected by RT-PCR and western blot, respectively. Compared with rats in the model controls, TGP (50 or 100 mg/kg/day)-treated rats with TNBS/ethanol-induced colitis showed significant improvements of DAI, CMDI, HPS, and MPO activity. Moreover, administration of TGP (50 or 100 mg/kg/day) decreased the up-regulated levels of serum TNF-α and IL-1β, and expression of TNF-α and IL-1β mRNA and protein in colonic tissues, and increased the serum IL-10 and colonic IL-10 mRNA and protein level. And there was no significant difference compared with administration of SASP (P > 0.05). TGP attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the adjustment of Th1/Th2 cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10.     

IMPLICATION OF TNF-α CONVERTASE (TACE/ADAM17) IN INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION AND INFLAMMATION IN AN EXPERIMENTAL MODEL OF COLITIS

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-α convertase (TACE; ADAM17). TNF-α plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-α levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-α and NO−x levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-α. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-α release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-α release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

Elucidation of colon-protective efficacy of diosgenin in experimental TNBS-induced colitis: inhibition of NF-κB/IkB-α and Bax/Caspase-1 signaling pathways

ABSTRACT

The aim of present investigation was to elucidate the unrevealed beneficial role of diosgenin against an experimental model of TNBS (2,4,6-trinitrobenzenesufonic acid)-induced ulcerative colitis (UC). Colitis was induced in Sprague-Dawley rats by intrarectal administration of TNBS (in 50% ethanol). Then animals were treated with diosgenin (50, 100, and 200 mg/kg) for 14 days. Various biochemical, behavioral, molecular, and histological analysis was performed. Diosgenin significantly decreased (p < 0.05) TNBS-induced elevated colonic oxido-nitrosative damage, myeloperoxidase, hydroxyproline, mRNA expressions of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IFN-γ) and inflammatory markers (iNOs and COX-2) induced by TNBS. Western blot analysis relevated that TNBS-induced up-regulated protein expressions of NF-κB, IκBα, Bax, and Caspase-1 were markedly decreased (p < 0.05) by diosgenin treatment. It also markedly ameliorated the histological insults induced in the colon by TNBS. In conclusion, diosgenin exerts its colon-protective efficacy probably through the inhibition of NF-κB/IkB-α and Bax/Caspase-1 signaling pathways to experimental TNBS-induced ulcerative colitis.     

Antibiotic treatment with ampicillin accelerates the healing of colonic damage impaired by aspirin and coxib in the experimental colitis. Importance of intestinal bacteria, colonic microcirculation and proinflammatory cytokines

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for their anti-inflammatory, analgesic and antipyretic effects, however their use is associated with the broad spectrum of side effects observed in human as well as the experimental animals. Despite damaging activity of NSAIDs in upper gastrointestinal (GI) tract, these drugs exert deleterious influence in lower GI tract, including colon. The role of GI microflora in the pathogenesis of NSAIDs-induced experimental colonic damage is not completely understood. The aim of this study was 1) to evaluate the relative importance of the GI microflora on the experimental colonic damage in the presence of caused by NSAID, and 2) to assess the efficacy of antibiotic treatment with ampicillin on the process of healing of colitis. We compared the effect of vehicle, ASA applied 40 mg/kg intragastrically (i.g.) or the selective cyclooxygenase (COX)-2 inhibitor, celecoxib (25 mg/kg i.g.) without or with ampicillin treatment (800 mg/kg i.g.) administered throughout the period of 10 days, on the intensity of TNBS-induced colitis in rats. The severity of colonic damage, the alterations in the colonic blood flow (CBF) and myeloperoxidase (MPO) activity, the mucosal expression of TNF-α, IL-1β, COX-2, VEGF and iNOS and the plasma concentration of TNF-α and IL-1β were assessed. In all rats, the faeces samples as well as those from the colonic mucosa, blood, liver and spleen underwent microbiological evaluation for intestinal bacterial species including Escherichia coli and Enterococcus spp. The administration of TNBS resulted in macroscopic and microscopic lesions accompanied by the significant fall in the CBF, an increase in tissue weight and 4-5-fold rise in the MPO activity and a significant increase in the plasma IL-1β and TNF-α levels. ASA or celecoxib significantly increased the area of colonic lesions, enhanced MPO activity and caused the marked increase in colonic tissue weight and plasma IL-1β and TNF-α levels, as well as an overexpression of mRNA for IL-1β and TNF-α, COX-2, VEGF and iNOS in the colonic tissue. ASA and coxib also resulted also in a significant increase of E. coli counts in the stool at day 3 and day 10 day of the observation compared with the intact rats. Moreover, E. coli translocation from the colon to the blood and extraintestinal organs such as liver and spleen in the group of rats treated without or with ASA and coxib. E. coli was the most common bacteria isolated from these organs. Treatment with ampicillin significantly attenuated the ASA- or celecoxib-induced increase in plasma levels of IL-1β and TNF-α and suppressed the mucosal mRNA expression for IL-1β and TNF-β, COX-2, iNOS and VEGF in the colonic mucosa. Ampicillin administration caused a significant fall in the number of E. coli in the faeces at day 3 and day 10 of observation in ASA- and coxib-treated rats with colitis. Antibiotic therapy markedly reduced bacterial translocation to the colonic tissue and the extraintestinal organs such as the liver and spleen. We conclude that administration of ASA and to lesser extent of celecoxib, delays the healing of experimental colitis and enhances the alterations in colonic blood flow, proinflammatory markers such as IL-1β, TNF-α, COX-2, iNOS and VEGF and increased intestinal mucosal permeability resulting in the intestinal bacterial translocation to the blood, spleen and liver. Antibiotic treatment with ampicillin is effective in the diminishing of the severity of colonic damage, counteracts both the NSAID-induced fall in colonic microcirculation and bacterial E.coli translocation to the extraintestinal organs.     

Is administration of n-3 fatty acids by mucosal enema protective against trinitrobenzene-induced colitis in rats?

We investigated the protective role of fish oil (FO-source of n-3 FA) enriched diet (in the first protocol) in 20 rats and FO administration intrarectally (in the second protocol) in 40 rats with trinitrobenzene (TNB) colitis. All colonic specimens were pathologically evaluated, myeloperoxidase enzyme activities were measured, leukotriene B4 (LTB4) and LTC4 levels were determined by radioimmunoassay. In the first protocol 10 rats (group A1) were fed with 8% sunflower and cotton oil enriched diet and (group A2) with 8% FO enriched diet for 6 weeks. At the end of this period, TNB (30 mg in 0.25 ml of 30% ethanol) were intrarectally administered. After 2 weeks, rats were sacrificed. MPO activities (2.47 versus 30.17), LTB4 (34.5 versus 903.3) and LTC4 (77.7 versus 456.0) levels were significantly reduced in group A2 compared with group A1 (P<0.005). There was also a significant difference in pathologic scores (1.55 versus 2.12, P<0.002) between two groups. In the first part of the second protocol, 20 male rats were randomized into two equal groups (B1 and B2) and TNB colitis was induced. After 1 day, 1 ml of saline (group B1) or n-3 FA enemas (group B2) were administered every day for 2 weeks. At the end of this period, rats were sacrificed and evaluated as done for previous groups. Although there was no significant difference between the two groups in comparison with MPO enzyme activities and pathologic scores, the LTB4 (130.1 versus 971.0) and LTC4 (126.0 versus 532.0) levels of FO group were significantly reduced (P<0.005). In the second part of the second protocol, 20 male rats were randomized into two groups. One millilitre of saline (group B3) or FO enemas (group B4) were administered to rats every day for 3 days. At the fourth day, TNB-colitis was induced and after 24 h rats were sacrificed. We could not find any significant difference in MPO activities, pathologic scores, LTB4 and LTC4 levels between groups B3 and B4. In conclusion, FO enriched diet decreased both pathologic damage and tissue LT levels. The second protocol of our study revealed that the long-term FO enemas decreased the LTB4 and LTC4 levels; however, did not have any beneficial effect on the tissue lesions. Short periods of FO enemas did not have a protective role in the occurrence of experimental colitis. The present study showed that FO enemas significantly decreased LT levels. The protective effect of FO (oral and enema) in TNB colitis may open a new insight into the treatment of inflammatory bowel disease.     

Lactobacillus reuteri Maintains a Functional Mucosal Barrier during DSS Treatment Despite Mucus Layer Dysfunction

Treatment with the probiotic bacterium Lactobacillus reuteri has been shown to prevent dextran sodium sulfate (DSS)-induced colitis in rats. This is partly due to reduced P-selectin-dependent leukocyte- and platelet-endothelial cell interactions, however, the mechanism behind this protective effect is still unknown. In the present study a combination of culture dependent and molecular based T-RFLP profiling was used to investigate the influence of L. reuteri on the colonic mucosal barrier of DSS treated rats. It was first demonstrated that the two colonic mucus layers of control animals had different bacterial community composition and that fewer bacteria resided in the firmly adherent layer. During DSS induced colitis, the number of bacteria in the inner firmly adherent mucus layer increased and bacterial composition of the two layers no longer differed. In addition, induction of colitis dramatically altered the microbial composition in both firmly and loosely adherent mucus layers. Despite protecting against colitis, treatment with L. reuteri did not improve the integrity of the mucus layer or prevent distortion of the mucus microbiota caused by DSS. However, L. reuteri decreased the bacterial translocation from the intestine to mesenteric lymph nodes during DSS treatment, which might be an important part of the mechanisms by which L. reuteri ameliorates DSS induced colitis.     

Effects of recombinant human intestinal trefoil factor on trinitrobenzene sulphonic acid induced colitis in rats

Intestinal trefoil factor (ITF) has been proved to be effective in treatment of ulcerative colitis. However, the mechanisms of it remain unclear. In this study, we observed the effects of combined treatment with 5-aminosalicylic acid (5-ASA) and recombinant human ITF (rhITF) on the expression of Myeloperoxidase (MPO), nuclear factor-κB (NF-κB) and epidermal growth factor (EGF) in trinitrobenzene sulphonic acid (TNBS) induced colitis in rats. Forty Sprague-Dawley (SD) male rats which were induced to distal colitis by the colonic administration of TNBS, were randomly divided into four groups and colonically treated with normal saline (A), 5-ASA (B), rhITF (C), respectively. The macroscopic and histological changes of the colon, activities of MPO, expressions of serum EGF and tissue NF-κB were detected. The results showed that manifestation, colonic damage score and MPO activities of the rats treated with 5-ASA or/and rhITFs were improved, serum EGF production was augmented and expression of tissue NF-κB was down-regulated. Single usage of 5-ASA or rhITF had no significant difference, but combined using of them had more significant and noticeable effects compared to any single treatment. It could be concluded that topical treatment with 5-ASA and rhITF had beneficial effects in treating TNBS-induced colitis of rats and combined treatment was better than single treatment. It was possibly related to suppression of neutrophil infiltration, down-regulation expression of NF-κB and up-regulation expression of EGF.     

Orally administered emu oil attenuates disease in a mouse model of Crohn’s-like colitis

Crohn’s disease is a severe, incurable inflammatory bowel disease. Orally administered emu oil has demonstrated anti-inflammatory properties in previous models of gastrointestinal disease. We aimed to determine whether orally administered emu oil could attenuate disease in a mouse model of Crohn’s-like colitis. Female ARC(s) mice (CD-1 equivalent, n = 10/group) were intra-rectally administered water (120 μL) or trinitrobenzene sulfonic acid (TNBS; 3 mg in 50% ethanol; 120 μL bolus) on day 0. Mice were orally administered water (80 μL) or emu oil (80 μL or 160 μL) daily for five days and euthanized on day six. Bodyweight and disease activity were recorded daily. Colonoscopy, burrowing activity, facial grimace, histological parameters (damage severity, small intestinal villus height/crypt depth and colonic crypt depth), myeloperoxidase activity and intestinal permeability were assessed. P < 0.05 was considered statistically significant. TNBS decreased bodyweight (days 1, 2, 4; P < 0.05) and increased disease activity (days 1–6; P < 0.01), compared to normal controls. Emu oil (80 μL) attenuated disease activity on days 5–6 (P < 0.05), although bodyweight loss was not significantly impacted (P > 0.05). Facial grimace and colonoscopy scores were significantly increased in TNBS-control mice; effects attenuated by both volumes of emu oil (P < 0.001). TNBS increased histological damage severity compared to normal controls (P < 0.05); an effect attenuated by 80 μL emu oil (proximal and distal colon; P < 0.05) and 160 μL emu oil (distal colon; P < 0.01). In the ileum, villus height and crypt depth were unaffected by TNBS or emu oil treatment compared to normal (P > 0.05). TNBS-induced distal colonic crypt lengthening was unaffected following emu oil administration (P > 0.05). Remaining parameters, including burrowing, myeloperoxidase activity and intestinal permeability, were unchanged across all treatment groups (P > 0.05). In normal mice, emu oil treatment did not significantly impact any parameter compared to normal controls. In conclusion, emu oil reduced overall disease severity and facial grimace scores in TNBS mice. These results suggest therapeutic potential for orally administered emu oil in the management of Crohn’s disease.     

Lactobacillus casei prevents the upregulation of ICAM-1 expression and leukocyte recruitment in experimental colitis

Lactobacillus casei has been shown to attenuate the severity of experimental colitis. The objective of the present study was to determine whether the effects of L. casei on colitis are related to modulation of leukocyte recruitment into the inflamed intestine. Rats with a colonic segment excluded from fecal transit were surgically prepared. The segment was decontaminated with antibiotics and recolonized with normal flora isolated from the inflamed rat colon, associated or not to L. casei. Control and colitic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] animals were studied. Leukocyte-endothelial cell interactions were characterized in the colonic microcirculation by intravital microscopy, and ICAM-1 and VCAM-1 expression was measured by the radiolabeled antibody technique. Compared with the noninflamed colonic segment, induction of colitis by TNBS provoked a marked increase in the number of leukocytes firmly adherent to the venular wall (0.5 +/- 0.1 vs. 2.1 +/- 0.6 leukocytes/100 mum, P < 0.01). Colonization with L. casei significantly reduced the number of adherent leukocytes (1.3 +/- 0.4 leukocytes/100 mum; P < 0.05) but did not affect the increased rolling interactions associated with the induction of colitis. Compared with the noncolitic group, induction of colitis was associated with a marked increase in ICAM-1 expression (117 +/- 4 vs. 180 +/- 3 ng antibody/g tissue) that was abrogated when the colitic segment was colonized by L. casei (117 +/- 3 ng antibody/g tissue, P < 0.05). However, L. casei administration did not modify VCAM-1 upregulation in colitic animals. L. casei attenuates leukocyte recruitment observed in experimental colitis induced by TNBS. This effect is possibly related to abrogation of ICAM-1 upregulation.     

Glutamine enema regulates colonic ubiquitinated proteins but not proteasome activities during TNBS‐induced colitis leading to increased mitochondrial activity

Ubiquitin proteasome system contributes to the regulation of intestinal inflammatory response as its inhibition is associated with tissue damage improvement. We aimed to evaluate whether glutamine is able to limit inflammation by targeting ubiquitin proteasome system in experimental colitis. Colitis was induced in male rats by intrarectal instillation of 2‐4‐6‐trinitrobenzen sulfonic acid (TNBS) at day 1. From day 2 to day 6, rats daily received either an intrarectal instillation of PBS (TNBS/PBS group) or glutamine (TNBS/Gln). Rats were euthanized at day 7 and colonic samples were taken to evaluate ubiqutinated proteins by proteomic approach combining 2D electrophoresis and immunoblots directed against ubiquitin. Results were then confirmed by evaluating total expression of proteins and mRNA levels. Survival rate, TNFα, and IL‐1β mRNA were improved in TNBS/Gln compared with TNBS/PBS (p < 0.05). Proteasome activities were affected by TNBS but not by glutamine. We identified eight proteins that were less ubiquitinated in TNBS/PBS compared with controls with no effect of glutamine. Four proteins were more ubiquitinated in TNBS/PBS group and restored in TNBS/Gln group. Finally, 12 ubiquitinated proteins were only affected by glutamine. Among proteins affected by glutamine, eight proteins (GFPT1, Gapdh, Pkm2, LDH, Bcat2, ATP5a1, Vdac1, and Vdac2) were involved in metabolic pathways. In conclusion, glutamine may regulate ubiquitination process during intestinal inflammation.     

Intestinal responsiveness to experimental colitis in young rats is altered by maternal diet

Increasing evidence suggests that fetal and neonatal nutrition impacts later health. Aims of the present study were to determine the effect of maternal dietary fat composition on intestinal phospholipid fatty acids and responsiveness to experimental colitis in suckling rat pups. Female rats were fed isocaloric diets varying only in fat composition throughout gestation and lactation. The oils used were high (8%) in n-3 [canola oil (18:3n-3)], n-6 (72%) [safflower oil (18:2n-6)], or n-9 (78%) [high oleic acid safflower oil (18:1n-9)] fatty acids, n = 6/group. Colitis was induced on postnatal day 15 by intrarectal 2,4-dinitrobenzene sulfonic acid (DNBS) administration with vehicle (50% ethanol) and procedure (0.9% saline) controls. Jejunal and colonic phospholipids and milk fatty acids were determined. The distal colon was assessed for macroscopic damage, histology, and MPO activity. The 18:2n-6 maternal diet increased n-6 fatty acids, whereas the 18:3n-3 diet increased n-3 fatty acids in milk and pup jejunal and colonic phospholipids. Maternal diet, milk, and pup intestinal n-6-to-n-3 fatty acid ratios increased significantly in order: high 18:3n-3 < high 18:1n-9 < high 18:2n-6. DNBS administration in pups in the high 18:2n-6 group led to severe colitis with higher colonic damage scores and MPO activity than in the 18:1n-9 and 18:3n-3 groups. High maternal dietary 18:3n-3 intake was associated with colonic damage scores and MPO activity, which were not significantly different from ethanol controls. We demonstrate that maternal dietary fat influences the composition of intestinal lipids and responsiveness to experimental colitis in nursing offspring.