F. Sánchez de Medina

ORAL ADMINISTRATION OF RUTOSIDE CAN AMELIORATE INFLAMMATORY BOWEL DISEASE IN RATS

Rutoside, a flavonoid with antioxidant properties, was tested for acute and chronic antiinflammatory activity in trinitrobenzenesulfonic acid-induced rat colitis. Pretreatment with 10 or 25 mg/kg of rutoside by the oral route reduced colonic damage at 2 days. Several mechanisms can be involved in this activity, and one of these may be related to its ability in preventing glutathione depletion of colitic animals, and this could result in mucosal protection against oxidative insult. When rutoside was tested for 1 and 2 weeks after colitis induction, it was able to promote colonic healing. The chronic effect of the flavonoid was also related with its ability to increase colonic glutathione levels and thus reduce the tissue damage derived from intestinal oxidative stress which characterizes inflammatory colitis.

Effect of quercitrin on the early stages of hapten induced colonic inflammation in the rat.

Quercitrin is a flavonoid with antiinflammatory activity in experimental colitis, associated with an antioxidative action and amelioration of water absorption in vivo. However, its mechanism of action is unclear. This study focuses on the effect of quercitrin (1 and 5 mg/kg) in the early stages (24 h) of trinitrobenzene sulfonic acid colitis. Treatment with the flavonoid prevented the increase in colonic malondialdehyde and inhibited nitric oxide synthase and alkaline phosphatase activity but had no significant effects on observable damage. No effect on neutrophil infiltration (myeloperoxidase) was observed. On the other hand, quercitrin exerted complex effects on colonic hydroelectrolytic transport, showing a slight potentiation of water absorption in vivo (5 mg/kg) as well as a normalization of carbachol stimulated electrogenic ionic transport in the proximal colon in vitro (5 mg/kg). It is concluded that the beneficial effects of quercitrin on trinitrobenzene sulfonic acid chronic colitis arise from an early downregulation of the inflammatory cascade that is associated with amelioration of the disturbances in hydroelectrolytic transport.

Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting

It is currently a routine practice to require a measurement of a housekeeping reference, including actin, glyceraldehyde-3-phosphate dehydrogenase, β-tubulin, among others, in Western blots, as it is the rule in RNA blots. Reversible Ponceau staining has been applied successfully to check equal loading of gels. Here we test a new technique, with the Stain-Free gels from Bio-Rad, against both Ponceau staining and housekeeping protein immunodetection under different conditions. Our results show that Stain-Free gels outperform Ponceau staining and that both are more consistent than housekeeping proteins as a loading control.

Bovine glycomacropeptide ameliorates experimental rat ileitis by mechanisms involving downregulation of interleukin 17.

Bovine glycomacropeptide (BGMP) is an inexpensive, non‐toxic milk peptide with anti‐inflammatory effects in rat experimental colitis but its mechanism of action is unclear. It is also unknown whether BGMP can ameliorate inflammation in proximal regions of the intestine. Our aim was therefore two‐fold: first, to determine the anti‐inflammatory activity of BGMP in the ileum; second, to characterise its mechanism of action.

Exogenous alkaline phosphatase treatment complements endogenous enzyme protection in colonic inflammation and reduces bacterial translocation in rats.

Alkaline phosphatase (AP) inactivates bacterial lipopolysaccharide and may therefore be protective. The small intestine and colon express intestinal (IAP) and tissue nonspecific enzyme (TNAP), respectively. The aim of this study was to assess the therapeutic potential of exogenous AP and its complementarity with endogenous enzyme protection in the intestine, as evidenced recently. IAP was given to rats by the oral or intrarectal route (700U/kgday). Oral budesonide (1mg/kgday) was used as a reference treatment. Treatment with intrarectal AP resulted in a 54.5% and 38.0% lower colonic weight and damage score, respectively, and an almost complete normalization of the expression of S100A8, LCN2 and IL-1β (p<0.05). Oral AP was less efficacious, while budesonide had a more pronounced effect on most parameters. Both oral and intrarectal AP counteracted bacterial translocation effectively (78 and 100%, respectively, p<0.05 for the latter), while budesonide failed to exert a positive effect. AP activity was increased in the feces of TNBS colitic animals, associated with augmented sensitivity to the inhibitor levamisole, suggesting enhanced luminal release of this enzyme. This was also observed in the mouse lymphocyte transfer model of chronic colitis. In a separate time course study, TNAP was shown to increase 2-3 days after colitis induction, while dextran sulfate sodium was a much weaker inducer of this isoform. We conclude that exogenous AP exerts beneficial effects on experimental colitis, which includes protection against bacterial translocation. AP of the tissue-nonspecific isoform is shed in higher amounts to the intestinal lumen in experimental colitis, possibly aiding in intestinal protection.

Disturbances of colonic ion secretion in inflammation: role of the enteric nervous system and cAMP

Abstract. We used the trinitrobenzenesulphonic acid (TNBS) rat model of experimental colitis to study the alterations in electrogenic ion transport in the inflamed distal colon. The distal colon exhibited decreased basal transport and reduced short-circuit current responses to carbachol and isobutylmethylxanthine (IBMX). The concentration/response curve for IBMX was also shifted to the right. Ion substitution experiments indicated that electrogenic transport was attributable chiefly to Cl–secretion. The mucosal layer of the inflamed distal colon (devoid of the submucosa) exhibited normal maximal responses to carbachol and IBMX, although the concentration/response curve for the latter was again shifted to the right. Tetrodotoxin markedly increased the response of the normal distal colon to both secretagogues and nullified the inhibition of the response to carbachol, but not that to IBMX, in the inflamed colon. The response of the mucosal preparation to 8-bromoadenosine 3′,5′-cyclic monophosphate was similar in the normal and inflamed intestine, while the G protein activator NaF had a greater effect in the latter. The expression of the cystic fibrosis transmembrane conductance regulator (CFTR), as assessed by Northern blotting, was unchanged. cAMP levels in isolated colonocytes were markedly reduced by inflammation. We conclude that colonic inflammation produces disturbances of the enteric nervous system resulting in defective mucosal cAMP production and inhibition of ionic secretion.

Persistent epithelial barrier alterations in a rat model of postinfectious gut dysfunction

Background  Mucosal mast cells (MMCs), epithelial barrier function (EBF) and the enteric nervous system (ENS) are interactive factors in the pathophysiology of functional gastrointestinal disorders. We characterized postinfectious EBF alterations in the Trichinella spiralis infection model of MMC‐dependent intestinal dysfunction in rats.

Experimental inflammation of the rat distal colon inhibits ion secretion in the proximal colon by affecting the enteric nervous system

Intestinal inflammation causes hyporesponsiveness of the inflamed tissue to secretagogues but little is known about the behaviour of the areas proximal to the site of inflammation. We studied the responses of the proximal segment of the colon to carbachol, histamine, isobutylmethylxanthine (IBMX) and vasoactive intestinal peptide (VIP) in rats with trinitrobenzenesulphonic acid (TNBS)-induced, chronic inflammation of the distal colon. Macroscopic and biochemical analysis ruled out the presence of inflammation in the proximal colon. When mounted in Ussing chambers under voltage-clamp conditions, basal transport and conductance were not affected. However, the maximum response in the concentration/response curves (short-circuit current) for carbachol and histamine was reduced in TNBS-treated rats, without changes in the EC50. This effect corresponded to reduced chloride secretion, as demonstrated by ion substitution experiments. The responses to IBMX and VIP were virtually unaffected. The inhibitory effect was abolished by pretreatment with the neural blockers tetrodotoxin and lidocaine but not indomethacin, suggesting that the enteric nervous system is responsible for the inhibition. In conclusion, chronic distal inflammation of the distal colon results in inhibition of calcium-dependent secretion in the proximal colon via a reduction of the contribution of the enteric nervous system.

FXR-dependent and -independent interaction of glucocorticoids with the regulatory pathways involved in the control of bile acid handling by the liver

Treatment with glucocorticoids (GCs) may cause adverse effects, including cholestasis. The ability of dexamethasone, prednisolone and budesonide to affect the liver handling of bile acids (BAs) has been investigated. In rats treated with GCs for 4 days, altered serum and bile BA levels, changed conjugation pattern, and delayed and decreased ability to conjugate/secrete exogenously administered deoxycholate, were found using HPLC-MS/MS. RT-QPCR analyses revealed that GC treatment also induced a down-regulation of liver nuclear receptors (Fxr, Gr and Shp), transporters (Ntcp, Mrp4 and Bcrp) and enzymes (Cyp7a1 and Baat), whereas Bsep, Mrp2 and Cyp27a1 were up-regulated. Human HepG2 and Alexander cell lines were used as in vitro models of liver cells with and without constitutive FXR expression, respectively. In HepG2 cells, GCs induced a decreased expression of FXR and SHP, and inhibited the regulatory effect of GW4064 on FXR-target genes. In Alexander cells, only when they were transfected with FXR+RXR, GW4064 caused up-regulation of SHP and OSTβ, and a down-regulation of CYP27A1. GCs had the opposite effect on these genes, both in the absence and in the presence of FXR expression. Co-transfection of Alexander cells with IR-1-Luc and FXR+RXR revealed that GCs did not inhibit but moderately enhanced FXR activity. Moreover, GCs have a synergistic effect on GW4064-induced FXR activation, whereas chenodeoxycholate and GW4064 have an additive effect. In conclusion, GCs are able to directly or indirectly activate FXR but they also antagonize, through FXR-independent mechanisms, the expression of FXR and FXR target genes involved in the hepatic handling of BAs.

The bisphosphonate alendronate improves the damage associated with trinitrobenzenesulfonic acid-induced colitis in rats

The nitrogen‐containing bisphosphonates are drugs used successfully in the treatment of osteoporosis. They act inhibiting farnesyl diphosphate synthase. This mechanism may also produce anti‐inflammatory effects. The therapeutic activity of alendronate was tested in vivo using a model of inflammatory bowel disease.