Pilar Utrilla

Vasodilatory effects of flavonoids in rat aortic smooth muscle. Structure-activity relationships

1. Flavonoids relaxed the contractions induced by noradrenaline, KCl or phorbol 12-myristate, 13-acetate in rat aortic strips, the order of potency being: flavonols (quercetin, kaempferol, pentamethylquercetin) > flavones(luteolin, apigenin) > flavanols((+)-catechin, (-)-epicatechin) which correlates with the reported order of potency to inhibit protein kinase C. 2. The relaxant effects of kaempferol and luteolin were slightly potentiated by isoprenaline and those of pentamethylquercetin, kaempferol and apigenin by sodium nitroprusside. 3. It is concluded that the main vasodilatory mechanism of flavonoids seems to be the inhibition of protein kinase C. Inhibition of cyclic nucleotide phosphodiesterases or decreased Ca2+ uptake may also contribute to their vasodilatory effects.

Melatonin counteracts inducible mitochondrial nitric oxide synthase-dependent mitochondrial dysfunction in skeletal muscle of septic mice

Abstract:  Mitochondrial nitric oxide synthase (mtNOS) produces nitric oxide (NO) to modulate mitochondrial respiration. Besides a constitutive mtNOS isoform it was recently suggested that mitochondria express an inducible isoform of the enzyme during sepsis. Thus, the mitochondrial respiratory inhibition and energy failure underlying skeletal muscle contractility failure observed in sepsis may reflect the high levels of NO produced by inducible mtNOS. The fact that mtNOS is induced during sepsis suggests its relation to inducible nitric oxide synthase (iNOS). Thus, we examined the changes in mtNOS activity and mitochondrial function in skeletal muscle of wild‐type (iNOS+/+) and iNOS knockout (iNOS−/−) mice after sepsis. We also studied the effects of melatonin administration on mitochondrial damage in this experimental paradigm. After sepsis, iNOS+/+ but no iNOS−/− mice showed an increase in mtNOS activity and NO production and a reduction in electron transport chain activity. These changes were accompanied by a pronounced oxidative stress reflected in changes in lipid peroxidation levels, oxidized glutathione/reduced glutathione ratio, and glutathione peroxidase and reductase activities. Melatonin treatment counteracted both the changes in mtNOS activity and rises in oxidative stress; the indole also restored mitochondrial respiratory chain in septic iNOS+/+ mice. Mitochondria from iNOS−/− mice were unaffected by either sepsis or melatonin treatment. The data suggest that inducible mtNOS, which is coded by the same gene as that for iNOS, is responsible for mitochondrial dysfunction during sepsis. The results also suggest the use of melatonin for the protection against mtNOS‐mediated mitochondrial failure.

The combination of fructooligosaccharides and resistant starch shows prebiotic additive effects in rats

Different types of dietary fiber can be distinguished considering their rate of fermentability, thus determining the location of the large intestine where they exert their beneficial effect. Their combination could be interesting to obtain health-promoting effects throughout the entire colon. The aim of the present study was to evaluate the synergistic effect of two dietary fibers with different fermentation patterns, fructooligosaccharides (FOS) (Beneo(®)-95) and resistant starch (Fibersol(®)-2), after their administration to healthy rats or in trinitrobenzenesulphonic acid-(TNBS) colitic rats, with an altered colonic immune response. In healthy rats, the administration of the combination of FOS and resistant starch induced changes in the intestinal microbiota, by increasing lactobacilli and bifidobacteria in caecum and colonic contents. Furthermore, its administration up-regulated the expression of the trefoil factor-3 and MUC-2 in comparison with untreated rats, thus improving the intestinal barrier function. The beneficial effects observed with this combination were confirmed in the TBNS model of rat colitis, since it was able to exert intestinal anti-inflammatory effect, associated with an increase of protective bacteria and up-regulation of epithelial defense mechanisms. In conclusion, the combination of two different dietary fibers may result in a synergistic prebiotic effect, and may confer greater health benefits to the host.

Butyrate in vitro immune-modulatory effects might be mediated through a proliferation-related induction of apoptosis

Survival and proliferation signals are two processes closely interrelated and finely controlled in most cell types, whose deregulation may lead to carcinogenesis. In the last decade, different studies have suggested that both cellular functions are also intimately associated with other cellular activities such as differentiation and cellular activation, especially in immune cells. The aim of this study was to evaluate the effects of the short-chain fatty acid (SCFA) butyrate on the proliferation and activation state of different cell types involved in inflammatory bowel disease. We focused on intestinal epithelial cells, macrophages and T-lymphocytes, using both primary non-transformed cultures and established cell lines. The results showed that low concentrations of butyrate inhibited the proliferation of all the immune cell types tested in this work, whereas it only induced apoptosis in activated T-lymphocytes, non-differentiated epithelial cells and macrophage cell lines, but not in differentiated epithelial cells or primary macrophages. Butyrate apoptosis induction was mediated by caspase-3/7 activation. This SCFA was only able to modify cell activation, measured as expression of inflammatory cytokines, in those cell types in which apoptosis was induced. In conclusion, our results suggest a cell type-specificity of the immune-modulatory effects of butyrate based on the proliferation/activation characteristic physiology of these processes in different cells types.

The association of minocycline and the probiotic Escherichia coli Nissle 1917 results in an additive beneficial effect in a DSS model of reactivated colitis in mice

Antibiotics have been empirically used for human inflammatory bowel disease, being limited to short periods. Probiotics are able to attenuate intestinal inflammation due to its immunomodulatory properties, being considered as safe when chronically administered. The aim was to test the association of minocycline, a tetracycline with immunomodulatory properties, and the probiotic Escherichia coli Nissle 1917 (EcN) in a mouse model of reactivated colitis. For this purpose, female C57BL/6J mice were assigned to different groups: non-colitic and dextran sodium sulfate (DSS)-control groups (without treatment), minocycline (50 mg/kg/day; p.o.), EcN (5×10(8) CFU/day; p.o.), and minocycline plus EcN treated groups. Colitis was induced by adding DSS in the drinking water (3%) for 5 days; 2 weeks later, colitis was reactivated by subsequent exposure to DSS. The inflammatory status was evaluated daily by a disease activity index (DAI); colonic damage was assessed histologically and biochemically by evaluating mRNA relative expression of different mediators by qPCR. Finally, a microbiological analysis of the colonic contents was performed. Minocycline and EcN exerted intestinal anti-inflammatory effect and attenuated the reactivation of the colitis, as shown by the reduced DAI values, being these effects greater when combining both treatments. This was evidenced histologically and biochemically, by reduced expression of TNFα, IL-1β, IL-2, MIP-2, MCP-1, ICAM-1, iNOS and MMP-9, together with increased MUC-3 and ZO-1 expression. Finally, the altered microbiota composition of colitic mice was partially restored after the different treatments. In conclusion, EcN supplementation to minocycline treatment improves the recovery of the intestinal damage and prevents the reactivation of experimental colitis.

A probiotic strain of Escherichia coli, Nissle 1917, given orally exerts local and systemic anti-inflammatory effects in lipopolysaccharide-induced sepsis in mice

Background and purpose:  Escherichia coli Nissle 1917 is a probiotic strain used in the treatment of intestinal immune diseases, including ulcerative colitis. The aim of the present study was to test if this probiotic bacterium can also show systemic immunomodulatory properties after oral administration.

Suppression of TNBS-induced colitis in rats by 4-methylesculetin, a natural coumarin: Comparison with prednisolone and sulphasalazine

The aim of the present study was to compare the effects of the 4-methylesculetin with those produced by prednisolone and sulphasalazine and to elucidate the mechanisms involved in its action. Colitis was induced in rat by instillation of trinitrobenzenesulphonic acid (TNBS). The colon damage was evaluated using macroscopic, microscopic and biochemical analysis. In addition, in vitro studies were performed to evaluate cytokine production in cell cultures using the murine macrophage cell line RAW264.7, mouse splenocytes and the human colonic epithelial cell line Caco-2. 4-Methylesculetin produced a reduction of the macroscopic damage score and the recovery of the intestinal cytoarchitecture. These effects were associated with a prevention of the GSH depletion and an inhibition in AP activity. After colitis relapse, 4-methylesculetin improved the colonic inflammatory status as evidenced by histological findings, with a reduction in apoptosis, as well as biochemically by inhibition of colonic myeloperoxidase, alkaline phosphatase and metalloproteinase 9 activities. Paired with this inhibitive activity, there was a decrease in malondialdehyde content and in IL-1β levels. In vitro assays revealed that 4-methylesculetin promoted an inhibition in IL-1β, IL-8, IL-2 and IFN-γ production in cell cultures. In conclusion, 4-methylesculetin showed similar efficacy to that obtained with either prednisolone or sulphasalazine, both in the acute phase of colitis as well as following a curative protocol. The intestinal anti-inflammatory activity by 4-methylesculetin is likely related to its ability in reduce colonic oxidative stress and inhibit pro-inflammatory cytokine production.

The intestinal anti-inflammatory effect of minocycline in experimental colitis involves both its immunomodulatory and antimicrobial properties

Some antibiotics, including minocycline, have recently been reported to display immunomodulatory properties in addition to their antimicrobial activity. The use of a compound with both immunomodulatory and antibacterial properties could be very interesting in the treatment of inflammatory bowel disease (IBD), so the aim of our study was to evaluate the anti-inflammatory effect of minocycline in several experimental models of IBD. Firstly, the immunomodulatory activity of the antibiotic was tested in vitro using Caco-2 intestinal epithelial cells and RAW 264.7 macrophages; minocycline was able to inhibit IL-8 and nitrite production, respectively. In vivo studies were performed in trinitrobenzenesulfonic acid (TNBS)-induced rat colitis and dextran sodium sulfate (DSS)-induced mouse colitis. The results revealed that minocycline exerted an intestinal anti-inflammatory effect when administered as a curative treatment in the TNBS model, modulating both immune and microbiological parameters, being confirmed in the DSS model; whereas none of the other antibiotics tested (tetracycline and metronidazole) showed anti-inflammatory effect. However, minocycline administration before the colitis induction was not able to prevent the development of the intestinal inflammation, thus showing that only its antimicrobial activity is not enough for the anti-inflammatory effect. In conclusion, minocycline displays an anti-inflammatory effect on different models of rodent colitis which could be attributed to the association of its antibacterial and immunomodulatory properties.

A shorter and more specific oral sensitization-based experimental model of food allergy in mice.

Cows milk protein allergy (CMPA) is one of the most prevalent human food-borne allergies, particularly in children. Experimental animal models have become critical tools with which to perform research on new therapeutic approaches and on the molecular mechanisms involved. However, oral food allergen sensitization in mice requires several weeks and is usually associated with unspecific immune responses. To overcome these inconveniences, we have developed a new food allergy model that takes only two weeks while retaining the main characters of allergic response to food antigens. The new model is characterized by oral sensitization of weaned Balb/c mice with 5 doses of purified cows milk protein (CMP) plus cholera toxin (CT) for only two weeks and posterior challenge with an intraperitoneal administration of the allergen at the end of the sensitization period. In parallel, we studied a conventional protocol that lasts for seven weeks, and also the non-specific effects exerted by CT in both protocols. The shorter protocol achieves a similar clinical score as the original food allergy model without macroscopically affecting gut morphology or physiology. Moreover, the shorter protocol caused an increased IL-4 production and a more selective antigen-specific IgG1 response. Finally, the extended CT administration during the sensitization period of the conventional protocol is responsible for the exacerbated immune response observed in that model. Therefore, the new model presented here allows a reduction not only in experimental time but also in the number of animals required per experiment while maintaining the features of conventional allergy models. We propose that the new protocol reported will contribute to advancing allergy research.

Luteolin 5-rutinoside from Salvia lavandulifolia ssp. oxyodon

The flavonoid, luteolin 5-rutinoside, has been isolated from Salvia lavandulifolia ssp. oxyodon and identified by enzymic hydrolysis and spectroscopic methods.