The lipopolysaccharide from Escherichia coli O127:B8 induces inflammation and motility disturbances in rabbit ileum

[EN] The aim of this work was to evaluate the effects of lipopolysaccharide (LPS) from Escherichia coli O127:B8 on the expression of toll-like receptor 4 (TLR4), the histology, and motor function in rabbit ileum. Rabbits were injected intravenously with saline or LPS (100 μg/kg, 2 h). The mRNA expression and localization of TLR4 were determined by reverse transcriptase-PCR and immunofluorescence, respectively. Histological damage induced by LPS was evaluated in sections of ileum stained with haematoxylin and eosin. Contractility studies of ileum were performed in an organ bath. The mRNA expression of TLR4 decreased in the muscular but not in the mucosal layer of rabbits treated with LPS. TLR4 was localised in both the mucosal and muscular layers of rabbit ileum. LPS induced intestinal inflammation and altered the spontaneous contractions and the serotonin-, acetylcholine- and KCl-induced contractions. In conclusion, LPS from E. coli O127:B8 induced a decrease in the mRNA expression of TLR4, an inflammatory response, and changes in the contractility of rabbit ileum.This work was funded by Gobierno de Aragón and European Regional Development Fund ERDF (B61/2012), Spain.Grasa, L.; Gonzalo, S.; De Martino, A.; Murillo, MD. (2017). The lipopolysaccharide from Escherichia coli O127:B8 induces inflammation and motility disturbances in rabbit ileum. World Rabbit Science. 25(2):185-191. https://doi.org/10.4995/wrs.2017.5160SWORD18519125

In-vitro toxicity of carbon nanotube/polylysine colloids to colon cancer cells

Single-walled carbon nanotubes (SWCNTs) are thoroughly purified and dispersed in an aqueous solution of high molecular weight poly-L-lysine (pLlys). Human intestinal epithelial Caco-2/TC7 cells are incubated with the SWCNT dispersions in pLlys, and their effects on cell viability are studied by image flow cytometry. No significant changes are observed in the cell culture wells up to pLlys concentrations of 10 g mL-1. However, high mortality is detected at pLlys concentrations of 100 g mL-1. The presence of oxygen-free SWCNTs does not modify the effects of pLlys on cell cultures at any of the tested concentrations (£ 1 g mL-1). In addition, SWCNTs having an 8 wt.% of surface oxygen are tested with identical results. Thus, purified SWCNTs, even bearing oxygen functional groups, act as inert particles in the cell culture medium. This result supports the applicability of SWCNTs as carriers in pharmacological formulations against digestive tract diseases.This work was funded by the Spanish Ministry MINECO and the European Regional Development Fund under the projects PRI-PIBAR-2011-1 and ENE2013-48816-C5-5-R, and the Government of Aragon and the European Social Fund (DGA-ESF-T66 Grupo Consolidado and DGA-ESF-B61 Grupo Consolidado).Peer reviewe

A novel pan-negative-gating modulator of KCa2/3 channels, fluoro-di-benzoate, RA-2, inhibits Endothelium-derived hyperpolarization–type relaxation in coronary artery and produces bradycardia in vivo

Small/intermediate conductance KCa channels (KCa2/3) are Ca2+/calmodulin regulated K+ channels that produce membrane hyperpolarization and shape neurologic, epithelial, cardiovascular, and immunologic functions. Moreover, they emerged as therapeutic targets to treat cardiovascular disease, chronic inflammation, and some cancers. Here, we aimed to generate a new pharmacophore for negative-gating modulation of KCa2/3 channels. We synthesized a series of mono- and dibenzoates and identified three dibenzoates [1,3-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate) (RA-2), 1,2-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate), and 1,4-phenylenebis(methylene) bis(3-fluoro-4-hydroxybenzoate)] with inhibitory efficacy as determined by patch clamp. Among them, RA-2 was the most drug-like and inhibited human KCa3.1 with an IC50 of 17 nM and all three human KCa2 subtypes with similar potencies. RA-2 at 100 nM right-shifted the KCa3.1 concentration-response curve for Ca2+ activation. The positive-gating modulator naphtho[1,2-d]thiazol-2-ylamine (SKA-31) reversed channel inhibition at nanomolar RA-2 concentrations. RA-2 had no considerable blocking effects on distantly related large-conductance KCa1.1, Kv1.2/1.3, Kv7.4, hERG, or inwardly rectifying K+ channels. In isometric myography on porcine coronary arteries, RA-2 inhibited bradykinin-induced endothelium-derived hyperpolarization (EDH)–type relaxation in U46619-precontracted rings. Blood pressure telemetry in mice showed that intraperitoneal application of RA-2 (=100 mg/kg) did not increase blood pressure or cause gross behavioral deficits. However, RA-2 decreased heart rate by ˜145 beats per minute, which was not seen in KCa3.1-/- mice. In conclusion, we identified the KCa2/3–negative-gating modulator, RA-2, as a new pharmacophore with nanomolar potency. RA-2 may be of use to generate structurally new types of negative-gating modulators that could help to define the physiologic and pathomechanistic roles of KCa2/3 in the vasculature, central nervous system, and during inflammation in vivo

Mitogen activated protein kinases blockade improves lipopolysaccharide-induced ileal motor disturbances El bloqueo de las proteínas cinasas activadas por mitógenos mejora las alteraciones motoras inducidas por el lipopolisacárido en íleon

Background: several diseases such as sepsis can affect the ileum. Lipopolysaccharide (LPS), an endotoxin present in the cell wall of gram negative bacteria, is a causative agent of sepsis. Objectives: the aims of this study were: a) to investigate the role of mitogen activated protein kinases (MAPKs) in the effect of LPS on the acetylcholine-induced contractions of rabbit ileum; and b) to study the localization of MAPKs in the ileum. Material and methods: ileal contractility was studied in an organ bath and MAPKs were localized by immunohistochemistry. Results: acetylcholine-induced contractions decreased with LPS. SB203580, SP600125 and U0126 blocked the effect of LPS on the acetylcholine-induced contractions. Phosphorylated p38 and ERK were detected in neurons of myenteric plexus and phosphorylated p38 and JNK in smooth muscle cells of ileum. Conclusion: we can suggest that p38, JNK, and ERK MAPKs are involved in the mechanism of action of LPS in the ileum.<br>Introducción: varias enfermedades como la sepsis pueden afectar al íleon. El lipopolisacárido (LPS), una endotoxina presente en la pared celular de las bacterias gram-negativas, es un agente causal de la sepsis. Objetivos: los objetivos del presente estudio fueron: a) investigar el papel de las proteína cinasas activadas por mitógenos (MAPKs) en los efectos del LPS en las contracciones inducidas por acetilcolina en el íleon de conejo; y b) estudiar la localización de las MAPKs en el íleon. Material y métodos: la contractilidad ileal se estudió en un baño de órganos y las MAPKs se localizaron mediante inmunohistoquímica. Resultados: el LPS disminuyó las contracciones inducidas por acetilcolina. El SB203580, el SP600125 y el U0126 bloquearon los efectos del LPS sobre las contracciones inducidas por acetilcolina. La p38 y la ERK fosforiladas se detectaron en las neuronas del plexo mientérico y la p38 y la JNK fosforiladas en las células del músculo liso del íleon. Conclusión: concluimos que la p38, la JNK y la ERK MAPKs parecen estar involucradas en el mecanismo de acción del LPS en el íleon

Evaluation of biological effects of single-walled carbon nanotubes (SWCNTs) in intestine

Resumen del trabajo presentado a la II Internacional Conference on Water Soluble Metal Complexes: Metals and Water, celebrada en Jaca, Huesca (España) del 13 al 15 de junio de 2017.Peer reviewe

Study of the biological effects of single-walled carbon nanotubes (SWNTs) on rabbit small intestine

Resumen del póster presentado al Joint International Neurogastroenterology and Motility Meeting, celebrado en Italia en 2012.[Objective]: Single-walled carbon nanotubes (SWNTs) are structures of carbon of nanometric scale. Biomedical research is exploring possible application of nanotubes (NTs) as drug delivery carriers. Studies about the biological effects of NTs are necessary to ensure that they are not toxic when they are linked to a drug to be delivered.The aim of this study was to investigate the effects of SWNTs systemic administration on the motility, generation of free radicals and expression of inflammatory mediators in rabbit ileum.[Methods]: Rabbits were injected intravenously with saline or SWNTs (0.02, 0.2 and 2 lg kg-1 body weight) for 24 h. Contractility studies were performed suspending pieces of ileum in an organ bath in the direction of longitudinal smooth muscle fibres. The ileum and plasma concentrations of malondialdehyde (MDA) + 4-hydroxyalkenals (4-HDA) and carbonyls were used as indexes of lipid and protein peroxidation respectively. The mRNA expression of iNOS and COX-2 in rabbit ileum was studied by RT-PCR.[Results]: The frequency of the spontaneous contractions was slightly reduced in the ileum of rabbits treated with SWNTs 0.02, 0.2 and 2 lg kg-1, while the amplitude of these contractions was increased only at the dose of 2 lg kg-1. Serotonin 10–4 mol L)1, ACh 10-4 mol L)1 and KCl 80 m mol L)1 induced contractile responses that were increased in ileum from rabbits treated with SWNTs 0.02, 0.2 and 2 lg kg-1. The levels of MDA+4-HDA and carbonyls were increased in plasma from rabbits treated with SWNTs 0.2 and 2 lg kg-1, while in ileum these levels were increased only at the dose of 2 lg kg-1. The mRNA expression of iNOS and COX-2 in rabbit ileum was not modified after any of the treatments with SWNTs.[Conclusion]: These results suggest that SWNTs 0.02 lg kg-1 does not modify the expression of inflammatory mediators and free radicals in ileum, although it has some slight effects on ileum motility. Thus, SWNTs 0.02 lg kg-1 could be use in future studies for drug delivery.Funding by Gobierno de Aragon (B61/2011).Peer reviewe

Diferentes mecanismos de acción de la genisteína y quercetina en las contracciones espontáneas del duodeno de conejo

Flavonoids are known to relax precontracted intestinal smooth muscle and delay intestinal transit or intestinal peristalsis. The aim of this study was to determine the effects of genistein and quercetin on spontaneous contractions of rabbit duodenum in vitro in an organ bath. Genistein and quercetin (0.1-10 µM) reduced the amplitude of spontaneous contractions in the longitudinal and circular smooth muscle of rabbit duodenum, but they did not modify the frequency. Bay K8644 (L-type Ca2+ channel activator), apamin, charybdotoxin, and tetraetylammonium (K+ channel blockers) reverted the inhibition of amplitude of spontaneous contractions induced by genistein in longitudinal and circular smooth muscle. H-89 (protein kinase A inhibitor) antagonized the reduction of the amplitude of spontaneous contractions induced by quercetin in longitudinal and circular smooth muscle of duodenum, while 2,5-dideoxiadenosine (adenylyl cyclase inhibitor) reverted only the reduction of the amplitude in circular smooth muscle. In conclusion, genistein and quercetin reduce the spontaneous contractions in the duodenum by different mechanisms of actions. The effect of genistein would be mediated by Ca2+ and K+ channels, while the effect of quercetin would be mediated by cAMP and protein kinase A. Los flavonoides son conocidos por relajar el músculo intestinal precontraído y retrasar el tránsito o la peristalsis intestinal. El objetivo de este estudio era determinar los efectos de la genisteína y quercetina sobre las contracciones espontáneas del duodeno de conejo in vitro en un baño de órganos. La genisteína o quercetina (0,1-10 µM) redujeron la amplitud de las contracciones espontáneas en el músculo liso longitudinal y circular de duodeno de conejo sin modificar la frecuencia. El Bay K8644 (activador del canal de Ca2+ tipo L), apamina, caribdotoxina y tetraetilamonio (inhibidores de los canales de K+) revertieron la inhibición de la amplitud de las contracciones espontáneas inducidas por la genisteína en el músculo longitudinal y circular. El H-89 (inhibidor de la proteína kinasa A) antagonizó la reducción inducida por la quercetina sobre la amplitud de las contracciones espontáneas del músculo longitudinal y circular de duodeno, mientras la 2,5-dideoxiadenosina (inhibidor de la adenil ciclasa) revertió solamente la reducción de la amplitud en el músculo circular. En conclusión, la genisteína y la quercetina reducen las contracciones espontáneas del duodeno por diferentes mecanismos de acción. El efecto de la genisteína actuaría sobre canales de Ca2+ y de K+, y el efec

Single-walled carbon nanotubes (SWCNTs) enhance KCl-, acetylcholine-, and serotonin-induced contractions and evoke oxidative stress on rabbit ileum

We examined the effects of intravenous administration of purified arc-discharge single-walled carbon nanotubes (SWCNTs) on rabbit ileum to establish the possibility of using these SWCNTs as cell markers or drug carriers for the treatment of intestinal diseases. The SWCNT purification process eliminated carbonaceous impurities and decreased the amount of metals. SWCNTs increased the contractile responses induced by KCl, acetylcholine (ACh), and serotonin (5-HT) in rabbit ileum. Verapamil, apamin, glibenclamide, quinine and charybdotoxin reduced the contractile responses induced by ACh and 5-HT in ileum from rabbits treated with SWCNTs, indicating that voltage-dependent Ca2+ channels and small, intermediate, and large-conductance Ca2+-activated, ATP-sensitive, and voltage-dependent K+ channels are involved in these effects. Atropine and hexamethonium reduced the ACh response, indicating that muscarinic and nicotinic receptors are involved in this effect. Ondansetron and GR 113808 reduced the 5-HT response, indicating that serotonin 5-HT3 and 5-HT4 receptors are involved in this effect. SWCNTs increased the malondialdehyde plus 4-hydroxyalkenals and carbonyl levels in rabbit plasma and ileum, indicating that SWCNTs produce oxidative stress. SWCNTs did not produce relevant histological changes or modify the levels of the inflammatory mediators iNOS and COX-2 in the ileum. In conclusion, this study demonstrates that the intravenous administration of SWCNTs can evoke oxidative stress and affect contractility in rabbit ileum. These effects could reduce the possibility of using the arc-discharge SWCNTs as cell markers or drug carriers to treat intestinal diseases.Peer reviewe

Spasmolytic effect of Jasonia glutinosa on rodent intestine

Introduction: Jasonia glutinosa is an endemic plant species of the Iberian Peninsula and Southern France traditionally used in infusions as a spasmolytic; this plant is also known as "te de roca" (rock tea) but there is no scientific evidence about the effects of this plant. Aim: To evaluate the spasmolytic effect of rock tea. Methods: We have studied the in vitro effect of a rock tea extract on rat duodenum spontaneous contractions and the in vivo effect on mice gastrointestinal transit. Results: Rock tea extract reduced the spontaneous contractions of rat duodenal smooth muscle, inhibited KCl-induced contractions and blocked the contractions invoked by both extracellular Ca2+ and the agonist of L-type calcium channels Bay K8644. This inhibitory effect was similar to the one observed after the addition of the antagonist of L-type calcium channels verapamil. Rock tea did not modify gastrointestinal transit in healthy mice. However, after the treatment with dextran sulfate sodium, an inducer of colitis, rock tea extract reverted the increase in the gastrointestinal transit associated with this treatment. Conclusion: Rock tea extract relaxed duodenal smooth muscle via L-type calcium channels and normalized gastrointestinal transit in a model of colitis. These results may validate the traditional use of Jasonia glutinosa in patients with gastrointestinal alterations. Thus, rock tea may be used as a spasmolytic agent to treat gastrointestinal disorders