Laura Grasa

Antibiotic-Induced Depletion of Murine Microbiota Induces Mild Inflammation and Changes in Toll-Like Receptor Patterns and Intestinal Motility

We examine the impact of changes in microbiota induced by antibiotics on intestinal motility, gut inflammatory response, and the function and expression of toll-like receptors (TLRs). Alterations in mice intestinal microbiota were induced by antibiotics and evaluated by q-PCR and DGGE analysis. Macroscopic and microscopic assessments of the intestine were performed in control and antibiotic-treated mice. TLR expression was determined in the intestine by q-RT-PCR. Fecal parameter measurements, intestinal transit, and muscle contractility studies were performed to evaluate alterations in intestinal motility. Antibiotics reduced the total bacterial quantity 1000-fold, and diversity was highly affected by treatment. Mice with microbiota depletion had less Peyer’s patches, enlarged ceca, and mild gut inflammation. Treatment with antibiotics increased the expression of TLR4, TLR5, and TLR9 in the ileum and TLR3, TLR4, TLR6, TLR7, and TLR8 in the colon, and it reduced the expression of TLR2, TLR3, and TLR6 in the ileum and TLR2 and TLR9 in the colon. Antibiotics decreased fecal output, delayed the whole gut and colonic transit, and reduced the spontaneous contractions and the response to acetylcholine (ACh) in the ileum and colon. Activation of TLR4 by lipopolysaccharide (LPS) reverted the reduction of the spontaneous contractions induced by antibiotics in the ileum. Activation of TLR4 by LPS and TLR5 by flagellin reduced the response to ACh in the ileum in control mice. Our results confirm the role of the microbiota in the regulation of TLRs expression and shed light on the microbiota connection to motor intestinal alterations.

Inhibition of p38 MAPK improves intestinal disturbances and oxidative stress induced in a rabbit endotoxemia model

Background  Lipopolysaccharide (LPS) decreases intestinal contractility and induces the release of reactive oxygen species, which play an important role in the pathogenesis of sepsis. p38 mitogen‐activated protein kinase (MAPK) can be activated by a variety of stimuli such as LPS. The aims of this study were: (i) to investigate the role of p38 MAPK in the effect of LPS on (a) the acetylcholine, prostaglandin E2 and KCl‐induced contractions of rabbit duodenum and (b) the oxidative stress status; (ii) to localize the active form of p38 in the intestine.

IL-10 modulates serotonin transporter activity and molecular expression in intestinal epithelial cells

Serotonin is a neuromodulator mainly synthesized by intestinal enterochromaffin cells that regulate overall intestinal physiology. The serotonin transporter (SERT) determines the final serotonin availability and has been described as altered in inflammatory bowel diseases. IL-10 is an anti-inflammatory cytokine that is involved in intestinal inflammatory processes and also contributes to intestinal mucosa homeostasis. The regulation of SERT by pro-inflammatory factors is well known; however, the effect of IL-10 on the intestinal serotoninergic system mediated by SERT remains unknown. Therefore, the aim of the present study is to determine whether IL-10 affects SERT activity and expression in enterocyte-like Caco-2 cells. Treatment with IL-10 was assessed and SERT activity was determined by 5-HT uptake. SERT mRNA and protein expression was analyzed using quantitative RT-PCR and western blotting. The results showed that IL-10 induced a dual effect on SERT after 6h of treatment. On one hand, IL-10, at a low concentration, inhibited SERT activity, and this effect might be explained by a non-competitive inhibition of SERT. On the other hand, IL-10, at a high concentration, increased SERT activity and molecular expression in the membrane of the cells. This effect was mediated by the IL-10 receptor and triggered by the PI3K intracellular pathway. Our results demonstrate that IL-10 modulates SERT activity and expression, depending on its extracellular conditions. This study may contribute to understand serotoninergic responses in intestinal pathophysiology.

K+ channels involved in contractility of rabbit small intestine

Most excitable cells, including gastrointestinal smooth muscle cells, express several types of K+ channels. The aim of this study was to examine the types of K+ channels involved in the contractility of longitudinal smooth muscle of rabbit small intestinein vitro. Spontaneous contractions and KCl-stimulated contractions were reduced by atropine, phentolamine, propranolol, suramin, tetrodotoxin and indomethacin. The amplitude and tone of spontaneous contractions were increased by apamin, charybdotoxin, iberiotoxin, E4031, tetraetylammonium (TEA) and BaCl2. The frequency of contractions was reduced in the presence of apamin and TEA and increased by charybdotoxin. It was found that 4-aminopyridine increased the tone of spontaneous contractions and reduced the amplitude and frequency of contractions. Glibenclamide did not modify the amplitude, frequency or tone of contractions. KCl-stimulated contractions were increased by E4031, were not modified by apamin, glibenclamide, NS1619 or diazoxide, and were reduced by charybdotoxin, TEA, 4-aminopyridine or BaCl2. These results suggest that both Ca2+-activated K+ channels of small and high conductance, and HERG K+ channels and inward rectifier K+ channels participate in spontaneous contractions of small intestine. On the other hand, voltage-dependent K+ channels, HERG K+ channels, inward rectifier K+ channels and high conductance Ca2+-activated K+ channels are involved in KCl-stimulated contractions.ResumenLa mayoría de las células excitables, incluyendo las células lisas gastrointestinales, expresan varios tipos de canales de K+. El objetivo de este estudio es examinar los tipos de canales de K+ que están involucrados en la contractilidad del músculo liso longitudinal del intestino delgado de conejoin vitro. Las contracciones espontáneas y las producidas por KCl se redujeron por atropina, fentolamina, propranolol, suramina, tetrodotoxina e indometacina. La amplitud y tono de las contracciones espontáneas aumentaron por apamin, charybdotoxina, iberiotoxina, E4031, tetraetilamonio (TEA) y BaCl2, mientras que la frecuencia de las contracciones se redujo en presencia de apamin, charybdotoxina y TEA. La 4-aminopiridina aumentó el tono de las contracciones espontáneas y redujo la amplitud y frecuencia de las contracciones. La glibenclamida no modificó la amplitud, frecuencia y tono de las contracciones. Las contracciones producidas por el KCl aumentaron en presencia de E4031, no fueron modificadas por el apamin, glibenclamida, NS1619 o diazóxida y disminuyeron en presencia de la charybdotoxina, TEA, 4-aminopiridina o BaCl2. Estos resultados sugieren que los canales de K+ activados por Ca2+ de pequeña y gran conductancia, canales de K+ HERG canales de K+ rectificadores de entrada participan en las contracciones espontáneas del intestino delgado. Por otra parte, los canales de K+ voltaje-dependientes, canales de K+ HERG, canales de K+ rectificadores de entrada y canales de K+ activados por Ca2+ de gran conductancia están implicados en las contracciones producidas por el KCl.

Neomycin and bacitracin reduce the intestinal permeability in mice and increase the expression of some tight-junction proteins

BACKGROUND Tight-junction (TJ) proteins regulate paracellular permeability. Gut permeability can be modulated by commensal microbiota. Manipulation of the gut microbiota with antibiotics like bacitracin and neomycin turned out to be useful for the treatment of diarrhoea induced by Clostridium difficile or chemotherapy drugs. AIM To evaluate the effects of the microbiota depletion evoked by the oral administration of neomycin and bacitracin on the intestinal permeability and expression of TJ proteins in mice. METHODS Mice received neomycin and bacitracin orally for 7 days. Intestinal permeability was measured by the fluorescein-isothiocyanate-dextran (FITC-dextran) method. The gene expression of TJ proteins in the intestine was determined by real time-PCR. RESULTS FITC-dextran levels in serum were reduced by half in antibiotic-treated mice, indicating a reduction of intestinal permeability. Antibiotics increased the expression of zonula occludens 1 (ZO-1), junctional adhesion molecule A (JAM-A, and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon. CONCLUSION The combination of neomycin and bacitracin reduce intestinal permeability and increase the gene expression of ZO-1, junctional adhesion molecule A (JAM-A), and occludin in the ileum and ZO-1, claudin-3, and claudin-4 in the colon.

Role of TLR4 and MAPK in the local effect of LPS on intestinal contractility

Objectives  Lipopolysaccharide (LPS) has been shown to alter intestinal contractility. Toll‐like receptor 4 (TLR4), K+ channels and mitogen‐activated protein kinases (MAPKs) have been proposed to be involved in the mechanism of action of LPS. The aim of this study was to determine the role of TLR4, K+ channels and MAPKs (p38, JNK and MEK1/2) in the local effect of LPS on the acetylcholine (ACh)‐induced contractions in rabbit small intestine in vitro.

Intestinal Serotonin Transporter Inhibition by Toll-Like Receptor 2 Activation. A Feedback Modulation.

TLR2 is a microbiota recognition receptor that has been described to contribute to intestinal homeostasis and to ameliorate inflammatory intestinal injury. In this context, serotonin (5-HT) has shown to be an essential intestinal physiological neuromodulator that is also involved in intestinal inflammatory diseases. Since the interaction between TLR2 activation and the intestinal serotoninergic system remains non-investigated, our main aim was to analyze the effect of TLR2 on intestinal serotonin transporter (SERT) activity and expression and the intracellular pathways involved. Caco-2/TC7 cells were used to analyze SERT and TLR2 molecular expression and SERT activity by measuring 5-HT uptake. The results showed that apical TLR2 activation inhibits SERT activity in Caco-2/TC7 cells mainly by reducing SERT protein level either in the plasma membrane, after short-term TLR2 activation or in both the plasma membrane and cell lysate, after long-term activation. cAMP/PKA pathway appears to mediate short-term inhibitory effect of TLR2 on SERT; however, p38 MAPK pathway has been shown to be involved in both short- and long-term TLR2 effect. Reciprocally, 5-HT long-term treatment yielded TLR2 down regulation in Caco-2/TC7 cells. Finally, results from in vivo showed an augmented intestinal SERT expression in mice Tlr2-/-, thus confirming our inhibitory effect of TLR2 on intestinal SERT in vitro. The present work infers that TLR2 may act in intestinal pathophysiology, not only by its inherent innate immune role, but also by regulating the intestinal serotoninergic system.

Extracellular signal-regulated kinase (ERK) is involved in LPS-induced disturbances in intestinal motility

Background  Lipopolysaccharide (LPS) is a causative agent of sepsis. A relationship has been described between LPS, free radicals, and cyclooxygenase‐2 (COX‐2). Here, we investigate the role of extracellular signal‐regulated kinase (ERK) mitogen‐activated protein kinases (MAPK) in the effect of LPS on intestinal motility, oxidative stress status, and COX‐2 expression.

Nuclear factor κB is a key transcription factor in the duodenal contractility alterations induced by lipopolysaccharide

Alterations in intestinal motility are one of the features of sepsis induced by lipopolysaccharide (LPS). This study investigated the role of the nuclear transcription factor κB (NF‐κB) in the LPS‐induced duodenal contractility alterations, generation of reactive oxygen species (ROS) and production of cytokines in rabbit duodenum. Rabbits were treated with saline, LPS, sulfasalazine + LPS, pyrrolidinedithiocarbamate (PDTC) + LPS or RO 106‐9920 + LPS. Contractility studies were performed in an organ bath. The formation of products of oxidative damage to proteins (carbonyls) and lipids (malondialdehyde and 4‐hydroxyalkenals) was quantified in intestinal tissue and plasma. The protein expression of NF‐κB was measured by Western blot. The DNA binding activity of NF‐κB was evaluated by transcription factor activity assay. The expression of interleukin‐1β, tumour necrosis factor α (TNF‐α), interleukin‐6, interleukin‐10 and interleukin‐8 mRNA was determined by RT‐PCR. Sulfasalazine, PDTC and RO 106‐9920 blocked the inhibitory effect of LPS on contractions induced by ACh in the longitudinal smooth muscle of rabbit duodenum. Sulfasalazine, PDTC and RO 106‐9920 reduced the increased levels of malondialdehyde and 4‐hydroxyalkenals and the carbonyls induced by LPS in plasma. Lipopolysaccharide induced the activation, translocation to the nucleus and DNA binding of NF‐κB. Lipopolysaccharide increased the mRNA expression of interleukin‐6 and TNF‐α in duodenal tissue, and this effect was partly reversed by PDTC, sulfasalazine and RO 106‐9920. In conclusion, NF‐κB mediates duodenal contractility disturbances, the generation of ROS and the increase in the expression of interleukin‐6 and TNF‐α induced by LPS. Sulfasalazine, PDTC and RO 106‐9920 may be therapeutic drugs to reduce these effects.

Lipopolysaccharide-induced intestinal motility disturbances are mediated by c-Jun NH2-terminal kinases

BACKGROUND Lipopolysaccharide (LPS) is a causative agent of sepsis. Many alterations, such as intestinal motility disturbances, have been attributed to LPS. AIMS Here we investigated the role of c-Jun NH(2)-terminal kinases (JNK) in the effect of LPS on intestinal motility, the oxidative stress status and the cyclooxygenese-2 (COX-2) expression. METHODS Rabbits were injected with either (1) saline, (2) LPS, (3) SP600125, a specific JNK inhibitor, or (4) SP600125+LPS. Duodenal contractility was studied in an organ bath. The formation of products of oxidative damage to proteins (carbonyls) and lipids [malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)] was quantified by spectrophotometry in the intestine and plasma. The protein expression of p-JNK, total JNK, and COX-2 was measured by Western blot, and p-JNK was localized by immunohistochemistry. RESULTS LPS decreased the contractions evoked by acetylcholine and prostaglandin E(2) and KCl-induced contractions. LPS increased phospho-JNK and COX-2 expressions and the levels of carbonyls and MDA+4-HDA. SP600125 blocked the effect of LPS on the acetylcholine, prostaglandin E(2), and KCl-induced contractions, the levels of carbonyls and MDA+4-HDA, and the p-JNK and COX-2 expressions. p-JNK was detected in the smooth muscle cells of duodenum. CONCLUSION Our results suggest that JNK is involved in the mechanism of action of LPS in the intestine.