Sergio Gonzalo

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.

Melatonin and Trolox ameliorate duodenal LPS-induced disturbances and oxidative stress

BACKGROUND AND AIMS Lipopolysaccharide evokes gastrointestinal motility disturbances and oxidative stress. The aims of the present study were to investigate the effect of melatonin and Trolox in the actions of lipopolysaccharide on duodenal contractility and on lipid peroxidation in rabbit duodenum. METHODS The in vitro duodenal contractility studies were carried out in organ bath and the levels of malondialdehyde were assayed by spectrophotometry. Duodenal segments were incubated with lipopolysaccharide (0.3 microg mL(-1)). RESULTS Lipopolysaccharide decreased acetylcholine-induced contractions and increased malondialdehyde and 4-hydroxyalkenals concentrations in homogenates of duodenum. Melatonin reduced the amplitude of spontaneous contractions in duodenal muscle. Acetylcholine-induced contractions were not altered by melatonin in longitudinal and circular muscles. Trolox decreased the amplitude of spontaneous contractions of duodenal muscle. Trolox (1.2 or 4 mM) did not alter acetylcholine-induced contractions in duodenal muscle, but the concentration of 12 mM diminished the frequency of contractions and acetylcholine-induced contractions. Melatonin (0.3 mM) or Trolox (4 mM) diminished malondialdehyde and 4-hydroxyalkenals levels induced by lipopolysaccharide in the duodenum. CONCLUSIONS Melatonin and Trolox reduce oxidative stress induced by lipopolysaccharide and ameliorate the effect of lipopolysaccharide on duodenal contractility.

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.

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.

Mitogen activated protein kinases blockade improves lipopolysaccharide-induced ileal motor disturbances

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.

Trolox reduces the effect of ethanol on acetylcholine-induced contractions and oxidative stress in the isolated rabbit duodenum

Trolox is a hydrophilic analogue of vitamin E and a free radical scavenger. Ethanol diminishes the amplitude of spontaneous contractions and acetylcholine (ACh)-induced contractions in rabbit duodenum. The aim of this work was to study the effect of Trolox on the alterations induced by ethanol on contractility and lipid peroxidation in the duodenum. The duodenal contractility studies in vitro were carried out in an organ bath and the levels of malondialdehyde and 4-hydroxyalkenals (MDA+4-HAD) were measured by spectrophotometry. Trolox increased the reduction induced by ethanol on the amplitude of spontaneous contractions in longitudinal muscle but not in circular muscle. Trolox 4 mM decreased the effects of ethanol on ACh-induced contractions and on MDA+4-HDA concentrations. We conclude that Trolox might prevent oxidative stress induced by ethanol in the duodenum.

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 (0.1-10µM) 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.

Roles of Toll-Like Receptor 4, IκB Kinase, and the Proteasome in the Intestinal Alterations Caused by Sepsis

BackgroundLipopolysaccharide decreases intestinal contractility and induces the production of cytokines, which play an important role in the pathogenesis of sepsis.AimThe objective of the present study was to examine the role of Toll-like receptor 4, IκB kinase, and the proteasome in the intestinal alterations induced by lipopolysaccharide.MethodsSepsis was induced in rabbits by intravenous injection of lipopolysaccharide. Contractility studies of rabbit duodenum were performed in an organ bath. Expressions of interleukin-1β, interleukin-6, interleukin-8, interleukin-10, IκB kinase-α, IκB kinase-β, IκB kinase-γ, and the proteasome mRNA were determined by RT-PCR on rabbit duodenum.ResultsNeomycin and polymyxin B (Toll-like receptor 4 inhibitors), IKK NBD peptide (IκB kinase complex inhibitor), and MG-132 (proteasome inhibitor) blocked partially the effects of lipopolysaccharide on the acetylcholine-, prostaglandin E2-, substance P-, and KCl-induced contractions in the longitudinal and circular smooth muscle of rabbit duodenum. Lipopolysaccharide increased the mRNA expression of interleukin-6 and interleukin-8 in duodenal tissue, and this effect was partly reversed by neomycin, polymyxin B, IKK NBD peptide, and MG-132. IκB kinase-α, IκB kinase-β, IκB kinase-γ, and the proteasome mRNA expressions was not affected by lipopolysaccharide treatment.ConclusionsToll-like receptor 4, the IκB kinase complex, and the proteasome could be therapeutic targets in the treatment of sepsis symptoms in the intestine.