Natural Product Chemistry and Biological Research.

Natural products are substances found in nature that have not been significantly modified by humans [...].

Involvement of NO/cGMP Signaling Pathway, Ca2+ and K+ Channels on Spasmolytic Effect of Everlasting Flower Polyphenolic Extract (Helichrysum stoechas (L.) Moench).

Functional gastrointestinal diseases (FGID) are worldwide prevalent conditions. Pharmacological treatments can be ineffective, leading the population to turn to herbal or traditional remedies. Helichrysum stoechas (L.) Moench is a medicinal plant traditionally used in the Iberian Peninsula to treat digestive disorders, but its effects on gastrointestinal motility have not been scientifically demonstrated. The aim of this work was to evaluate the antispasmodic effect of a polyphenolic extract of H. stoechas (HSM), its mechanism of action and its antioxidant activity. Isometric myography studies were performed in rat ileum, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) levels were measured in rat jejunum. HSM reduced the integrated mechanical activity of spontaneous contractions. In Ca2+-free medium, HSM reduced the concentration-response curve of CaCl2 similarly to verapamil. Pre-incubation with the extract blocked the contraction induced by Bay K8644, KCl and carbachol. L-NAME, ODQ, Rp-8-Br-PET-cGMPS, KT-5823, apamin, TRAM-34 and charybdotoxin reduced the relaxant effect of the extract on spontaneous contractions. MDA+4-HDA levels in LPS-treated tissue were reduced by the extract, showing antioxidant activity. In conclusion, HSM showed antispasmodic activity through inhibition of Ca2+ influx, activation of the NO/PKG/cGMP pathway and opening of Ca2+-activated K+ channels. The results suggest that H. stoechas could help in the prevention or treatment of FGIDs.

Intestinal serotonergic system is modulated by Toll-like receptor 9

Intestinal serotonergic system is a key modulator of intestinal homeostasis; however, its regulation is still unclear. Toll-like receptor 9 (TLR9), an innate immune receptor, detects different external agents in the intestine, preserving intestinal integrity. Since little is known about TLR9 role in the intestine, our aim was to address the potential regulation between TLR9 and intestinal serotonergic system. Caco-2/TC7 cell line and intestinal tract of Tlr9−/− mice were used in this study. Serotonin uptake studies were performed, and molecular expression of different serotonergic components was analyzed by western blot and real-time PCR. Our results show that TLR9 activation inhibits serotonin transporter activity and expression, involving p38/MAPK and ERK/MAPK intracellular pathways, and reciprocally, serotonin increases TLR9 expression. Supporting this interaction, serotonin transporter, serotonin receptors and serotonin producer enzymes were found altered in intestinal tract of Tlr9−/− mice. We conclude that TLR9 could contribute to intestinal homeostasis by modulation of intestinal serotonergic system. (AU)

Intestinal serotonergic system is modulated by Toll-like receptor 9.

Intestinal serotonergic system is a key modulator of intestinal homeostasis; however, its regulation is still unclear. Toll-like receptor 9 (TLR9), an innate immune receptor, detects different external agents in the intestine, preserving intestinal integrity. Since little is known about TLR9 role in the intestine, our aim was to address the potential regulation between TLR9 and intestinal serotonergic system. Caco-2/TC7 cell line and intestinal tract of Tlr9-/- mice were used in this study. Serotonin uptake studies were performed, and molecular expression of different serotonergic components was analyzed by western blot and real-time PCR. Our results show that TLR9 activation inhibits serotonin transporter activity and expression, involving p38/MAPK and ERK/MAPK intracellular pathways, and reciprocally, serotonin increases TLR9 expression. Supporting this interaction, serotonin transporter, serotonin receptors and serotonin producer enzymes were found altered in intestinal tract of Tlr9-/- mice. We conclude that TLR9 could contribute to intestinal homeostasis by modulation of intestinal serotonergic system.

The Potential Role of Everlasting Flower (Helichrysum stoechas Moench) as an Antihypertensive Agent: Vasorelaxant Effects in the Rat Aorta.

Helichrysum stoechas (L.) Moench (H. stoechas) is a medicinal plant traditionally used in the Iberian Peninsula to treat different disorders such as arterial hypertension. The aim of this study was to investigate the vascular effects of a polyphenolic methanolic extract of H. stoechas, which has high antioxidant activity, and its mechanism of action. Isometric myography studies were performed in an organ bath with rat aortic rings with intact endothelium. The H. stoechas extract produced vasorelaxation in the aortic rings that were precontracted by phenylephrine or KCl. L-NAME and Rp-8-Br-PET-cGMPS but not indomethacin or H-89; it also reduced the relaxant response evoked by H. stoechas extract on the phenylephrine-induced contractions. H. stoechas extract reduced the response to CaCl2 similar to verapamil and reduced the phenylephrine-induced contractions comparable with heparin. TRAM-34, apamin and glibenclamide reduced relaxation induced by the H. stoechas extract. The combination of L-NAME+TRAM-34+apamin almost completely inhibited the H. stoechas-induced effect. In conclusion, the relaxant effect of the H. stoechas extract is partially mediated by endothelium through the activation of the NO/PKG/cGMP pathway and the opening of Ca2+-activated K+ channels. Furthermore, the decrease in the cytosolic Ca2+ by the inhibition of Ca2+ influx through the L-type Ca2+ channels and by the reduction of Ca2+ release from the sarcoplasmic reticulum via the IP3 pathway is also involved.

Different mechanisms of actions of genistein, quercetin on spontaneous contractions of rabbit duodenum.

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.

Diferentes mecanismos de acción de la genisteína y quercetina en las contracciones espontáneas del duodeno de conejo / Different mechanisms of actions of genistein and quercetin on spontaneous contractions of rabbit duodenum

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 efecto de la quercetina actuaría sobre el AMPc y la proteína kinasa A

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

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

BACKGROUND: Lipopolysaccharide decreases intestinal contractility and induces the production of cytokines, which play an important role in the pathogenesis of sepsis. AIM: The 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. METHODS: Sepsis was induced in rabbits by intravenous injection of lipopolysaccharide. Contractility studies of rabbit duodenum were performed in an organ bath. Expressions of interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, IκB kinase-α, IκB kinase-ß, IκB kinase-γ, and the proteasome mRNA were determined by RT-PCR on rabbit duodenum. RESULTS: Neomycin 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-ß, IκB kinase-γ, and the proteasome mRNA expressions was not affected by lipopolysaccharide treatment. CONCLUSIONS: Toll-like receptor 4, the IκB kinase complex, and the proteasome could be therapeutic targets in the treatment of sepsis symptoms 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.

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

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(AU)

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 therole 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(AU)

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-1ß, 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.

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. METHODS: Segments of rabbit duodenum were suspended in the direction of longitudinal or circular smooth muscle fibres in a thermostatically controlled organ bath. KEY FINDINGS: LPS (0.3 µg/ml) reduced the contractions induced by ACh (100 µm) in the longitudinal and circular smooth muscle of the duodenum after 90 min of incubation. Polymyxin (TLR4 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor) and U0126 (MEK1/2 inhibitor) antagonized the effects of the LPS on ACh-induced contractions in duodenal smooth muscle. Incubation with the blockers of K(+) channels, TEA, apamin, charybdotoxin, iberiotoxin, glibenclamide or quinine, did not reverse the effect of LPS on ACh-induced contractions. CONCLUSIONS: These results suggest that the effect of LPS on ACh-induced contractions in the rabbit duodenum might be mediated by TLR4 and p38, JNK1/2 and MEK1/2 MAPKs.

Contractile effect of tachykinins on rabbit small intestine.

AIM: To study the role of the tachykinin receptors in spontaneous contractions of longitudinal and circular smooth muscle from rabbit small intestine and to determine the mechanism of action of Substance P (SP). METHODS: Rabbit duodenum, jejunum and ileum segments were prepared. The spontaneous contractions of longitudinal and circular smooth muscle were recorded using a computer via an isometric force transducer. The specific agonists and antagonists of tachykinin receptors were added into the organ bath. RESULTS: The agonists of tachykinin NK1 receptor (SP and [Sar9] SP), NK2 receptor (NKA and (ß-Ala8)-NKA), and NK3 receptor (NKB and Senktide) all induced contractions in the small intestine. The contractions were diminished by NK1 receptor antagonist L-733,060, NK2 receptor antagonist GR-94800, and NK3 receptor antagonist SB 218795. Contractions caused by SP were also reduced by atropine, verapamil, PKC inhibitor staurosporine, and PLC inhibitor U73122. CONCLUSION: Ttachykinin NK1, NK2, and NK3 receptors mediate the contractions of the smooth muscle in rabbit intestine. Furthermore, SP acts directly on smooth muscle cells through the tachykinin NK1 receptor.

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.

Intestinal effects of lipopolysaccharide in rabbit are mediated by cyclooxygenase-2 through p38 mitogen activated protein kinase.

The mediators of the pathophysiological symptoms of septic shock are not completely understood. The intracellular signalling mechanisms of lipopolysaccharide (LPS)-induced effects need further investigation. This study investigates (1) the role of COX-2 in the effect of LPS on (a) the KCl, acetylcholine and prostaglandin E2-induced contractions of rabbit duodenum and (b) the oxidative stress status in plasma and intestine and (2) the relationship between p38 MAPK and COX-2 expression in rabbit duodenum. Rabbits were injected i.v. with either (1) saline, (2) LPS, (3) SB203580, a p38 MAPK inhibitor, (4) SB203580+LPS, (5) NS-398, a COX-2 inhibitor or (6) NS-398+LPS. Contractility studies were performed by suspending pieces of duodenum in an organ bath in the direction of longitudinal and circular smooth muscle fibres. The formation of products of oxidative damage to proteins (carbonyls) and lipids [malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA)] was quantified in intestinal tissue and plasma. The protein expression of COX-2 was measured by western blot. The KCl, acetylcholine and prostaglandin E2-induced contractions decreased with LPS. In addition, LPS increased the levels of carbonyls and MDA+4-HDA in plasma and duodenum as well as COX-2 expression in duodenal tissue. All these effects were blocked by NS-398. The p38 MAPK inhibitor SB203580 blocked the effect of LPS on COX-2 expression. These results suggest that the effect of LPS on KCl, acetylcholine and prostaglandin E2-induced contractions in the rabbit duodenum and oxidative stress might be mediated by an increase in COX-2 expression through the p38 MAPK pathway.