Diverticular Disease Worsening Is Associated with Increased Oxidative Stress and Gut Permeability: New Insights by Circulating Biomarkers.

Diverticular disease (DD) management is impaired by its pathogenesis, which is still not completely defined, with an unmet clinical need for improved therapies. Ex vivo DD human models demonstrated the presence of a transmural oxidative imbalance that supports an ischemic pathogenesis. This study aimed to assess, with the use of circulating biomarkers, insights into DD pathogenesis and possible therapeutic targets. Nox2-derived peptide, H2O2, antioxidant capacity, isoprostanes, thromboxanes, TNF-α, LPS and zonulin were evaluated by ELISA in healthy subjects (HS) and asymptomatic and symptomatic DD patients. Compared to HS, DD patients presented low antioxidant capacity and increase in sNox2-dp, H2O2 and isoprostanes paralleled to a TNFα increase, lower than that of oxidative markers. TxB2 production correlated to Nox2 and isoprostanes, suggesting platelet activation. An increase in zonulin and LPS highlighted the role of gut permeability and LPS translocation in DD pathogenesis. The increase of all the markers statistically correlated with DD severity. The present study confirmed the presence of a main oxidative imbalance in DD and provides evidence of platelet activation driven by LPS translocation. The use of circulating biomarkers could represent a new clinical tool for monitoring disease progression and validate therapeutic strategies never tested in DD as antioxidant supplementation.

Pharmacological effects of Pugionium cornutum (L.) Gaertn. extracts on gastrointestinal motility are partially mediated by quercetin.

BACKGROUND: The majority of global population suffer from various functional gastrointestinal disorders. Pugionium cornutum (L.) Gaertn. (PCG) is used to relieve indigestive symptoms in traditional Chinese medicine. However, little is known about the effects of bioactive components from PCG extracts on gastrointestinal motility. METHODS: Crude ethanol extract of PCG (EEP) was prepared from Pugionium cornutum (L.) Gaertn. Different solvents were used to prepare fine extracts from EEP, including water extract of PCG (WEP), petroleum ether extract of PCG (PEEP), dichloromethane extract of PCG (DEP) and ethyl acetate extract of PCG (EAEP). Smooth muscle cell model and colonic smooth muscle stripe model were used to test the bioactive effects and mechanisms of different PCG extracts on contraction and relaxation. Diverse chromatographic methods were used to identify bioactive substances from PCG extracts. RESULTS: EEP was found to promote the relaxation of gastric smooth muscle cell and inhibit the contraction of colonic smooth muscle strip. Among the fractions of EEP, EAEP mainly mediated the relaxation effect by stimulating intracellular calcium influx. Further evidences revealed that EAEP was antagonistic to acetylcholine. In addition, COX and NO-GC-PKC pathways may be also involved in EAEP-mediated relaxation effect. Quercetin was identified as a bioactive compound from PCG extract for the relaxation effect. CONCLUSION: Our research supports the notion that PCG extracts promote relaxation and inhibits contraction of gastrointestinal smooth muscle at least partially through the effect from quercetin.

Amelioration of gut dysbiosis and gastrointestinal motility by konjac oligo-glucomannan on loperamide-induced constipation in mice.

OBJECTIVE: Konjac oligo-glucomannan (KOG) is a non-digestible dietary fiber that is resistant to digestion and absorption in gastrointestinal (GI) tract. Thus, it might be used as an alternative management for constipation. The aim of this study was to evaluate the effects of KOG on gut motility and microbiota to relieve constipation in mice. METHODS: Mice received Bifidobacterium animalis, lactulose, konjac glucomannan (KGM), or KOG for 14 d. Constipation was induced by 5 mg/kg loperamide days 12 through 14 in all groups except the control. Defecation frequency, small intestinal transit, and total gut transit time were indicated by counting the number of feces, and using charcoal meal and Evans blue as markers, respectively. Smooth muscle (SM) contraction and gut motility were evaluated by organ bath and GI motility monitor system. Gut microbiota were measured by fluorescence in situ hybridization technique. RESULTS: KOG significantly (P < 0.01) increased defecation frequency and small intestinal transit but decreased total gut transit time when compared with the constipation-without-treatment group. These results were similar to the effects of Bifidobacterium animalis, lactulose, and KGM. KOG ameliorated the effect of loperamide on contraction frequency of distal colonic circular SM. The motility patterns were changed in the KOG group from non-propagation to propagation contraction. KOG significantly inhibited the effects of loperamide on gut microbiota by increasing the numbers of Bifidobacterium spp. and decreasing the numbers of Clostridium spp. and Bacteroides spp. CONCLUSION: These results suggest that KOG acts as a prebiotic and stimulant laxative for relief and prevention of constipation.

Altered ryanodine receptor gene expression in Hirschsprung's disease.

AIM OF THE STUDY: Ryanodine receptors are the largest of all ion channels, named after their exogenous ligand, ryanodine. The ryanodine receptor calcium release channel is central to cytoplasmic Ca2+ signalling in skeletal muscle, the heart, and many other tissues, playing a vital role in muscular contraction. Three ryanodine receptors exist, Ryr1, Ryr2 and Ryr3. The ryanodine receptor, Ryr3, is encoded by the Ryr3 gene, which has been reported to be highly specific to colonic smooth muscle cells in mice. We designed this study to investigate Ryr1, Ryr2 and Ryr3 gene expression in the normal human colon and in Hirschsprung's disease (HSCR). METHODS: HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify Ryr1, Ryr2 and Ryr3 gene expression, and immunolabelling of Ryr1, Ryr2 and Ryr3 proteins was visualised using confocal microscopy. MAIN RESULTS: qRT-PCR analysis revealed a significant downregulation of the Ryr1 and Ryr3 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed Ryr1, Ryr2 and Ryr3 protein expressions within the smooth muscle, with a reduction in aganglionic and ganglionic HSCR colon compared to controls. CONCLUSIONS: Ryr1 and Ryr3 gene expression is significantly downregulated in HSCR colon, suggesting a role for these genes in colonic smooth-muscle motility. Ryr1 and Ryr3 downregulations within ganglionic specimens highlight the physiologically abnormal nature of this segment which may explain the occurrence of persistent bowel symptoms in some HSCR patients following a properly performed pull-through operation.

Prebiotic oligosaccharides from dragon fruits alter gut motility in mice.

Dragon fruit oligosaccharide (DFO) has a prebiotic property which improves gut health by selectively stimulating the colonic microbiota. Altering microbiota composition may affect intestinal motility. However, no study has been done to understand the DFO effects on gut motor functions. This research thus aimed to investigate the DFO effects on mice colons compared to the prebiotic fructo-oligosaccharide (FOS) and probiotic bifidobacteria. The mice in this study received distilled water; 100, 500, and 1000 mg/kg DFO; 1000 mg/kg FOS; or 109 CFU Bifidobacterium animalis daily for 1 week and some treatments for 2 weeks. Gastrointestinal transits were analysed and motility patterns, smooth muscle (SM) contractions and morphological structures of the colons were assessed. Administration of FOS, 500 and 1000 mg/kg DFO significantly increased fecal output when compared to the control group. In mice treated with FOS and bifidobacteria, gut transit time was reduced, while upper gut transit was increased in comparison to DFO groups. Spatiotemporal maps of colonic wall motions showed that DFO increased the number of colonic non-propagation contractions and fecal pellet velocity, consistent with the results from groups treated with FOS and bifidobacteria. DFO also increased the amplitude and duration of colonic SM contractions. Histological stains showed normal epithelia, crypts, goblet cells, and SM thickness in all groups. In conclusion, DFO increased colonic SM contractions without morphological change and acted as a bulk-forming and stimulant laxative to increase fecal output and intestinal motility. Thus, DFO as a dietary supplement may promote gut health and correct gastrointestinal motility disorders.

Persistent mechanical stretch-induced calcium overload and MAPK signal activation contributed to SCF reduction in colonic smooth muscle in vivo and in vitro.

Gastrointestinal (GI) distention is a common pathological characteristic in most GI motility disorders (GMDs), however, their detail mechanism remains unknown. In this study, we focused on Ca2+ overload of smooth muscle, which is an early intracellular reaction to stretch, and its downstream MAPK signaling and also reduction of SCF in vivo and in vitro. We successfully established colonic dilation mouse model by keeping incomplete colon obstruction for 8 days. The results showed that persistent colonic dilation clearly induced Ca2+ overload and activated all the three MAPK family members including JNK, ERK and p38 in smooth muscle tissues. Similar results were obtained from dilated colon of patients with Hirschsprung's disease and stretched primary mouse colonic smooth muscle cells (SMCs). Furthermore, we demonstrated that persistent stretch-induced Ca2+ overload was originated from extracellular Ca2+ influx and endoplasmic reticulum (ER) Ca2+ release identified by treating with different Ca2+ channel blockers, and was responsible for the persistent activation of MAPK signaling and SCF reduction in colonic SMCs. Our results suggested that Ca2+ overload caused by smooth muscle stretch led to persistent activation of MAPK signaling which might contribute to the decrease of SCF and development of the GMDs.

In vitro spontaneous contractile activity of colonic smooth muscle in naive Lewis rats: Acute effect of gonadal hormones.

OBJECTIVE: Functional gastrointestinal disorders affect females more often. Changes in colonic motility may be etiological co-factors for the clinical symptoms. The aim of the present study was to analyze the influence of gonadal hormones on colonic contractile activity. METHODS: In vitro measurements of colonic contractile activity in longitudinal smooth muscle strips of female and male Lewis rats were performed in an organ chamber experiment. After the administration of a gonadal hormone estradiol [EST], progesterone [PROG] and testosterone [TEST]) or ethanol solution as control, stimulation with acetylcholine (ACh) or inhibition with norepinephrine (NE) was performed. RESULTS: Compared to the smooth muscle strips of male rats, significantly higher spontaneous colonic contractile activity (SCCA) was observed in female animals. Increasing doses of ACh showed the progressive stimulation of SCCA whereas rising doses of NE resulted in a stepwise inhibition of SCCA, respectively. EST superfusion displayed an inhibitory effect on SCCA in both sexes and inhibited the ACh effect in female rats. Similarly, acute superfusion with high-dose PROG inhibited SCCA in females. Acute TEST superfusion inhibited SCCA in males and led to significant higher colonic contractile activity in males following subsequent stimulation with ACh. In female rats, the inhibitory effect of NE was reduced by prior exposure to TEST. CONCLUSION: In our in vitro study the acute exposure of colonic smooth muscle tissue to gonadal hormones led to sex-dependent changes in SCCA and translated in a modified response of smooth muscle strips to both pro-contractile and anti-contractile neurotransmitters.

Potential role of the gaseous mediator hydrogen sulphide (H2S) in inhibition of human colonic contractility.

BACKGROUND: Hydrogen sulphide (H2S) is an endogenous signalling molecule that might play a physiologically relevant role in gastrointestinal motility. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are two enzymes responsible for H2S production. d,l-Propargylglycine (PAG) is a CSE inhibitor whereas both aminooxyacetic acid (AOAA) and hydroxylamine (HA) are CBS inhibitors. The characterization of H2S responses and its mechanism of action are crucial to define H2S function. METHODS: Human colonic strips were used to investigate the role of H2S on contractility (muscle bath) and smooth muscle electrophysiology (microelectrodes). NaHS was used as a H2S donor. RESULTS: Combination of PAG and AOAA depolarized the smooth muscle (5-6mV, n=4) and elicited a transient increase in tone (260.5±92.8mg, n=12). No effect was observed on neural mediated inhibitory junction potential or relaxation. In the presence of tetrodotoxin 1µM, NaHS concentration-dependently inhibited spontaneous contractions (EC50=329.2µM, n=18). This effect was partially reduced by the guanylyl cyclase inhibitor ODQ 10µM (EC50=2.6µM, n=12) and by l-NNA 1mM (EC50=1.4mM, n=8). NaHS reversibly blocked neural mediated cholinergic (EC50=2mM) and tachykinergic (EC50=5.7mM) contractions. NaHS concentration-dependently reduced the increase in spontaneous mechanical activity (AUC) induced by carbachol (EC50=1.9mM) and NKA (EC50=1.7mM AUC). CONCLUSIONS: H2S might be an endogenous gasomediator regulating human colonic contractility. Its inhibitory effect is observed at high concentrations and could be mediated by a direct effect on smooth muscle with a possible synergistic effect with NO, as well as by an interaction with the cholinergic and tachykinergic neural mediated pathways.

EP2 and EP4 receptors mediate PGE2 induced relaxation in murine colonic circular muscle: pharmacological characterization.

BACKGROUND: Prostaglandin E2 (PGE2) is a regulator of gastrointestinal motility that might be involved in impaired motor function associated to gut inflammation. The aim of the present work is to pharmacologically characterize responses to exogenous and endogenous PGE2 in the mouse colon targeting EP2 and EP4 receptors. METHODS: Wild type (WT) and EP2 receptor knockout (EP2-KO) mice were used to characterize PGE2 and butaprost (EP2 receptor agonist) effects on smooth muscle resting membrane potential and myogenic contractility in circularly oriented colonic preparations. RESULTS: In WT animals, PGE2 and butaprost concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. Combination of both EP2 (PF-04418948 0.1µM) and EP4 receptor antagonists (L-161,982 10µM) was needed to block both electrical and mechanical PGE2 responses. Butaprost inhibitory responses (both electrical and mechanical) were totally abolished by PF-04418948 0.1µM. In EP2-KO mice, PGE2 (but not butaprost) concentration-dependently inhibited spontaneous contractions and hyperpolarized smooth muscle cells. In EP2-KO mice, PGE2 inhibition of spontaneous contractility and hyperpolarization was fully antagonized by L-161,982 10µM. In WT animals, EP2 and EP4 receptor antagonists caused a smooth muscle depolarization and an increase in spontaneous mechanical activity. CONCLUSIONS: PGE2 responses in murine circular colonic layer are mediated by post-junctional EP2 and EP4 receptors. PF-04418948 and L-161,982 are selective EP2 and EP4 receptor antagonists that inhibit PGE2 responses. These antagonists might be useful pharmacological tools to limit prostaglandin effects associated to dismotility in gut inflammatory processes.

Experimental evidence and mathematical modeling of thermal effects on human colonic smooth muscle contractility.

It has been shown, in animal models, that gastrointestinal tract (GIT) motility is influenced by temperature; nevertheless, the basic mechanism governing thermal GIT smooth muscle responses has not been fully investigated. Studies based on physiologically tuned mathematical models have predicted that thermal inhomogeneity may induce an electrochemical destabilization of peristaltic activity. In the present study, the effect of thermal cooling on human colonic muscle strip (HCMS) contractility was studied. HCMSs were obtained from disease-free margins of resected segments for cancer. After removal of the mucosa and serosa layers, strips were mounted in separate chambers. After 30 min, spontaneous contractions developed, which were measured using force displacement transducers. Temperature was changed every hour (37, 34, and 31°C). The effect of cooling was analyzed on mean contractile activity, oscillation amplitude, frequency, and contraction to ACh (10(-5) M). At 37°C, HCMSs developed a stable phasic contraction (~0.02 Hz) with a significant ACh-elicited mean contractile response (31% and 22% compared with baseline in the circular and longitudinal axis, respectively). At a lower bath temperature, higher mean contractile amplitude was observed, and it increased in the presence of ACh (78% and 43% higher than the basal tone in the circular and longitudinal axis, respectively, at 31°C). A simplified thermochemomechanical model was tuned on experimental data characterizing the stress state coupling the intracellular Ca(2+) concentration to tissue temperature. In conclusion, acute thermal cooling affects colonic muscular function. Further studies are needed to establish the exact mechanisms involved to better understand clinical consequences of hypothermia on intestinal contractile activity.

Lactobacillus rhamnosus protects human colonic muscle from pathogen lipopolysaccharide-induced damage.

BACKGROUND: Lactobacillus species might positively affect gastrointestinal motility. These Gram-positive bacteria bind Toll-like receptor 2 (TLR2) that elicits anti-inflammatory activity and exerts protective effects on damage induced by lipopolysaccharide (LPS). Whether such effect occurs in gastrointestinal smooth muscle has not been established yet. Aim of this study was to characterize the effects of Lactobacillus rhamnosus GG (LGG) and of supernatants harvested from LGG cultures on human colonic smooth muscle and to explore their protective activity against LPS-induced myogenic morpho-functional alterations. METHODS: The effects of LGG (ATCC 53103 strain) and of supernatants have been tested on both human colonic smooth muscle strips and isolated cells in the absence or presence of LPS obtained from a pathogenic strain of Escherichia coli. Their effects on myogenic morpho-functional properties, on LPS-induced NFκB activation, and on cytokine production have been evaluated. Toll-like receptor 2 expression has been analyzed by qPCR and flow cytometry. KEY RESULTS: Lactobacillus rhamnosus GG exerted negligible transient effects per se whereas it was capable of activating an intrinsic myogenic response counteracting LPS-induced alterations. In particular, both LGG and supernatants significantly reduced the LPS-induced morpho-functional alterations of muscle cells, i.e. cell shortening and inhibition of contractile response. They also hindered LPS-induced pro-inflammatory effects by decreasing pro-inflammatory transcription factor NFκB activation and pro-inflammatory cytokine IL-6 secretion, and restored the secretion levels of anti-inflammatory cytokine IL10. CONCLUSIONS & INFERENCES: Taken together these data demonstrate that LGG protects human colonic smooth muscle from LPS-induced myogenic damage and might be beneficial on intestinal motor disorders due to bacterial infection.

HEF-19-induced relaxation of colonic smooth muscles and the underlying mechanisms.

AIM: To investigate the relaxant effect of chromane HEF-19 on colonic smooth muscles isolated from rabbits, and the underlying mechanisms. METHODS: The relaxant effect and action mechanisms of HEF-19 were investigated using descending colon smooth muscle of the rabbits. Preparations 1 cm long were mounted in 15-mL tissue baths containing Tyrode's solution, maintained at 37 ± 0.5 °C and aerated with a mixture of 5% CO2 in oxygen (Carbogen). The tension and amplitude of the smooth muscle strips were recorded after adding HEF-19 (10(-6), 10(-5) and 10(-4) mol/L). After cumulative administration of four antispasmodic agents, including acetylcholine chloride (Ach) (10(-4) mol/L), histamine (10(-4) mol/L), high-K(+) (60 mmol/L) and BaCl2 (8.2 mmol/L), HEF-19 (3 × 10(-7)-3 × 10(-4) mol/L) was added to investigate the relaxant effect of HEF-19. CaCl2 (10(-4)-2.5 × 10(-3) mol/L) was added cumulatively to the smooth muscle preparations pretreated with and without HEF-19 (1 × 10(-6) or 3 × 10(-6) mol/L) and verapamil (1 × 10(-7) mol/L) to study the mechanisms involved. Finally, phasic contraction was induced with ACh (15 × 10(-6) mol/L), and CaCl2 (4 × 10(-3) mol/L) was added to the smooth muscle preparations pretreated with and without HEF-19 (3 × 10(-6) mol/L or 1 × 10(-5) mol/L) and verapamil (1 × 10(-7) mol/L) in calcium-free medium to further study the underlying mechanisms. RESULTS: HEF-19 (1 × 10(-6), 1 × 10(-5) and 1 × 10(-4) mol/L) suppressed spontaneous contraction of rabbit colonic smooth muscles. HEF-19 (3 × 10(-7)-3 × 10(-4) mol/L) relaxed in a concentration-dependent manner colonic smooth muscle preparations pre-contracted with BaCl2, high-K(+) solution, Ach or histamine with respective EC50 values of 5.15 ± 0.05, 5.12 ± 0.08, 5.58 ± 0.16 and 5.25 ± 0.24, thus showing a spasmolytic activity. HEF-19 (1 × 10(-6) mol/L and 3 × 10(-6) mol/L) shifted the concentration-response curves of CaCl2 to the right and depressed the maximum response to CaCl2. The two components contracted by Ach were attenuated with HEF-19 (3 × 10(-6) mol/L or 10(-5) mol/L) in calcium-free medium. CONCLUSION: HEF-19 inhibited rabbit colonic smooth muscle contraction, probably through inhibiting opening of voltage-dependent Ca(2+) channels. HEF-19 reduced inflow and intracellular release of Ca(2+) ions.

Effects of Tamoxifen on the Voltage-dependent Ionic Currents in Mouse Colonic Smooth Muscle Cells / 대한소화기학회지

BACKGROUND/AIMS: Tamoxifen is a widely used anticancer drug for breast cancer with frequent gastrointestinal side effects. Changes in gastrointestinal motility is associated with altered activities of membrane ion channels. Ion channels have important role in regulating membrane potential and cell excitability. This study was performed to investigate the effects of tamoxifen on the membrane ionic currents in colonic smooth muscle cells. METHODS: Murine colonic smooth muscle cells were isolated from the proximal colon using collagenase, and the membrane currents were recorded using a whole-cell patch clamp technique. RESULTS: Two types of voltage-dependent K+ currents were recorded (A-type and delayed rectifier K+ currents). Tamoxifen inhibited both types of voltage-dependent K+ currents in a dose-dependent manner. However, tamoxifen did not change the half-inactivation potential and the recovery time of voltage-dependent K+ currents. Chelerythrine, a protein kinase C inhibitor or phorbol 12, 13-dibutyrate, a protein kinase C activator did not affect the voltage-dependent K+ currents. Guanosine 5'-O-(2-thio-diphosphate) did not affect the tamoxifen-induced inhibition of voltage-dependent K+ currents. Tamoxifen inhibited voltage-dependent Ca2+ currents completely in whole-test ranges. CONCLUSIONS: These results suggest that tamoxifen can alter various membrane ionic currents in smooth muscle cells and cause some adverse effects on the gastrointestinal motility.

Effects of berberine on potassium current of guinea pig colonic smooth muscle cells / 中国药理学通报

AIM To clarify the mechanism of berberine on mobility diarrhea by investigating the effects of berberine on calcium-activated potassium current I K(Ca) and delayed-rectifier potassium channel current I K(V) of guinea pig colonic smooth muscle cells. METHODS Single guinea pig colonic smooth muscle cells was isolated by collagenase; The effects of berberine on I K(Ca) and I K(V) were detected by using patch clamp technique, under the conventional whole cell patch clamp mode. RESULTS 10, 50, 100 ?mol?L -1 berberine inhibited I K(Ca) of guinea pig colonic smooth muscle cells significantly (P