Delay In Gastrointestinal Transit Research Articles

Piezo1 mediated calcium-calcineurin signaling pathway regulates gastrointestinal smooth muscle cell phenotype in a stiffness dependent manner

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A polyphenol-rich açaí seed extract protects against 5-fluorouracil-induced intestinal mucositis in mice through the TLR-4/MyD88/PI3K/mTOR/NF-κBp65 signaling pathway

Açaí seed extract (ASE) is obtained from Euterpe oleracea Mart. (açaí) plant (Amazon region) has high nutritional and functional value. ASE is rich in polyphenolic compounds, mainly proanthocyanidins. Proanthocyanidins can modulate the immune system and oxidative stress by inhibiting the toll-like receptor-4 (TLR-4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) pathway. A great deal of evidence suggests that inflammatory cytokines and oxidative stress contribute to the pathogenesis of intestinal mucositis, and these events can lead to intestinal dysmotility. We hypothesized that ASE acts as an anti-inflammatory and antioxidant compound in intestinal mucositis induced by 5-fluorouracil (5-FU) through modulation of the TLR-4/MyD88/phosphatidylinositol-3-kinase α/mechanistic target of rapamycin/NF-κBp65 pathway. The animals were divided into linear 5-FU (450 mg/kg) and 5-FU + ASE (10, 30, and 100 mg/kg) groups. The weight loss of the animals was evaluated daily. Samples from duodenum, jejunum, and ileum were obtained for histopathological, biochemical, and functional analyses. ASE reduced weight loss, inflammatory parameters (interleukin-1β; tumor necrosis factor-α; myeloperoxidase activity) and the gene expression of mediators involved in the TLR-2/MyD88/NF-κB pathway. ASE prevented histopathological changes with beneficial effects on gastrointestinal transit delay, gastric emptying, and intestinal absorption/permeability. In conclusion, ASE protects the integrity of the intestinal epithelial barrier by inhibiting the TLR/MyD88/PI3K/mechanistic target of rapamycin/NF-κBp65 pathway.

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility

Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Slowed gastrointestinal transit is associated with an altered caecal microbiota in an aged rat model.

Gastrointestinal (GI) motility is largely dependent upon activity within the enteric nervous system (ENS) and is an important part of the digestive process. Dysfunction of the ENS can impair GI motility as is seen in the case of constipation where gut transit time is prolonged. Animal models mimicking symptoms of constipation have been developed by way of pharmacological manipulations. Studies have reported an association between altered GI motility and gut microbial population. Little is known about the changes in gut microbiota profile resulting specifically from pharmacologically induced slowed GI motility in rats. Moreover, the relationship between gut microbiota and altered intestinal motility is based on studies using faecal samples, which are easier to obtain but do not accurately reflect the intestinal microbiome. The aim of this study was to examine how delayed GI transit due to opioid receptor agonism in the ENS modifies caecal microbiota composition. Differences in caecal microbial composition of loperamide-treated or control male Sprague Dawley rats were determined by 16S rRNA gene amplicon sequencing. The results revealed that significant differences were observed at both genus and family level between treatment groups. Bacteroides were relatively abundant in the loperamide-induced slowed GI transit group, compared to controls. Richness and diversity of the bacterial communities was significantly lower in the loperamide-treated group compared to the control group. Understanding the link between specific microbial species and varying transit times is crucial to design interventions targeting the microbiome and to treat intestinal motility disorders.

Acute Endothelial Cell Dysfunction Caused by a Protein Extract from Damaged Skeletal Muscle

INTRODUCTION: Trauma is the fourth leading cause of death. “DAMPs” initiate a delayed SIRS. However, the blockade of inflammatory responses only minimally alters mortality. Our aim was to investigate the acute effects of a select protein extract from damaged skeletal muscle on endothelial cell function. METHODS: A murine trauma model was constructed consisting of the dorsal subcutaneous implantation of a syngeneic donor skeletal muscle matrix. A specific skeletal muscle matrix protein extract was isolated by centrifugation and size exclusion. Animals were studied immediately or 24 hours after resuscitation. Intestinal microvascular leak was assessed by vessel fluorophore illuminated confocal microscopy. Endothelial cell dysfunction was studied in human umbilical vein endothelial cells, fura-2–labeled human intestinal microvascular endothelial cells, and GCamp8 jejunal vessel endothelial cells. Gastrointestinal transit assays generated distribution histograms and calculated geometric centers (n = 3 to 4). RESULTS: Muscle matrix 15% body weight and protein extract IV injection (100 µL) caused a perfuse microvascular leak of the 70-kDa fluorophore and additionally caused a significant delay in gastrointestinal transit (GC = 4.8 ± 0.89 and 4.3 ± 1.34). Extract IV injection greater than 100 µL caused death without recovering mobility. Human umbilical vein endothelial cells acutely contracted by 20% to the trauma extract. Human intestinal microvascular endothelial cells incubated 6 hours in 25% normal human serum developed robust rapid calcium transients to platelet activating factor (50 nM). Human intestinal microvascular endothelial cells incubated in human trauma serum had slowly developing, muted transients to platelet aggregating factor. GCamp8 endothelial cell calcium sparks acutely increased in frequency on trauma extract exposure. CONCLUSION: Damaged skeletal muscle releases a protein that acutely causes endothelial cell dysfunction leading to microvascular leak, ileus, and a precipitous death.

Mas-related G protein-coupled receptor D is involved in modulation of murine gastrointestinal motility.

What is the central question of this study? The physiological function of Mas-related G protein-coupled receptor D (MrgprD) in gastrointestinal motility is unknown. The aim of this study was to assess the effects of MrgprD and its receptor agonists on murine gastrointestinal motility. What is the main finding and its importance? Mrgprd deficiency improved murine gastrointestinal motility in vivo but had no effects on the spontaneous contractions of murine intestinal rings ex vivo. Systemic administration of the MrgprD ligand, either β-alanine or alamandine, delayed gastrointestinal transit in vivo and attenuated the spontaneous contractions of isolated intestinal rings ex vivo. Mas-related G protein-coupled receptor D (MrgprD) was first identified in sensory neurons of mouse dorsal root ganglion and has been demonstrated to be involved in sensations of pain and itch. Although expression of MrgprD has recently been found in the gastrointestinal (GI) tract, its physiological role in GI motility is unknown. To address this question, we used Mrgprd knockout (Mrgprd-/- ) mice and MrgprD agonists to examine the effects of Mrgprd gene deletion and MrgprD signalling activation, respectively, on murine intestinal motility, both in vivo and ex vivo. We observed that the deletion of Mrgprd accelerated the transmission of charcoal through the mouse GI tract. But Mrgprd deficiency did not affect the mean amplitudes and frequencies of spontaneous contractions in ileum ex vivo. Colonic motor complexes in the proximal and the distal colon were recorded from wild-type and Mrgprd-/- mice, but their control frequencies were not different. Moreover, in wild-type mice, systemic administration of an MrgprD agonist, either β-alanine or alamandine, delayed GI transit in vivo and suppressed spontaneous contractions in the ileum and colonic motor complexes in the colon ex vivo. Our results suggest that MrgprD and its agonist are involved in the modulation of GI motility in mice.

Corticotrophin-releasing factor-mediated effects of DA-9701 in Postoperative Ileus Guinea Pig Model.

Postoperative ileus (POI) is abdominal surgery-induced impaired gastrointestinal (GI) motility. We aimed to investigate the effects of DA-9701, a prokinetic agent formulated from Pharbitis Semen and Corydalis tuber, likely mediated via corticotrophin-releasing factor (CRF) pathways, in a POI model. A laparotomy with cecal manipulation was performed to induce POI in guinea pigs. GI transit was measured based on charcoal migration after intragastric administration of DA-9701 1, 3, and 10mgkg-1 . CRF1 receptor antagonist, CP-154526 (subcutaneous) or agonist, human/rat (h/r) CRF (intraperitoneal) was injected. Then, plasma adrenocorticotropic hormone (ACTH) levels were measured, and the average intensity of the CRF expression was analyzed in the proximal colon and hypothalamus, and c-Fos in the hypothalamus. DA-9701 significantly increased delayed GI transit in POI in a dose-dependent manner and decreased plasma ACTH levels at 10mgkg-1 . CP-154526 significantly decreased plasma ACTH levels but was not as effective on GI transit as DA-9701 was. h/r CRF did not significantly affect GI transit and plasma ACTH levels. No significant difference was observed in GI transit and plasma ACTH levels in both groups administered DA-9701 with h/r CRF and h/r CRF alone. CRF expression in the proximal colon decreased after DA-9701 administration, but not significantly, compared with levels in POI alone. However, CRF expression in the hypothalamus was significantly lower in the DA-9701-pretreated POI than in the untreated POI. The DA-9701-induced improvement in GI transit and inhibition of plasma ACTH levels was mediated by the central CRF pathway.

Evaluation of the effect of GLP‐1 agonists on quality of bowel preparation for colonoscopy in patients with diabetes

AbstractPeople with diabetes mellitus warrant a greater need for colonoscopy owing to the increased risk of colorectal cancer and autonomic neuropathy. Unfortunately, this population is reported to have a higher proportion of inadequate bowel preparation during colonoscopy, which translates into lower adenoma detection rates and repeat colonoscopies. Glucagon‐like peptide‐1 (GLP‐1) agonists commonly used in the management of diabetes are well known to cause delayed gastrointestinal (GI) transit times, yet it is unknown if they contribute to the higher frequency of inadequate bowel preparation in people with diabetes.Therefore, we conducted a retrospective medical record analysis of type 2 diabetic patients aged 45–75 who had an outpatient colonoscopy between 2012 and 2015. Our patients were taking either Byetta, Victoza or Bydureon and the method of bowel preparation used was polyethylene glycol electrolyte lavage solution. Patients who had a diagnosis of conditions known to cause delayed GI transit times (autonomic neuropathy, GI surgery, stroke and multiple sclerosis) were excluded. The final pool consisted of 126 diabetic patients taking a GLP‐1 agonist and a control group of 129 diabetic patients who were not taking this class of medication. The percentage of satisfactory bowel preparations in the group taking a GLP‐1 agonist was 92.06% and in the control group was 92.25%. Chi‐squared p‐value for significant difference between these two groups was p = 1.0 (95% confidence interval −0.066 to 0.069). Hence there were no significant differences in bowel preparation among diabetic patients taking GLP‐1 agonists vs the control group; GLP‐1 agonists can therefore continually be used according to the current standard protocols for colonoscopy before and after colonoscopy. Copyright © 2017 John Wiley & Sons.

Development of A Novel Murine Model of Combined Radiation and Peripheral Tissue Trauma Injuries.

Detonation of a 10-kiloton nuclear bomb in an urban setting would result in >1 million casualties, the majority of which would present with combined injuries. Combined injuries, such as peripheral tissue trauma and radiation exposure, trigger inflammatory events that lead to multiple organ dysfunction (MOD) and death, with gastrointestinal (GI) and pulmonary involvement playing crucial roles. The objective of this study was to develop an animal model of combined injuries, peripheral tissue trauma (TBX animal model) combined with total body irradiation with 5% bone marrow shielding (TBI/BM5) to investigate if peripheral tissue trauma contributes to reduced survival. Male C57BL/6J mice were exposed to TBX10%, irradiation (TBI/BM5), or combined injuries (TBX10% + TBI/BM5). Experiments were conducted to evaluate mortality at day 7 after TBI/BM5. Serial euthanasia was performed at day 1, 3 and 6 or 7 after TBI/BM5 to evaluate the time course of pathophysiologic processes in combined injuries. Functional tests were performed to assess pulmonary function and GI motility. Postmortem samples of lungs and jejunum were collected to assess tissue damage. Results indicated higher lethality and shorter survival in the TBX10% +T BI/BM5 group than in the TBX10% or TBI/BM5 groups (day 1 vs. day 7 and 6, respectively). TBI/BM5 alone had no effects on the lungs but significantly impaired GI function at day 6. As expected, in the animals that received severe trauma (TBX10%), we observed impairment in lung function and delay in GI transit in the first 3 days, effects that decreased at later time points. Trauma combined with radiation (TBX10% + TBI/BM5) significantly augmented impairment of the lung and GI function in comparison to TBX10% and TBI/BM5 groups at 24 h. Histologic evaluation indicated that combined injuries caused greater tissue damage in the intestines in TBX10% + TBI/BM5 group when compared to other groups. We describe here the first combined tissue trauma/radiation injury model that will allow conduction of mechanistic studies to identify new therapeutic targets and serve as a platform for testing novel therapeutic interventions.

The Effect of DA-9701 in Opioid-induced Bowel Dysfunction of Guinea Pig.

Background/AimsOpioid induced bowel dysfunction (OIBD) is associated with decreased gastrointestinal (GI) propulsive activity due to intake of opioid analgesics. DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber has promising effects on GI motor function. Therefore, we aim to evaluate the prokinetic effects of DA-9701 in an OIBD model of guinea pig.MethodsThe ileal and distal colon muscle contraction in presence of different doses of DA-9701, morphine, and combination (morphine + DA-9701) was measured by tissue bath study. The prokinetic effect of DA-9701 was assessed by charcoal transit and fecal pellet output assay in an OIBD model of guinea pig.ResultsDA-9701 significantly increased the amplitude and area under the curve of ileal muscle contraction, while there was insignificant effect on the distal colon compared to the control. The maximal amplitude of ileal muscle contraction was acquired at a concentration of 10 μg/mL of DA-9701. In contrast, morphine significantly decreased the amplitude of ileal and distal colon muscle contraction compared to the control. Morphine delayed both upper (P < 0.01) and lower (P < 0.05) GI transit, and delayed GI transit was restored by the administration of DA-9701. Morphine induced reduction of contractility was significantly ameliorated by addition of DA-9701 in both ileal and distal colon muscles.ConclusionsDA-9701 significantly increased the amplitude of contraction of the ileal muscle, however the distal colon muscle contraction was insignificant. Additionally, it restored delayed upper and lower GI transit in an OIBD model of guinea pig, and it might prove to be a useful candidate drug in a clinical trial for OIBD.

The Wireless Motility Capsule: a One-Stop Shop for the Evaluation of GI Motility Disorders.

The wireless motility and pH capsule (WMC) provides an office-based test to simultaneously assess both regional and whole gut transit. Ingestion of this non-digestible capsule capable of measuring temperature, pH, and the pressure of its immediate surroundings allows for the measurement of gastric, small bowel, and colonic transit times in an ambulatory setting. Approved by the US Food and Drug Administration for the evaluation of suspected conditions of delayed gastric emptying and the evaluation of colonic transit in chronic idiopathic constipation, WMC should be considered in suspected gastrointestinal motility disorders as it provides a single study capable of simultaneously assessing for regional, multiregional, or generalized motility disorders. Specific indications for testing with the WMC should include the evaluation of suspect cases of gastroparesis, small bowel dysmotility, and slow transit constipation, as well as symptom syndromes suggestive of a multiregional or generalized gastrointestinal transit delay.

Surgical intestinal manipulation increases gene expression of TrkA, CGRP, and PAR-2 IN dorsal root ganglia in the rat.

Surgical handling of the bowel evokes degranulation of peritoneal mast cells (PMC). Nonetheless, role of PMCs in postoperative ileus (POI) is somewhat controversial. We aimed to investigate if intestinal manipulation elicits changes in afferent mediators related to MC activation and alteration of gastrointestinal (GI) motility. Postoperative ileus was induced by intestinal manipulation in Sprague-Dawley rats. Additionally, compound 48/80 (C48/80) and ketotifen were used to modulate MC activity. Rat mast cell protease 6 (RMCP-6, ELISA) release was determined in peritoneal lavage 20 min after intestinal manipulation. At 24 h, GI transit was determined. Gene expression of calcitonin gene-related peptide (CGRP), protease-activated receptor-2 (PAR-2), nerve growth factor (NGF), and TrkA receptor was determined (PCR) in dorsal root ganglia (DRG). Ileal wall inflammation was assessed by myeloperoxidase (MPO) activity, interleukin-6 expression (IL-6). Intestinal manipulation and exposure to C48/80-induced degranulation of PMCs delayed GI transit and up-regulated IL-6 and MPO activity. Intestinal manipulation, but not C48/80, up-regulated CGRP, PAR-2, and NGF/TrkA in DRGs. Ketotifen only improved gastric emptying and fecal output. Up-regulation of CGRP and TrkA expression in DRG was not prevented by ketotifen. Postoperative ileus is accompanied by activation of CGRP, NGF-TrkA, and PAR-2 in DRGs. Our results suggest that these mediators could be a target in further POI studies in order to find new therapeutic targets for this medical condition.

The Pharmacological Effects of Benachio-F(®) on Rat Gastrointestinal Functions.

Functional dyspepsia (FD) is a prevalent idiopathic upper gastrointestinal (GI) disorder characterized by diverse symptomatology including epigastric pain or discomfort, postprandial fullness, and early satiety. Although its pathophysiological mechanisms have not yet been fully established, the available studies suggest that the etiology of FD is invariably multifactorial. Benachio-F® (BF) is a proprietary liquid formulation of 7 herbal extracts that has been proposed to address this multifactorial etiology using multi-drug phytotherapy. The pharmacological effects of BF, in comparison with those of two other herbal products (Whalmyungsu®; WM and Iberogast®; IB) were evaluated in rats. In a laparotomy-induced rat model of delayed GI transit, BF significantly accelerated the delayed gastric emptying caused by morphine, apomorphine, and cisplatin, and also significantly increased mean gastric transit, as compared to the control animals. BF markedly increased gastric accommodation in rats and produced higher gastric volume values than did the control treatment. The effects of BF were generally comparable or superior to those of WM and IB in these models. Furthermore, BF significantly stimulated biliary flow, as compared to the control treatment. These results indicated that BF might have great potential as an effective phytotherapeutic agent capable of reducing GI symptoms and increasing quality of life in FD patients.

75 In Combat Soldiers Resting Urinary Metabotypes Correlate With the Development of Gastrointestinal Symptoms During Stress

Introduction: Western-diet (WD) is associated with reduced stool frequency in humans. High-fat fed mice exhibit delayed gastrointestinal (GI) transit associated with reduced number of nitrergic myenteric neurons. Mechanisms of these alterations are still unknown. Our aim was to investigate if WD feeding leads to modifications of fecal metabolites associated with the development of enteric neurodegeneration and the role played by the gut microbiota in such phenomenon. Methods: C57Bl/6 mice were fed a WD (34.5% kcal from fat, representative of the typical American diet fat calorie intake, n=5) or a Regular diet (RD, 16.9% kcal from fat, n=5). 6 weeks post feeding, fecal metabolites were analyzed by high-resolution mass spectrometry (LTQ-FT). 12 weeks post feeding, nitrergic myenteric neurons were quantified in the proximal colon by NADPH-diaphorase staining and GI and colonic transit were measured. Citrulline was measured by ELISA in germ-free (GF) mice stools fed a WD or a RD for 6 weeks (n=3 per group). Data are presentedas mean ± SEM and are significant to p < 0.05. Results: After 12 weeks of feeding, WD mice exhibited a reduced stool frequency in comparison to RD mice (1.5±0.5 and 4.4±0.3 stools/min, respectively) and this was associated with a delayed intestinal transit time (281.5±57.5 and 152.8±15.7 min). In addition, WD mice had a longer colonic transit time (as measured by bead expulsion time: WD: 127.5±52.5 min; RD: 13.2±2.0 min) associated with reduced number of nitrergic myenteric neurons in the proximal colon (113.0±5.6 and 64.2±7.4 neurons/field). Together, these data demonstrate an altered colonic motor function after 12 weeks of WD. We quantified 3730 metabolites in the feces but only 185 were significantly higher in WD mice compared to RD mice. In addition to palmitate, we found increased concentrations of citrulline (associated with enhanced nitric oxide synthase activity), 3-hydroxykynurenine (reflecting neuroinflammation) and bacterial muramic acid [Figure 1]. In order to understand the possible role of the gut microbiota in these changes, we measured fecal citrulline in GF mice fed a WD or a RD for 6 weeks and found similar concentrations (3.0±0.5 and 2.6±0.3 ng/mg). Conclusion: Our results show that long-term WD consumption is associated with nitrergic myenteric neuronal loss contributing to delayed GI transit. These alterations were preceded by gut dysbiosis and elevated fecal citrulline, potentially reflecting higher plasma concentrations that lead to oxidative stress and apoptosis in NO producing neurons. Importantly, fecal citrulline was not affected by a WD feeding under germfree conditions, suggesting that the gut microbiota is required for such effects. Fecal citrulline may be considered as a marker of myenteric neuronal impairments responsible for the motor alterations observed during WD feeding.

74 Fecal Metabolites Associated With Nitrergic Myenteric Neuronal Impairment and Gastrointestinal Motility Disorders in Western Diet-Fed Mice

Introduction: Western-diet (WD) is associated with reduced stool frequency in humans. High-fat fed mice exhibit delayed gastrointestinal (GI) transit associated with reduced number of nitrergic myenteric neurons. Mechanisms of these alterations are still unknown. Our aim was to investigate if WD feeding leads to modifications of fecal metabolites associated with the development of enteric neurodegeneration and the role played by the gut microbiota in such phenomenon. Methods: C57Bl/6 mice were fed a WD (34.5% kcal from fat, representative of the typical American diet fat calorie intake, n=5) or a Regular diet (RD, 16.9% kcal from fat, n=5). 6 weeks post feeding, fecal metabolites were analyzed by high-resolution mass spectrometry (LTQ-FT). 12 weeks post feeding, nitrergic myenteric neurons were quantified in the proximal colon by NADPH-diaphorase staining and GI and colonic transit were measured. Citrulline was measured by ELISA in germ-free (GF) mice stools fed a WD or a RD for 6 weeks (n=3 per group). Data are presentedas mean ± SEM and are significant to p < 0.05. Results: After 12 weeks of feeding, WD mice exhibited a reduced stool frequency in comparison to RD mice (1.5±0.5 and 4.4±0.3 stools/min, respectively) and this was associated with a delayed intestinal transit time (281.5±57.5 and 152.8±15.7 min). In addition, WD mice had a longer colonic transit time (as measured by bead expulsion time: WD: 127.5±52.5 min; RD: 13.2±2.0 min) associated with reduced number of nitrergic myenteric neurons in the proximal colon (113.0±5.6 and 64.2±7.4 neurons/field). Together, these data demonstrate an altered colonic motor function after 12 weeks of WD. We quantified 3730 metabolites in the feces but only 185 were significantly higher in WD mice compared to RD mice. In addition to palmitate, we found increased concentrations of citrulline (associated with enhanced nitric oxide synthase activity), 3-hydroxykynurenine (reflecting neuroinflammation) and bacterial muramic acid [Figure 1]. In order to understand the possible role of the gut microbiota in these changes, we measured fecal citrulline in GF mice fed a WD or a RD for 6 weeks and found similar concentrations (3.0±0.5 and 2.6±0.3 ng/mg). Conclusion: Our results show that long-term WD consumption is associated with nitrergic myenteric neuronal loss contributing to delayed GI transit. These alterations were preceded by gut dysbiosis and elevated fecal citrulline, potentially reflecting higher plasma concentrations that lead to oxidative stress and apoptosis in NO producing neurons. Importantly, fecal citrulline was not affected by a WD feeding under germfree conditions, suggesting that the gut microbiota is required for such effects. Fecal citrulline may be considered as a marker of myenteric neuronal impairments responsible for the motor alterations observed during WD feeding.

Effects of GC7101, a Novel Prokinetic Agent on Gastric Motor Function: Ex Vivo Study

Background/AimsGC7101, an extract of Lonicera Flos, is a novel developing drug for reflux esophagitis and functional dyspepsia. However, the drug’s exact pharmacological mechanism of action remains unclear. This study assessed the effects of GC7101 on gastrointestinal (GI) motor function.MethodsWe used male guinea pigs to evaluate the effects of GC7101 on GI motility. The contraction of antral circular muscle in the presence of different doses of GC7101 was measured in a tissue bath. The prokinetic effects of GC7101 were tested using the charcoal transit assay from the pylorus to the most distal point of migration of charcoal mixture. To clarify the mechanism of action of GC7101, atropine, dopamine and the selective 5-hydroxytryptamine 4 receptor antagonist, GR113808 were used.ResultsThe maximal amplitude of circular muscle contraction was induced by 5 mg mL−1 GC7101. The area under the curve of contraction was significantly increased at 5 mg mL−1 GC7101. Addition of 10−6 M atropine, 10−8 M dopamine or 10−7 M GR 113808 to GC7101 5 mg mL−1 decreased the amplitude and area under curve compared to GC7101 5 mg mL−1 alone. GC7101 accelerated GI transit in a dose dependent manner except 100 mg kg−1. Delayed GI transit caused by atropine, dopamine and GR 113808 was restored by GC7101 50 mg kg−1.ConclusionsGC7101, an extract of Lonicera Flos, exerts a gastric prokinetic effect in guinea pig through cholinergic, antidopaminergic and serotonergic mechanisms. Therefore, GC7101 might be a novel drug for the treatment of functional dyspepsia.

Systemic and anti-nociceptive effects of prolonged lidocaine, ketamine, and butorphanol infusions alone and in combination in healthy horses

BackgroundProlonged drug infusions are used to treat horses with severe signs of pain, but can be associated with altered gastrointestinal transit. The purpose of this study was to determine the effects of prolonged constant rate infusions (CRI) of lidocaine (L), butorphanol (B), and ketamine (K) alone and in combination on gastrointestinal transit, behavior, and thermal nociceptive threshold in healthy horses.MethodsEight healthy adult horses were used in a randomized, cross-over, blinded, prospective experimental trial. Interventions were saline, L, K, B, LK, LB, BK, and LBK as an intravenous CRI for 96 hours. Drugs were mixed or diluted in saline; following a bolus, CRI rate was 0.15mL/kg/hr with drug doses as follows: L – 1.3 mg/kg then 3 mg/kg/hr; B – 0.018 mg/kg then 0.013 mg/kg/hr; K – 0.55 mg/kg then 0.5 mg/kg/hr. Two-hundred plastic beads were administered intragastrically by nasogastric tube immediately prior to the bolus. Feces were collected every 2 hours, weighed, and beads manually retrieved. Behavior was scored every 2 hours, vital parameters every 6 hours, and thermal nociceptive threshold every 12 hours for 96 hours. Drug concentrations in the LBK solution were tested every 6 hours for 72 hours.ResultsFour of 64 trials (3 LBK, 1 BK) were discontinued early due to signs of abdominal discomfort. There were no apparent differences between groups in vital parameters or thermal threshold. Transit time was delayed for LB and LBK with a corresponding decrease in fecal weight that was most severe in the final 24 hours of infusion. Significant changes in behavior scores, vital parameters, or thermal threshold were not observed. The concentration of each drug in the combined solution declined by less than 31% over the sampling period.ConclusionsDrug combinations containing butorphanol cause an apparent delay in gastrointestinal transit in healthy horses without substantially affecting somatic nociception at the doses studied. Combinations of lidocaine and ketamine may have less impact on gastrointestinal transit than infusions combined with butorphanol. Further work is needed to determine the effects of these drugs in painful or critically ill patients.

Absence of intestinal inflammation and postoperative ileus in a mouse model of laparoscopic surgery.

Postoperative ileus (POI) is characterized by impaired gastrointestinal motility resulting from intestinal handling-associated inflammation. The introduction of laparoscopic surgery has dramatically reduced the duration of POI. However, it remains unclear to what extent this results in a reduction of intestinal inflammation. The aim of the present study is to compare the degree of intestinal inflammation and gastrointestinal transit following laparoscopic surgery and open abdominal surgery. Mice were subjected to laparoscopic surgery or laparotomy alone or, in combination with standardized intestinal manipulation of the small bowel (IM). Gastrointestinal transit and intestinal inflammation were assessed 24 h after surgery by the number of myeloperoxidase (MPO) positive cells and the level of cytokine expression. The recovery time and the degree of inflammation were also analyzed in patients subjected to colectomy under open conditions (laparotomy) or laparoscopic conditions. Mice undergoing IM by laparotomy (open IM), but not by laparoscopy (Lap IM) developed a significant delay in gastrointestinal transit compared to laparotomy or laparoscopy alone. In addition, there was significant intestinal inflammation only after open IM. Similarly, cytokine levels in peritoneal lavage fluid were lower while recovery time was faster in patients subjected to colectomy under laparoscopic conditions compared to open colectomy. Our data confirms that intestinal inflammation is underlying the delayed gastrointestinal transit observed after open surgery. Most importantly, we demonstrate that intestinal inflammation under laparoscopic conditions is significantly lower compared to open surgery, most likely explaining the faster recovery following laparoscopic surgery.

Drug‐induced constipation alters gut microbiota stability leading to physiological changes in the host (1118.2)

Gut microbes play a significant role in development and maintenance of intestinal functions, but can contribute to pathogenesis of gastrointestinal (GI) disorders when host‐microbiome relationship is perturbed. In this study we sought to examine how constipation affects microbiome, and how these alterations in turn affect the host. Adult C57Bl/6 mice were treated with 0.1% Loperamide in drinking water for 7 days to induce constipation. Loperamide‐treated mice had increased GI transit time, indicating constipation. Bacterial DNA analysis by HiSeq sequencing showed decreased Firmicutes and increased Bacteroidetes in constipated mice. Microbiome metabolic potential was measured with tetrazolium dye assay and showed shift toward increased sugar metabolism in Loperamide‐treated mice. Microbial metabolites were measured by GC‐MS, and showed lower butyrate concentration in constipated mice. To determine constipation effects on the host, we introduced microbiota from constipated donors to germ‐free recipients. These mice had delayed GI transit time. To determine mechanism for delayed GI transit we analyzed colon tissues by qRT‐PCR and western blotting analysis, and determined lower expression of nNos mRNA and protein levels in these mice. Overall, these results suggest that delayed GI transit shifts microbiome taxonomic and metabolic profile, and these changes further contribute to development of constipation.Grant Funding Source: NIH NIDDK T32 DK07074

The cannabinoid‐1 receptor inverse agonist taranabant reduces abdominal pain and increases intestinal transit in mice

Constipation-predominant irritable bowel syndrome (IBS-C) is a common functional gastrointestinal (GI) disorder with abdominal pain and decreased motility. Current treatments of IBS-C are insufficient. The aim of this study was to evaluate the potential application of taranabant, a cannabinoid type 1 (CB1) inverse agonist using mouse models mimicking the symptoms of IBS-C. Changes in intestinal contractile activity were studied in vitro, using isolated mouse ileum and colon and intracellular recordings. In vivo, whole gastrointestinal transit (WGT) and fecal pellet output (FPO) were measured under standard conditions and with pharmacologically delayed GI transit. The antinociceptive effect was evaluated in mustard oil- and acetic acid-induced models of visceral pain. Forced swimming and tail suspension tests were performed and locomotor activity was measured to evaluate potential central side effects. In vitro, taranabant (10(-10) -10(-7) mol L(-1)) increased contractile responses in mouse ileum and blocked the effect of the CB agonist WIN 55,212-2. Taranabant had no effect on the amplitude of electrical field stimulation (EFS)-evoked junction potentials. In vivo, taranabant (0.1-3 mg kg(-1), i.p. and 3 mg kg(-1), p.o.) increased WGT and FPO in mice and reversed experimental constipation. The effect of taranabant was absent in CB1(-/-) mice. Taranabant significantly decreased the number of pain-related behaviors in animal models. At the doses tested, taranabant did not display mood-related adverse side effects typical for CB1 receptor inverse agonists. Taranabant improved symptoms related to slow GI motility and abdominal pain and may become an attractive template in the development of novel therapeutics targeting IBS-C.