deoxycholic-acid and Gastroesophageal-Reflux

Dysregulation of toll-like receptor (TLR) signaling within the gastrointestinal epithelium has been associated with uncontrolled inflammation and tumorigenesis. We sought to evaluate the role of TLR4 in the development of gastroesophageal reflux-mediated inflammation and mucosal changes of the distal esophagus. Verified human esophageal Barrett's cells with high grade dysplasia (CPB) and esophageal adenocarcinoma cells (OE33) were treated with deoxycholic acid for 24 hours. Cells were pretreated with a TLR4-specific inhibitor peptide 2 hours prior to deoxycholic acid treatment. Inflammatory markers were evaluated using immunoblotting and enzyme-linked immunosorbent assay. A surgical reflux mouse model was generated by performing a side-to-side anastomosis between the second portion of the duodenum and the gastroesophageal junction. Control animals underwent laparotomy with incision and closure of the esophagus superior to the gastroesophageal junction (sham procedure). Esophageal sections were evaluated using hematoxylin and eosin staining and immunohistochemistry. Deoxycholic acid increased expression of inflammatory markers including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and interleukin 8. Pretreatment with a TLR4 inhibitor significantly decreased deoxycholic acid-induced inflammatory marker expression. C3H/HeNCrl mice demonstrated a significant increase in mucosal hyperplasia and proliferation following DGEA compared to sham procedure. TLR4 mutant mice (C3H/HeJ) undergoing DGEA demonstrated an attenuated hyperplastic and proliferative response compared to C3H/HeNCrl mice. Inhibition of TLR4 signaling attenuates reflux-induced inflammation in vivo. These findings identify TLR4 inhibition as a potential therapeutic target to halt the progression of pathologic esophageal changes developing in the setting of chronic gastroesophageal reflux disease.

Topics: Animals; Barrett Esophagus; Deoxycholic Acid; Esophageal Neoplasms; Gastroesophageal Reflux; Humans; Inflammation; Mice; Mice, Inbred C3H; Toll-Like Receptor 4; Treatment Outcome

Obesity is a known risk factor for the development of gastroesophageal reflux disease (GERD), Barrett's Esophagus (BE) and the progression to esophageal adenocarcinoma. The mechanisms by which obesity contributes to GERD, BE and its progression are currently not well understood. Recently, changes in lipid metabolism especially in the context of a high fat diet have been linked to GERD and BE leading us to explore whether fatty acid oxidation plays a role in the disease progression from GERD to esophageal adenocarcinoma. To that end, we analyzed the expression of the rate-limiting enzyme, carnitine palmytoyltransferase 1A (CPT1A), in human tissues and cell lines representing different stages in the sequence from normal squamous esophagus to cancer. We determined uptake of palmitic acid, the most abundant fatty acid in human serum, with fluorescent dye-labeled lipids as well as functional consequences of stimulation with palmitic acid relevant to Barrett's tumorigenesis, e.g., proliferation, characteristics of stemness and IL8 mediated inflammatory signaling. We further employed different mouse models including a genetic model of Barrett's esophagus based on IL1β overexpression in the presence and absence of a high fat diet and deoxycholic acid to physiologically mimic gastrointestinal reflux in the mice. Together, our data demonstrate that CPT1A is upregulated in Barrett's tumorigenesis and that experimental palmitic acid is delivered to mitochondria and associated with increased cell proliferation and stem cell marker expression.

Topics: Adenocarcinoma; Animals; Barrett Esophagus; Carcinogenesis; Carnitine; Carnitine O-Palmitoyltransferase; Cell Proliferation; Cell Transformation, Neoplastic; Deoxycholic Acid; Esophageal Neoplasms; Fluorescent Dyes; Gastroesophageal Reflux; Humans; Interleukin-8; Mice; Obesity; Palmitic Acid

Various adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), have been shown to play a role in inflammation as well as contribute to tumor progression and prognosis. We hypothesized that gastroduodenal reflux upregulates ICAM-1 and VCAM-1 expression in the distal esophagus, serving as possible early markers of pathologic esophageal disease.. Normal human esophageal epithelial cells (HET1A), Barrett cells (CPB), and esophageal adenocarcinoma cells (FLO1 and OE33) were treated with deoxycholic acid at increasing concentrations for 24 hours. Adhesion molecule expression was assessed using immunoblotting. A surgical mouse reflux model was generated by performing a side-to-side anastomosis between the gastroesophageal junction and the first portion of the duodenum (duodenum-gastroesophageal anastomosis). Esophageal sections were evaluated using hematoxylin and eosin staining, immunohistochemistry, and immunofluorescence.. Deoxycholic acid induced a significant increase in ICAM-1 and VCAM-1 expression in HET1A, CPB, FLO1, and OE33 cells. Animals undergoing duodenum-gastroesophageal anastomosis demonstrated a significant increase in mucosal hyperplasia (P < .0001) and cellular proliferation (P < .0001) compared with control animals. Immunofluorescence and Western blot analysis of the lower esophagus demonstrated significant upregulation of ICAM-1 (P = .005), with no change in VCAM-1 expression (P = .82).. Our results reveal that ICAM-1 and VCAM-1 are upregulated in response to in vitro reflux treatment of normal esophageal epithelial cells. However, our investigation using a mouse reflux model found ICAM-1 is noticeably upregulated without a concomitant increase in VCAM-1. These findings identify ICAM-1, but not VCAM-1, as a potential player in early esophageal disease developing from chronic reflux exposure.

Topics: Animals; Cell Adhesion Molecules; Deoxycholic Acid; Disease Models, Animal; Gastroesophageal Reflux; Humans; Intercellular Adhesion Molecule-1; Mice; Vascular Cell Adhesion Molecule-1

The bile acid component of gastric refluxate has been implicated in inflammation of the oesophagus including conditions such as gastro-oesophageal reflux disease (GORD) and Barrett's Oesophagus (BO). Here we demonstrate that the hydrophobic bile acid, deoxycholic acid (DCA), stimulated the production of IL-6 and IL-8 mRNA and protein in Het-1A, a model of normal oesophageal cells. DCA-induced production of IL-6 and IL-8 was attenuated by pharmacologic inhibition of the Protein Kinase C (PKC), MAP kinase, tyrosine kinase pathways, by the cholesterol sequestering agent, methyl-beta-cyclodextrin (MCD) and by the hydrophilic bile acid, ursodeoxycholic acid (UDCA). The cholesterol-interacting agent, nystatin, which binds cholesterol without removing it from the membrane, synergized with DCA to induce IL-6 and IL-8. This was inhibited by the tyrosine kinase inhibitor genistein. DCA stimulated the phosphorylation of lipid raft component Src tyrosine kinase (Src). while knockdown of caveolin-1 expression using siRNA resulted in a decreased level of IL-8 production in response to DCA. Taken together, these results demonstrate that DCA stimulates IL-6 and IL-8 production in oesophageal cells via lipid raft-associated signaling. Inhibition of this process using cyclodextrins represents a novel therapeutic approach to the treatment of inflammatory diseases of the oesophagus including GORD and BO.

Topics: Barrett Esophagus; beta-Cyclodextrins; Bile Acids and Salts; Caveolin 1; Cell Line, Tumor; Cholesterol; Cytokines; Deoxycholic Acid; Esophagus; Gastroesophageal Reflux; Gene Expression; Humans; Inflammation; Interleukin-6; Interleukin-8; Lipids; Membrane Microdomains; Neoplasms; NF-kappa B; Phosphorylation; Signal Transduction; src-Family Kinases

The fundamental mechanisms underlying erosive oesophagitis and subsequent development of Barrett's oesophagus (BO) are poorly understood. Here, we investigated the contribution of specific components of the gastric refluxate on adhesion molecules involved in epithelial barrier maintenance. Cell line models of squamous epithelium (HET-1A) and BO (QH) were used to examine the effects of bile acids on cell adhesion to extracellular matrix proteins (Collagen, laminin, vitronectin, fibronectin) and expression of integrin ligands (α

Topics: Animals; Barrett Esophagus; Cell Adhesion; Cell Line; Collagen; Deoxycholic Acid; Disease Models, Animal; Epithelial Cells; Esophagitis; Fibronectins; Gastroesophageal Reflux; Gene Expression Profiling; Gene Expression Regulation; Humans; Integrin alphaV; Integrins; Laminin; Permeability; Protein Transport; Rats; Tissue Array Analysis; Vitronectin

Impaired esophageal mucosal integrity may be an important contributor in the pathophysiology of gastroesophageal reflux disease (GERD). Nevertheless, the effect of potentially harmful agents on epithelial integrity is mainly evaluated in vitro for a short period of time and the possible induction of epithelial apoptosis has been neglected. Our objective was to assess the effect of an acidic and weakly acidic solution containing deoxycholic acid (DCA) on the esophageal epithelium in an in vivo rabbit model of esophageal perfusion and to evaluate the role of the epithelial apoptosis. The esophagus of 55 anesthetized rabbits was perfused for 30 min with different solutions at pH 7.2, pH 5.0, pH 1.0, and pH 5.0 containing 200 and 500 μM DCA. Thereafter, animals were euthanized immediately or at 24 or 48 h after the perfusion. Transepithelial electrical resistance, epithelial dilated intercellular spaces, and apoptosis were assessed in Ussing chambers, by transmission electron microscopy, and by TUNEL staining, respectively. No macroscopic or major microscopic alterations were observed after the esophageal perfusions. The acidic and weakly acidic solution containing DCA induced similar long-lasting functional impairment of the epithelial integrity but different ultrastructural morphological changes. Only the solution containing DCA induced epithelial apoptosis in vivo and in vitro in rabbit and human tissue. In contrast to acid, a weakly acidic solution containing DCA induces epithelial apoptosis and a long-lasting impaired mucosal integrity. The presence of apoptotic cells in the esophageal epithelium may be used as a marker of impaired integrity and/or bile reflux exposure.

Topics: Animals; Apoptosis; Deoxycholic Acid; Electric Impedance; Esophageal Mucosa; Extracellular Space; Gastroesophageal Reflux; Humans; Hydrogen-Ion Concentration; Male; Middle Aged; Models, Animal; Perfusion; Rabbits; Time Factors

BACKGROUND AND AIMS. Gastroesophageal reflux disease (GERD) is associated with impaired epithelial barrier function. However, the influence of acid and/or bile acids on human esophageal epithelial barrier function and the tight junction (TJ) proteins has not been fully elucidated. The aim of the study is to investigate the esophageal barrier function and TJ expression in healthy subjects and patients with GERD. The functionality of esophageal mucosa exposed to bile salt deoxycholic acid (DCA) and trypsin has been studied in vitro. MATERIAL AND METHODS. Endoscopic biopsies from healthy controls and patients with GERD-related symptom with endoscopic erosive signs, as well as esophageal mucosa taken from patients undergoing esophagectomy were evaluated in Ussing chambers and by western blot and immunohistochemistry. RESULTS. The esophageal epithelium from GERD patients had lower electrical resistance and higher epithelial currents than controls. Claudin-1 and -4 were significantly decreased in GERD patients. The bile salt DCA in the low concentration of 1.5 mM and trypsin increased the resistance and claudin-1 expression, while the higher concentration of 2.5 mM DCA and trypsin decreased the resistance and the claudin-3, -4 and E-cadherin expressions. CONCLUSION. In addition to acidic reflux, duodenal reflux components, such as bile salts and trypsin, have the potential to disrupt the esophageal barrier function, partly by modulating the TJ proteins. However, the expression of TJ is dependent on both the refluxed material as well as the concentration of the bile salt.

Topics: Adult; Biomarkers; Biopsy; Blotting, Western; Cadherins; Case-Control Studies; Claudins; Deoxycholic Acid; Electric Impedance; Esophagoscopy; Esophagus; Female; Gastroesophageal Reflux; Humans; Immunohistochemistry; Male; Middle Aged; Mucous Membrane; Permeability; Tight Junctions; Trypsin

Barrett's oesophagus is an acquired precancerous condition that develops from mucosal injury incurred due to chronic gastro-oesophageal reflux. The aim of this study was to determine if bile and/or acid components of the refluxate can induce DNA damage in vitro. The oesophageal cell lines FLO-1 and HET1-A were exposed to primary bile salts, individually or as a mixture, and the secondary bile salt sodium deoxycholate, in neutral or acidified media. Cells were then examined in the comet assay to measure DNA strand breaks. Cell viability was also monitored. Acidified media induced DNA damage in a pH- and time-dependent manner. The primary bile compounds sodium glycocholate, glycocholic acid, sodium taurocholate and taurochenodeoxycholate, as an equimolar mixture (100 microM), caused a small but significant (P < 0.028) elevation in DNA damage, but only at neutral pH in FLO-1 cells. Sodium deoxycholate (100 microM) caused a significant (P < 0.008) elevation in DNA damage in both cell lines, but again only at neutral pH. These data suggest that specific components of gastro-oesophageal refluxate are capable of causing DNA damage and may participate in the genesis and progression of Barrett's oesophagus via this mechanism.

Topics: Barrett Esophagus; Bile Acids and Salts; Cell Line; Cell Survival; Comet Assay; Deoxycholic Acid; DNA Damage; Esophagus; Gastroesophageal Reflux; Humans; Hydrogen-Ion Concentration

C-myc over expression is implicated in malignancy although to date this has not been studied in Barrett's metaplasia. We sought to determine c-myc expression in the malignant progression of Barrett's metaplasia and whether it may be induced by bile acids seen in gastro-oesophageal refluxate.. C-myc protein and mRNA levels were assessed in 20 Barrett's metaplasia and 20 oesophageal adenocarcinoma samples by western blotting and real time polymerase chain reaction. Levels of c-myc and proliferation were also assessed in cell lines OE21, OE33, SW-480, and TE-7 stimulated with pulses or continuous exposure to the bile acids deoxycholic acid and chenodeoxycholic acid.. C-myc protein was upregulated in 50% of Barrett's metaplasia and 90% of oesophageal adenocarcinoma samples compared with squamous, gastric, and duodenal controls. C-myc immunolocalisation in Barrett's metaplasia revealed discrete nuclear localisation, becoming more diffuse with progression from low to high grade dysplasia to adenocarcinoma. Both continual and pulsed bile acid induced c-myc at pH 4, with no effect at pH 7 or with acidified media alone. Pulsed bile acid treatment induced proliferation (p<0.05); in contrast, continuous exposure led to suppression of proliferation (p<0.05).. We have shown upregulation of c-myc with malignant progression of Barrett's metaplasia and suggest that acidified bile may be a novel agent responsible for induction of this oncogene.

Topics: Adenocarcinoma; Barrett Esophagus; Bile Acids and Salts; Blotting, Western; Cell Division; Chenodeoxycholic Acid; Deoxycholic Acid; Esophageal Neoplasms; Fluorescent Antibody Technique; Gastroesophageal Reflux; Gene Expression Regulation; Genes, myc; Humans; Immunohistochemistry; Polymerase Chain Reaction; Tumor Cells, Cultured; Up-Regulation

The pathogenesis of alkaline reflux esophagitis was investigated in an experimental model by assessing individually the influence of different bile salt moieties and trypsin on esophageal mucosa. An isolated segment of rabbit esophagus was perfused at pH 7 with a solution containing the test agent under study, and the severity of mucosal damage was assessed by using as indicators of mucosal integrity transmucosal potential difference, net flux of Na+, and mucosal permeability to two neutral molecules of different sizes, 3H-H2O and 14C-erythritol. The data indicate that the secondary dihydroxy bile salt, deoxycholate, in its deconjugated form was highly injurious to esophageal mucosa; it was the only test agent that caused gross mucosal lesions during the experiment. The respective conjugated bile salt moiety, taurodeoxycholate, had a weaker effect. Also the primary dihydroxy bile salt, chenodeoxycholate, in its deconjugated form caused moderate damage to the mucosa, whereas its conjugated form, taurochenodeoxycholate, had no effect. The effect of the other three bile salts tested--cholate, taurocholate, and taurolithocholate--was negligible. Trypsin also adversely affected the mucosa, but its effect was weaker than that of deoxycholate. The results suggest that the deconjugated bile salts deoxycholate and chenodeoxycholate (which are formed following bacterial colonization of the upper gastrointestinal tract in the absence of gastric acid), the conjugated bile salt taurodeoxycholate, and the proteolytic enzyme trypsin may have significant roles in the pathogenesis of alkaline reflux esophagitis.

Topics: Alkalies; Animals; Bile Acids and Salts; Chenodeoxycholic Acid; Cholic Acids; Deoxycholic Acid; Esophagus; Gastroesophageal Reflux; Rabbits; Taurochenodeoxycholic Acid; Taurocholic Acid; Taurodeoxycholic Acid; Taurolithocholic Acid; Trypsin

Ten patients who had undergone total or subtotal gastrectomy for carcinoma 1 to 8 years earlier were evaluated in terms of subjective symptoms, endoscopy and the presence of bile in esophagojejunal aspirates obtained by direct aspiration during endoscopy. The concentrations of individual bile acids were determined by means of gas chromatography. Six of the patients had macroscopic esophagitis and all of them also had bile in their aspirate. The remaining four patients with normal esophageal mucosa did not have positive specimens. Neither did the total bile acid concentration nor any of the individual bile acids, regardless of whether they were free or conjugated, correlate with the severity of symptoms or the degree of endoscopic esophagitis. Esophagitis healed in all three patients who underwent conversion of loop esophagojejunostomy to a long Roux-Y reconstruction.

Topics: Adult; Aged; Bile Acids and Salts; Chenodeoxycholic Acid; Cholic Acids; Deoxycholic Acid; Esophagitis; Esophagoscopy; Female; Gastrectomy; Gastroesophageal Reflux; Humans; Male; Middle Aged