Lipid solubilization during bile salt-induced esophageal mucosal barrier disruption in the rabbit

Bile salts in the gastric contents are one of the harmful agents that may contribute to the mucosal injury found in clinical reflux esophagitis. To clarify the mechanism by which bile salts can injure the esophageal mucosa, we studied the effects of exposure to the bile salts taurocholate and deoxycholate in an in vivo perfused rabbit model of esophagitis. Specifically we examined the roles of accumulation of bile salt by the mucosa and solubilization of mucosal lipid into the lumen during bile salt-induced mucosal injury. Results showed a consistent association between bile salt accumulation by the esophageal mucosa and bile salt-induced disruption of the physiologic barrier to diffusion. Under certain conditions, bile salts caused barrier disruption with no release of lipid from the mucosa. Whenever exposure to bile salt did cause release of mucosal lipid into the lumen, a significant amount of morphologic injury was also observed by light microscopy. The findings suggest that the presence of bile salt in the mucosa is an important aspect of the mechanism of bile salt-induced barrier disruption. Solubilization of mucosal lipids into the lumen occurs when bile salts cause an injury that is severe enough to result in light microscopic evidence of morphologic damage.     

Inflammation and oxidative stress in gastroesophageal reflux disease

The etiology of esophageal mucosal injury is complex, since it may involve the reflux of gastric acid, bile acid, and pancreatic juice, external factors such as drugs and alcohol, or functional factors such as esophagogastric motility. The mechanism of esophageal mucosal injury has gradually been understood at the molecular biological level. It is particularly important that pro-inflammatory factors, such as inflammatory cytokines (interleukin-6 and -8), leukocytes and oxidative stress, have been demonstrated to be involved in the development of gastroesophageal reflux disease (GERD) including nonerosive reflux disease (NERD). In addition, nociceptors such as acid-sensitive vanilloid receptors, protease-activated receptors and substance P have also been implicated in the pathogenesis of neurogenic inflammation in NERD patients with esophageal hypersensitivity. The development of new therapy with anti-inflammatory and anti-oxidant effects is expected to assist in the treatment of intractable NERD/GERD and the prevention of carcinogenesis.     

Effect of cimetidine on gastric mucosal barrier in dogs

The effect of cimetidine on the gastric mucosal barrier (GMB) was investigated in adult mongrel dogs with a Heidenhain pouch. A linear correlation was found between the net flux of Na+ and H+, and transmucosal electrical potential difference (PD). The PD was thus considered to be a good indicator for the degree of impairment of the GMB which can be determined easily with high reproducibility. When irrigated with HCl alone, there was a slight, though not significant, upward tendency of the PD following intravenous cimetidine. The destruction of the GMB during perfusion with HCl plus sodium taurocholate resulted in a marked reduction of the PD. This response of the PD was significantly reduced by the injection of cimetidine. These results indicate that cimetidine protects the GMB from impairments by bile acid.     

Pathophysiology of acute acid injury in rabbit esophageal epithelium.

To increase our understanding of the pathophysiology of reflux esophagitis, we sought the early sequence of changes in mucosal structure and function in acutely acid-damage rabbit esophagus. Using a perfused catheter technique esophageal potential difference (PD) profiles were obtained in anesthetized rabbits before, during, and after perfusion of the lower one-half of the esophagus with phosphate-buffered saline or 80 mM NaCl. When acid perfusion reduced the lower esophageal PD by 40-50% or 80-100% of the initial values, the esophagus was removed, sectioned, and the mucosa studied with light microscopy, transmission electron microscopy, and Ussing chamber technique for evaluation of sodium and mannitol transport. The earlier stage of acid damage (PD 40-50%) was associated with reduced mucosal resistance fom 2,180 +/- 199 to 673 +/- 157 ohm cm2 and increased passive transport of sodium (0.10 +/- 0.06 to 1.82 +/- 0.48 microeq/h.cm2) and mannitol (0.008 +/- 0.003 to 0.051 +/- 0.012 microM/h.cm2) (p less than 0.05). There was no significant change in shirt circuit current (0.35 +/- 0.05 to 0.35 +/- 0.04) or net sodium transport (0.32 +/- 0.06 to 0.37 +/- 0.12) at this stage, and the only morphologic finding was dilated intercellular spaces on electron microscopy. The later stage of acid damage (PD 80-100%) exhibited a further reduction in resistance to 299 +/- 65 ohm.cm2 (p less than 0.05), a finding now accompanied by a reduction in short circuit current (0.35 +/- 0.05 to 0.21 +/- 0.04 microeq/h.cm2) and complete inhibition of net sodium transport (0.32 +/- 0.06 to 0.01 +/- 0.13) (p less than 0.05). Morphologic studies at this time revealed cellular necrosis, edema, and vesicle formation in the stratum spinosum. Both gross mucosal changes and transmural necrosis were notably absent. When esophageal perfusion was performed with a combination of acid (80 mM HCl-80 mM NaCl) and pepsin (100 microgram/ml), the morphologic and physiologic findings were essentially the same as with acid alone; however, the time of perfusion to reach either the 50 or 100% reduction in PD was shortened. The findings in this model can be explained on an initial increase in cellular and/or paracellular permeability followed by inhibition of active sodium transport. The resulting loss of osmolar regulation leads to cell necrosis in the stratum spinosum.     

Cytokine expression in GERD

The mechanism of esophageal mucosal injury has gradually been understood at the microbiological level. It is particularly important that pro-inflammatory factors, such as inflammatory cytokines, leukocytes and oxidative stress, have been demonstrated to be involved in the development of gastroesophageal reflux disease (GERD) including nonerosive reflux disease (NERD). Our present study reveals that expression of IL-8 mRNA, a potent neutrophil chemotactic and activating peptide, is correlated with the endoscopic grade of esophagitis or with inflammatory cell infiltration. In addition, it has been shown that bile acids and trypsin can promote IL-8 production from human esophageal epithelial cells via NFkappaB-and AP-1-dependent mechanism. Nociceptors such as acid-sensitive vanilloid receptors, protease-activated receptors and neuropeptides such as substance P have also been implicated in the pathogenesis of neurogenic inflammation in NERD patients with esophageal hypersensitivity. The development of new therapy with antiinflammatory and anti-oxidant effects is expected to assist in the treatment of intractable NERD/GERD and the prevention of carcinogenesis.     

Effect of 16,16-dimethyl PGE2 and indomethacin on bile acid-induced intestinal injury and restitution in rats

Topically administered 16,16-dimethyl prostaglandin E2 reduced bile acid-induced small intestinal mucosal injury; however, the time course of restitution after such injury and whether either exogenous or endogenous prostaglandins affect this restitution are unknown. To explore these questions, mucosal injury was produced in 50 cm small intestinal segments of anesthetized male Sprague-Dawley rats perfused in vivo for 0, 5, 15, 30, or 45 minutes with buffer containing 5 mmol/L chenodeoxycholic acid, and to assess mucosal restitution, additional rats were perfused for 45 minutes with chenodeoxycholic acid followed by 15, 30, 60 or 120 minutes with chenodeoxycholate-free buffer. The above studies were then repeated in rats receiving either intraperitoneal indomethacin (10 mg/kg) or 15 minutes of preperfusion with buffer containing 1.4 mumol/L (0.5 microgram/ml) 16,16-dimethyl prostaglandin E2. Prostaglandin pretreatment reduced and indomethacin pretreatment increased significantly the morphologic (as measured by quantitative histology) and functional (as measured by mannitol and water absorption) mucosal injury caused by chenodeoxycholic acid. However, neither pretreatment had a major impact on the time course of functional or morphologic mucosal restitution, with nearly complete restitution occurring within 1 hour. Thus, although both endogenous and exogenous prostaglandins have a significant impact on bile acid-induced small intestinal mucosal injury, this effect is not caused by an acceleration of the rate of mucosal restitution.     

Mechanism by which bile salt disrupts the gastric mucosal barrier in the dog.

Bile salts disrupt a functional "gastric mucosal barrier" increasing net forward-diffusion (+) of Na+ and back-diffusion (-) of H+. Studying canine Heidenhain pouches, we attempted to distinguish between two possible mechanisms for this effect: (a) mucosal uptake of bile salt with subsequent cellular injury or (b) dissolution of mucosal lipids by intralumenal bile salt. A 10 mM mixture of six conjugated bile salts simulating the proportions found in human bile induced net Na+ flux of 15.5 +/- 3.2 and net H+ flux of -9.9 +/- 3.3 mueq/min. This change was accompanied by an increase in phospholipid efflux out of gastric mucosa from a base-line value of 13.2 +/- 2.7 to 54.8 +/- 2.8 nmol/min (P < 0.001) and an increase in cholesterol efflux from 11.7 +/- 3.8 to 36.3 +/- 3.2 nmol/min (P < 0.001). Saturation with lecithin (25 mM) and cholesterol (50 mM) blocked disruption of the gastric mucosal barrier by bile salt (Na+ flux - 1.2 +/- 0.9, H+ flux 0.6 +/- 1.8 mueq/min). A 10 mM solution of taurodehydrocholate, a bile salt that does not form micelles, induced no net Na+ (-0.3 +/- 0.8) or H+ flux (-0.7 +/- 1.4) and did not increase efflux of phospholipid (11.3 +/- 1.7) or cholesterol (10.4 +/- 2.0) over base line. Bile salt was absorbed from the mixture of six conjugates at 752 +/- 85 nmol/min. Addition of subsaturation amounts of lecithin (4 mM) reduced bile salt absorption three fold to 252 +/- 57 (P < 0.001), but abnormal Na+ flux (14.1 +/- 3.4) and H+ flux (-15.6 +/- 3.5) persisted. Taurodehydrocholate was absorbed to an intermediate extent (467 +/- 116). Dissolution of mucosal lipids is apparently the mechanism by which bile salt disrupts the gastric mucosal barrier, and presumably at least one mechanism by which bile salt can injure the gastric mucosa.     

Cytotoxicity of ricinoleic acid (castor oil) and other intestinal secretagogues on isolated intestinal epithelial cells.

Epithelial cells were isolated from hamster small intestine by a technique of vibration and used to measure cytotoxicity in vitro of certain substances known to stimulate intestinal fluid secretion. These secretagogues have laxative properties and produce mucosal damage in vivo. Compounds tested were ricinoleic acid (caster oil), dioctyl sodium sulfosuccinate, oleic acid, sodium deoxycholate and sodium cholate. Cytotoxicity was assessed by: 1) exclusion of trypan blue; 2) release of intracellular (prelabeled) 51Cr; and 3) inhibition of cellular uptake of 3-O-methylglucose. Ricinoleate produced a dose-dependent (0.1-2.0 mM) cytotoxicity as assessed by all three methods. Oleic acid, a nonhydroxylated analog of ricinoleate, was less potent. The dihydroxy bile acid, deoxycholate, was equipotent with ricinoleate, was less potent. The dihydroxy bile acid, deoxycholate, was equipotent with ricinoleate but its trihydroxy congener, cholate, was less potent. Dioctyl sodium sulfosuccinate had cytotoxicity similar in magnitude to that of ricinoleate and deoxycholate. Cytotoxicity of these agents to isolated cells may relate to their secretory potential in vivo, their abilities to produce structural change at the mucosal surface and their laxative properties.     

Intestinal morphometry and bile acid-induced mucosal injury in chronic experimental renal failure

To examine whether the intestinal mucosa in uremia is more prone to injury, we studied acute intestinal mucosal injury in rats with experimental chronic renal failure (RF) and sham-operated and starved control animals. Intestinal injury was produced by perfusing intestinal segments in vivo with 5 mmol/L chenodeoxycholic acid. Histologic specimens were then taken from the proximal and distal perfused and unperfused intestinal segments. Quantitative morphometry was done with computerized image analysis, and samples of the unperfused intestine were assayed for protein and DNA content. Chronic RF did not significantly affect the functional or morphologic injury caused by chenodeoxycholic acid. However, it was noted that RF rats had consistently taller villi and deeper crypts in all the samples studied. The protein content and the ratio of DNA to protein was similar among the three groups. The mechanism of the increase in villus height and crypt depth in the RF rats was not related to increases in tissue water content or to alterations in protein or DNA content, and the mechanism thus remains unexplained. This study clearly demonstrates, however, that the intestinal mucosa of rats with chronic renal insufficiency is not more susceptible to mucosal injury by bile acids than is the mucosa of appropriate control animals.     

Effect of molecular structure on bile acid-induced alterations in absorptive function, permeability, and morphology in the perfused rabbit colon.

An in vivo intestinal perfusion system was used to study the effects of different bile acids on fluid secretion, mucosal permeability, and mucosal morphology in the rabbit colon. To define the structure-activity relationships of the bile acids, nine unconjugated bile acids were used, varying only in the number (two or three) or position (3, 7, or 12 or various combinations) of hydroxy or keto nuclear substituents. Results showed that bile acids with two hydroxy groups in the alpha configuration at the 3,7 position, 3,12 position, or 7,12 position induced fluid secretion, increased mucosal permeability, and produced mucosal damage as assessed by light and scanning electron microscopy and quantitated by DNA loss during perfusion. Replacement of hydroxy groups by keto groups or a change from alpha to beta configuration for the hydroxylic substituent in the 7 position abolished all three activities. Trisubstituted derivatives, whether hydroxy or keto, did not affect fluid secretion permeability or cause mucosal damage. These studies indicate that of the major primary and secondary bile acids in man, only deoxycholic and chenodeoxycholic acids alter colonic structure and function in the rabbit. They show further that the cathartic effects of bile acids have specific structural requirements; and they show that bile acid-induced secretion was invariably associated with increased mucosal permeability and epitheliolysis.     

Pathogenesis of gastro-esophageal reflux disease

Gastro-esophageal reflux disease (GERD) refers a condition that develops when the reflux of stomach contents causes troublesome symptoms and/or complications. The disease was subclassified into esophageal and extraesophageal syndromes in the new Montreal Definition. Hiatal hernia, decreased lower esophageal sphincter, transient lower esophageal sphincter relaxation, esophageal acid clearance, and delayed gastric emptying might be implicated as the pathogenesis of esophageal syndrome. Although non-erosive reflux disease (NERD) is included in the esophageal syndrome, it might be different from reflux esophagitis because of the lower response rates to acid suppression with proton pump inhibitors. Esophageal visceral hypersensitivity, sustained esophageal contractions, and abnormal tissue resistance are thought to be the mechanisms of NERD. Further investigations for the pathogenesis of each classification are expected.     

Protective effects of nonionic triblock copolymers on bile acid-mediated epithelial barrier disruption

Translocation of bacteria and other luminal factors from the intestine following surgical injury can be a major driver of critical illness. Bile acids have been shown to play a key role in the loss of intestinal epithelial barrier function during states of host stress. Experiments to study the ability of nonionic block copolymers to abrogate barrier failure in response to bile acid exposure are described. In vitro experiments were performed with the bile salt sodium deoxycholate on Caco-2 enterocyte monolayers using transepithelial electrical resistance to assay barrier function. A bisphenol A coupled triblock polyethylene glycol (PEG), PEG 15-20, was shown to prevent sodium deoxycholate-induced barrier failure. Enzyme-linked immunosorbent assay, lactate dehydrogenase, and caspase 3-based cell death detection assays demonstrated that bile acid-induced apoptosis and necrosis were prevented with PEG 15-20. Immunofluorescence microscopic visualization of the tight junctional protein zonula occludens 1 (ZO-1) demonstrated that PEG 15-20 prevented significant changes in tight junction organization induced by bile acid exposure. Preliminary transepithelial electrical resistance-based studies examining structure-function correlates of polymer protection against bile acid damage were performed with a small library of PEG-based copolymers. Polymer properties associated with optimal protection against bile acid-induced barrier disruption were PEG-based compounds with a molecular weight greater than 10 kd and amphiphilicity. The data demonstrate that PEG-based copolymer architecture is an important determinant that confers protection against bile acid injury of intestinal epithelia.     

Endohyperthermia--experimental evaluation of a new therapeutic approach for treatment of biliary carcinoma

BACKGROUND AND STUDY AIMS: To improve the prognosis of patients with unresectable, locally advanced bile duct carcinoma, new treatment strategies need to be evaluated. Hyperthermia has been successfully applied as part of multimodal therapy in esophageal and rectal carcinoma. We performed in-vitro and in-vivo experiments with a new intraluminal hyperthermia system in the biliary tract. METHODS: A radiofrequency system (13.56 MHz, Endoradiotherm XERT-200A; Olympus Optical Co., Tokyo, Japan) was used with a special intraluminal microelectrode (diameter 4.5 mm, length 40 mm) covered by a silicone balloon with cooling water and a large counter electrode for focusing the electromagnetic field around the electrode. The heating capacity of the endohyperthermia unit was examined in vitro in a muscle-equivalent phantom (agar 4 %), in isolated livers of pigs and cows, as well as in vivo in anesthetized sheep. Continuous thermometry was done with thermosensors at the applicator surface, and with multichannel thermocouple probes in the environment of the applicator. RESULTS: Endohyperthermia induced a homogeneous heating of the phantom and the isolated liver bile duct preparation to a temperature > or = 40 degrees C in an area at least 10 mm in depth. After placement of the applicator into the common bile duct of anesthetized sheep, endohyperthermia led to a consistent and repeatable heating of the surrounding tissue to 40.5 +/- 0.5 degrees C at 1 cm distance, and 39.9 +/- 0.7 degrees C at 2 cm distance. Blood pressure, heart rate, and systemic temperature did not change in vivo. Histological examination of the bile duct showed superficial mucosal necrosis (depth 100-200 microm), microvascular damage with petechiae, congestion and edema of the bile duct wall and adventitia after hyperthermia treatment in vivo. CONCLUSIONS: The intraluminal endohyperthermia system produces consistent and repeatable heating of the surrounding tissue. Since effective thermal power can reach a depth of up to 2 cm, tumors may also be heated adequately.     

Estimation of Cholesterol and Bile Acid Turnover in Man by Kinetic Analysis

The kinetics of cholesterol and bile acid turnover were determined from an analysis of the biliary lipids after a single intravenous injection of labeled cholesterol. A compartmental model was designed for the system, and the fractional metabolic rates and fluxes were determined in one lean and two obese normal humans. Each of the normals converted about 3% per day of their rapidly miscible cholesterol pool to cholic acid and 1% per day or less to chenodeoxycholate. Cholate was catabolized at about twice the rate of the dihydroxy bile acids in these normals. Two of the normals were fed corn oil with little change in their kinetic parameters from the control state. The other normal received cholestyramine and dramatically increased the bile acid flux with little change in neutral sterol catabolism. A cirrhotic patient was also studied by this technique and noted to have kinetic parameters quite different from the normals.     

呃逆的诱因分析及其对食管功能的影响

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Biliary glutathione promotes the mucosal metabolism of luminal peroxidized lipids by rat small intestine in vivo.

We previously found that exogenous GSH enhances mucosal GSH and promotes lipid hydroperoxide metabolism by rat small intestine (AW, T. Y., and M. W. WIlliams, 1992. Am. J. Physiol. 263:G665-G672). In this study, we have developed an in vivo bile and lymph fistula rat model to test the hypothesis that biliary GSH is an important luminal source of GSH. Peroxidized fish oil was infused into the proximal intestine, and hydroperoxide accumulation in lumen, mucosa, and lymph was determined. Diversion of bile decreased mucosal GSH and increased hydroperoxide accumulation in all fractions. Supplementation with GSH, but not with GSSG, increased tissue GSH and attenuated hydroperoxide accumulation (50-60%), consistent with enhancement of hydroperoxide removal by exogenous GSH. Addition of native bile deficient in GSH, but not cysteine, cystine, or GSSG, decreased luminal and lymph hydroperoxide levels by 20-30%. Amino acid supplementation concurrently attenuated hydroperoxide recoveries in these fractions by 30-40% and increased mucosal GSH by 40%, indicating a role for biliary amino acids in hydroperoxide elimination. The effect of amino acids was abolished by buthionine sulfoximine, confirming their role in GSH biosynthesis. Collectively, the results demonstrate that bile is a rich source of reductant for maintaining mucosal GSH to promote intestinal metabolism of luminal peroxidized lipids.     

Influence of obstructive jaundice on gastric mucosal barrier in dogs

The influence of obstructive jaundice on the gastric mucosal barrier was studied before and after ligation of the common bile duct using 21 mongrel dogs with a Heidenhain pouch. The gastric mucosal barrier was examined using gastric mucosal permeability of ionic net fluxes of Na+ and H+ (delta Na+, delta H+), and transmucosal electrical potential difference (PD) during the irrigation of the pouch with various test solutions. No significant differences in delta Na+ or delta H+ during the irrigation with 100 mN HCl were found between before and after obstructive jaundice. Almost the same results were observed during the irrigation with 150 mN HCl. Obvious increases in delta Na+ and delta H+ were found, when 20 mM sodium taurocholate (taurocholate) was used in addition to 100 mN HCl. No differences were, however, found between before and after obstructive jaundice. The PD during the irrigation with 100 and 150 mN HCl showed about the same levels of -50 mV. The PD decreased early during the irrigation with 20 mM taurocholate in addition to 100 mN HCl, but no differences were found in these values between before and after obstructive jaundice. Dose-response relationships were found between the concentration of taurocholate in addition to 100 mN HCl, and delta Na+, delta H+ and sigma PD/15 min, while no differences in ED50 of taurocholate for delta Na+, delta H+ or sigma PD/15 min were observed between before and after obstructive jaundice. These results suggest that obstructive jaundice itself exerts no influence on the gastric mucosal barrier with respect to gastric mucosal permeability and PD in dogs.     

Inhibition of active hexose and amino acid transport by conjugated bile salts in rat ileum

Abstract. The effect of conjugated trihydroxy bile salts, tauro- and glycocholate, and of deoxycholate on tissue uptake and mucosal to serosal transfer of actively transported hex-oses and amino acids has been examined in rat small intestine in vitro. Conjugated trihydroxy bile salts and deoxycholate markedly inhibited active transport of hexoses and amino acids in the ileum of rat small intestine, whereas in the jejunum, deoxycholate alone was inhibitory. The inhibitory effect of tauro- and glycocholate increased with incubation time. It persisted after washing of the tissue and reincubation with hexoses in a bile salt free medium, and could be observed with only 2 × 10^-4 M taurocholate. Taurocholate was able to evoke an increase of transmural potential difference (PD) in the ileum, but did not affect PD in the jejunum. Prein cubation of ileal small intestine with taurocholate depressed subsequent glucose-induced PD-increments. In the jejunum, however, taurocholate did not affect PD-increments induced by D-glucose. It is concluded that conjugated trihydroxy bile salts have to enter intestinal mucosal epithelial cells to an appreciable extent in order to affect other active, energy-requiring transport systems in rat small intestine. Previous results showing a failure of conjugated bile salts to inhibit active transport of hexoses and amino acids are explained by the fact that only jejunal transport had been examined.     

Pro-inflammatory chemokine IL-8 in gastroesophageal reflux disease

Recent reports have demonstrated significantly higher expression levels of interleukin-8 (IL-8) in patients with gastroesophageal reflux disease (GERD) including non-erosive reflux disease (NERD). The levels of IL-8 mRNA expression were significantly decreased after proton pump inhibitor. The esophageal expression of CINCs, rat IL-8-like chemokines, was markedly enhanced in the models of acute or chronic esophagitis in rats. The production of IL-8 from esophageal mucosal cells was enhanced by the exposure to bile acid. These results suggest that IL-8 chemokine may play a major role in the pathogenesis of esophageal inflammation in GERD.     

Is the ex vivo rat gastric chamber model suitable for studying the gastrotoxicity of refluxed duodenal contents? Initial results using deoxycholic acid

Duodenogastric reflux has been implicated in the pathogenesis of gastric mucosal disease but the relative toxicities of its constituents are not known. The suitability of the ex vivo rat gastric chamber model for systematic studies of bile acid gastrotoxicity was assessed using deoxycholic acid (DCA 0.2-5.0 mmol/l). Acute challenge with 5.0 mmol/l DCA produced significant loss of gastric transmucosal potential difference (delta PD = 25.9 +/- 3.3 mV: mean +/- SEM; p less than 0.01) compared with saline challenge (1.5 +/- 0.5 mV). Significant delta PD values were produced by DCA concentrations down to 0.5 mmol/l (16.0 +/- 2.8 mV). Challenge with 5.0 mmol/l DCA also caused significant increases in chamber fluid concentrations of nucleic acid (2.8 +/- 0.3 micrograms/ml; p less than 0.05) and acid phosphatase (130 +/- 23.4 microU/ml; p less than 0.01). This study demonstrates DCA gastrotoxicity at concentrations comparable to human intragastric total bile acid concentrations and the suitability of this model for studying the toxic components of refluxed duodenal contents.