Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats.

Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10-100 µM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 µM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 µM), and a P2Y receptor antagonist, cibacron blue F3GA (200 µM), inhibited the ATP (100 µM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 µM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 µM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 µM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.

Resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into esophageal fibroblasts via AKT signaling pathway.

Objectives: Resveratrol has been implicated in the differentiation and development of human umbilical cord mesenchymal stem cells. The differentiation of into esophageal fibroblasts is a promising strategy for esophageal tissue engineering. However, the pharmacological effect and underlying mechanism of resveratrol on human umbilical cord mesenchymal stem cells differentiation are unknown. Here, we investigated the effects and mechanism of resveratrol on the differentiation of human umbilical cord mesenchymal stem cells. Methods: Using a transwell-membrane coculture system to culture human umbilical cord mesenchymal stem cells and esophageal fibroblasts, we examined how resveratrol act on the differentiation of human umbilical cord mesenchymal stem cells. Immunocytochemistry, Sirius red staining, quantitative real-time PCR, and Western blotting were performed to examine collagen synthesis and possible signaling pathways in human umbilical cord mesenchymal stem cells. Results: We found that resveratrol promoted collagen synthesis and AKT phosphorylation. However, co-treatment of cells with resveratrol and the PI3K inhibitor LY294002 inhibited collagen synthesis and AKT phosphorylation. We demonstrated that resveratrol down-regulated the expression of IL-6, TGF-ß, caspase-9, and Bax by activating the AKT pathway in human umbilical cord mesenchymal stem cell. Furthermore, resveratrol inhibited phosphorylated NF-ĸB in human umbilical cord mesenchymal stem cells. Conclusion: Our data suggest that resveratrol promotes the differentiation of human umbilical cord mesenchymal stem cells into fibroblasts. The underlying mechanism is associated with the downregulation of IL-6 and TGF-ß via the AKT pathway and by inhibiting the NF-ĸB pathway. Resveratrol may be useful for esophageal tissue engineering.

Resolvin D1 attenuates acid-induced DNA damage in esophageal epithelial cells and rat models of acid reflux.

The role of resolvin D1 (RvD1) in gastroesophageal reflux disease (GERD) remains largely unknown. Here, we investigated the potential role of RvD1 in acid-induced DNA damage in esophageal epithelial cells, patients with refractory GERD and a rat model of acid reflux. Weak acid exposure induced longer comet tails, reactive oxygen species (ROS) generation, oxidative DNA damage and DNA double-strand breaks (DSBs) in cells and RvD1 (0.1 µM) blocked all these effects. Mechanistic analyses showed that apart from ROS-reducing effects, RvD1 possessed a strong capacity to promote DNA damage repair, augmenting cell cycle checkpoint activity and DSB repair by modulating phosphatase and tensin homolog (PTEN) in cells. We also detected the surface expression of formyl peptide receptor 2 (FPR2), a receptor for RvD1, in the esophageal epithelial cells, and inhibition of FPR2 abrogated the protective effects of RvD1 on cells. Furthermore, a positive correlation between RvD1 and PTEN was observed predominantly in the esophageal epithelium from patients with refractory GERD (r = 0.67, P < 0.05). Additionally, RvD1 administration upregulated PTEN, suppressed DNA DSBs and alleviated microscopic damage in the rat model of gastric reflux. FPR2 gene silencing abolished the therapeutic effects of RvD1 on the rat model. Taken together, RvD1 binding to FPR2 protects the esophageal epithelium from acid reflux-induced DNA damage via a mechanism involving the inhibition of ROS production and facilitation of DSB repair. These findings support RvD1 as a promising approach that may be valuable for the treatment of GERD.

Deoxycholic acid induces proinflammatory cytokine production by model oesophageal cells via lipid rafts.

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.

Effect of low-threshold versus high-threshold genitalia stimuli on the cystometry parameters in male rats.

The cross talk between external genitalia and urinary bladder could be used as part of management to certain pathological conditions affecting urinary bladder. Since urinary bladder function is also affected by pathologies of other organs (e.g., colon and esophagus), the effect of genitalia stimuli on parameters of bladder function in normal or under different pathological conditions needs to be characterized. Cystometry recordings in male rats were used to examine the effect of low-threshold (LT) and high-threshold (HT) stimulation of the scrotum and penis on urinary bladder function. These effects were studied in intact, colon irritation (CI), and esophagus irritation (EI) groups. Although HT penile stimulation had a significant inhibitory effect on micturition reflex in all groups, CI hypersensitized the penile-bladder inhibitory reflex. In addition, LT penile stimulation had a significant inhibitory effect on micturition, which was significant in CI group only. On the other hand, HT penile stimulation in CI group significantly increased the timing parameters of cystometry. Whereas LT and HT penile stimuli in EI group had a significantly increasing effect on all pressure parameters of cystometry. The scrotal stimuli had minimal effect on bladder function in all groups except for HT scrotal stimulation in the CI group, where it had a significant inhibitory effect on micturition reflex and significantly increased the maximum pressure and pressure amplitude of micturition cycles. These results show that CI and EI exacerbate the effects of genitalia stimuli, especially penile stimuli, on urinary bladder function.

Effects of codeine on esophageal peristalsis in humans using high resolution manometry.

BACKGROUND AND AIM: Opioid receptors agonists have been demonstrated to impair lower esophageal sphincter (LES) relaxation and induce spastic esophageal dysmotility, but little was known for their impact on distension-induced secondary peristalsis. The aim of the study was to investigate the hypothesis whether acute administration of codeine can influence physiological characteristics of primary and secondary peristalsis in healthy adults. METHODS: Eighteen healthy volunteers (13 men, mean age 27.5 years, aged 20-43 years) underwent high resolution manometry (HRM) with a catheter containing an injection port in mid-esophagus. Secondary peristalsis was performed with 10 and 20 mL rapid air injections. Two different sessions including acute administration of codeine (60 mg) or the placebo were randomly performed. RESULTS: Codeine significantly increased 4-s integrated relaxation pressure (IRP-4s) (P = 0.003) and shortened distal latency (DL) (P = 0.003) of primary peristalsis. The IRP-4s of secondary peristalsis was also significantly higher after codeine than the placebo during air injections with 10 mL (P = 0.048) and 20 mL (P = 0.047). Codeine significantly increased the frequency of secondary peristalsis during air injections with 10 mL than the placebo (P = 0.007), but not for air injection with 20 mL (P = 0.305). CONCLUSIONS: In addition to impair LES relaxation and reduce distal latency of primary peristalsis, codeine impairs LES relaxation of secondary peristalsis and increases secondary peristaltic frequency. Our study supports the notion in human esophagus that the impact of opioids on peristaltic physiology appears to be present in both primary and secondary peristalsis.

FBX4 mediates rapid cyclin D1 proteolysis upon DNA damage in immortalized esophageal epithelial cells.

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

Daidzein ameliorates experimental acute reflux esophagitis in rats via regulation of cytokines.

Context: Daidzein is a secondary metabolite derived from plants, has a flavonoid structure and is known for its protective activity in gastrointestinal disorders. Objective: The current work determines the preventive effect of daidzein against injury in the esophagus mucosa induced by esophageal reflux (RE) in an animal model. Methods: Adult male Wistar rats were classified into six groups: normal control, ER + different doses of daidzein and ER + omeprazole. RE was induced in all animals except controls and supplemented with daidzein and standard drugs orally for 6 hours. Serum and tissue were used for further biochemical parameters. Results: Daidzein as a flavonoid has antioxidant properties and shows in vitro antioxidant activity. The outcomes also reveal an elevation in lipid peroxidation and a decline in the levels of sulphhydryl groups and glutathione, along with the depletion in the activities of enzymatic antioxidants in the oxidative stress state. In a dose-dependent manner daidzein and omeprazole amended all macroscopic and biochemical variations and protected against the raised level of hydrogen peroxide (H2O2), calcium and free iron levels in esophageal tissue induced during RE. It also improved the expression and level of proinflammatory cytokines. Conclusion: The finding reports that daidzein has a potential to show a shielding effect against esophagus damage induced by RE in rats, at least in part via alteration of inflammatory cytokines.

Protective Effects of Inflammation of Curcumae Longae Rhizoma 30% EtOH Extract on Acute Reflux Esophagitis Rats.

Gastroesophageal reflux disease (GERD) is induced by the reflux of stomach contents or gastric acid, pepsin into the esophagus for prolonged periods of time due to defection of the lower esophageal sphincter. Reflux esophagitis is a disease found in less than 50% of GERD patients. This study is aimed at evaluating the protective effect of Curcumae longae Rhizoma 30% EtOH extract (CLR) in acute reflux esophagitis (ARE) rats. CLR measured antioxidant activity through in vitro experiments. Based on the results, we performed experiments in vivo. Before 90 min ARE induction, CLR was administered orally by concentration. ARE was derived by linking the metastatic junction between pylorus and forestomach and corpus in Sprague-Dawley rats. And rats were sacrificed 5 h after surgery. We analyzed the expression of antioxidant and inflammatory-related markers by western blot and observed the production of alanine aminotransferase (ALT), aspartate aminotransferase (AST), reactive oxygen species (ROS), peroxynitrite (ONOO-), and thiobarbituric acid reactive substance (TBARS). The administration of CLR reduced esophagus tissue damage in rats with acute reflux esophagitis and decreased the elevated ALT, AST, ROS, ONOO-, and TBARS. In addition, CLR effectively increased antioxidant-related factors and reduced inflammatory protein. Overall, these results suggest that CLR would be used as a therapeutic material in protection and treatment for ARE. Overall, CLR treatment informed that markedly ameliorated inactivation of NF-κB led to the inhibition of the expressions of proinflammatory proteins. These results suggest that CLR would be used as a therapeutic material in protection and treatment for ARE.

Effect of prucalopride on sildenafil-induced inhibition of esophageal peristalsis in healthy adults.

BACKGROUND AND AIM: Prucalopride, a high-affinity 5-hydroxytryptamine 4 receptor agonist, promotes esophageal peristalsis, while phosphodiesterase type 5 inhibitor sildenafil inhibits esophageal peristalsis. The present study was aimed to evaluate whether prucalopride would augment esophageal peristalsis subsequent to the application of sildenafil. METHODS: Seventeen healthy adults underwent high-resolution manometry by a catheter with one injection port located in the mid-esophagus. Secondary peristalsis was assessed by rapid air injections after water swallows. Two sessions were randomly performed including acute administration of sildenafil 50 mg after pretreatment with prucalopride or the placebo. RESULTS: The frequency of primary peristalsis subsequent to the administration of sildenafil was significantly increased by prucalopride (P = 0.02). Prucalopride also significantly increased distal contractile integral of primary peristalsis subsequent to the administration of sildenafil (P = 0.03). No difference in the frequency of secondary peristalsis subsequent to the administration of sildenafil for air injects of 10 mL (P = 0.14) or 20 mL (P = 0.21) was found between prucalopride and placebo. Prucalopride did not change distal contractile integral of secondary peristalsis subsequent to the administration of sildenafil for air injections of 10 mL (P = 0.09) or 20 mL (P = 0.12). CONCLUSIONS: Prucalopride modulates sildenafil-induced inhibition of primary peristalsis by increasing its effectiveness and peristaltic wave amplitude. Our findings suggest that activation of 5-hydroxytryptamine 4 receptors plays a role in mediating sildenafil-induced inhibition of esophageal primary peristalsis rather than secondary peristalsis.

Effects of phosphodiesterase-5 inhibitor sildenafil on esophageal secondary peristalsis: Studies with high-resolution manometry.

BACKGROUND AND AIM: Secondary peristalsis contributes to the clearance of retained refluxate from the esophagus. Sildenafil, a phosphodiesterase-5 inhibitor, inhibits primary esophageal peristalsis, but its effects on secondary peristalsis remain unknown. This study sought to investigate whether sildenafil could influence physiological characteristics of secondary peristalsis by applying high-resolution manometry (HRM). METHODS: Seventeen healthy volunteers (15 men and 2 women, aged 30.2 ± 6.4 years) underwent two HRM studies on separate days following the administration of either a placebo or 50 mg of sildenafil in a random order. Both studies were performed using a water-perfused HRM catheter containing one air injection channel positioned in the mid-esophagus. Secondary peristalsis was stimulated by a rapid mid-esophageal injection of 10 or 20 mL of air 1 h after the administration of either the placebo or sildenafil. The frequency and distal contractile integral of secondary peristalsis were then compared. RESULTS: Complete secondary peristalsis triggered by the 20-mL air injection was more frequent than observed with the 10-mL air injection (P < 0.001). The vigor of secondary peristalsis triggered by the injection of either volume of air was lower than that of primary peristalsis (P < 0.001). Sildenafil significantly reduced the success rate (P ≤ 0.001) and vigor (P < 0.001) of secondary peristalsis relative to the effects of the placebo at both distension volumes. CONCLUSIONS: Secondary peristalsis can be successfully triggered by rapid air injection during HRM. Sildenafil reduces both the success rate and the vigor of secondary peristalsis, similar to that seen with primary peristalsis.

Cellular Nrf2 Levels Determine Cell Fate during Chemical Carcinogenesis in Esophageal Epithelium.

Nrf2 is essential for cytoprotection against carcinogens, and through systemic Nrf2 knockout mice, Nrf2-deficient cells were shown to be susceptible to chemical carcinogens and prone to developing cancers. However, the oncogenic potential of Nrf2-deficient epithelial cells surrounded by normal cells in the esophagus could not be assessed by previous models, and the fate of Nrf2-deficient cells in such situations remains elusive. In this study, therefore, we generated mice that harbor almost equal levels of cells with Nrf2 deleted and those with Nrf2 intact in the basal layer of the esophageal epithelium, utilizing inducible Cre-mediated recombination of Nrf2 alleles in adults through moderate use of tamoxifen. In this mouse model, epithelial cells with Nrf2 deleted were maintained with no obvious decrease or phenotypic changes for 12 weeks under unstressed conditions. Upon exposure to the carcinogen 4-nitroquinoline-1-oxide (4NQO), the cells with Nrf2 deleted accumulated DNA damage and selectively disappeared from the epithelium, so almost all 4NQO-induced tumors originated from cells with Nrf2 intact and not from those with Nrf2 deleted. We propose that cells with Nrf2 deleted do not undergo carcinogenesis due to selective elimination upon exposure to 4NQO, indicating that cellular Nrf2 abundance and the epithelial environment determine the cell fate or oncogenic potential of esophageal epithelial cells in 4NQO-induced carcinogenesis.

Same-day opioid administration in opiate naïve patients is not associated with opioid-induced esophageal dysfunction (OIED).

BACKGROUND: Opioid-induced esophageal dysfunction (OIED) is a recognized complication of chronic opioid use. However, the impact of acute opioid administration on esophageal motility remains unclear. METHODS: Opioid naïve patients with high-resolution manometry (HRM) <480 min following esophagogastroduodenoscopy (EGD) (opioid-HRM) and a control group with HRM <36 h prior to EGD between January 1, 2016, and November 10, 2018, from a single institution were identified. EGDs were performed exclusively with versed and fentanyl. KEY RESULTS: One hundred and seventy-four patients were identified, with 83 (47.7%) opioid-HRM and 91 (52.3%) controls. Mean time from EGD to HRM was 229 (78-435) min. Baseline clinical features and HRM indications were similar between opioid-HRM and controls. Chicago classification v3.0 defined HRM findings were similar between groups. Major motility disorders as defined by the Chicago classification v3.0 occurred at a similar frequency among opioid-HRM and controls (27.7% vs. 36.3%, p = 0.23). Mean distal contractile integrity (DCI) was higher in opioid-HRM (1939.3 ± 1318.9 vs. 1792.2 ± 2062.3 mmHg∙cm∙s, p = 0.043), but maximum DCI, distal latency, and integrated relaxation pressure did not differ between groups. Subgroup analysis assessing time and dose dependency did not identify differences in individual manometric parameters and Chicago classification v3.0 diagnosis between patients with HRM <240 min after EGD, >240 min after EGD, ≥125 mcg of IV fentanyl, <125 mcg IV fentanyl and controls. CONCLUSIONS AND INFERENCES: Same-day acute opioid administration did not affect HRM findings in opioid naïve patients. Studies assessing the pathophysiology of and duration-dependent relationship with opioids in OIED are needed.

PROSPECTS FOR CREATION OF RADIOPROTECTIVE MEANS BASED ON NATURAL POLYPHENOLS AND POLYSACCHARIDES. / PERSPEKTYVY STVORENNIa ZASOBIV RADIOZAKhYSNOÏ DIÏ NA OSNOVI KOMPOZYTsII PRYRODNYKh POLIFENOL'NYKh SPOLUK I POLISAKhARYDIV.

The high level of nuclear radiation threats in the modern world determines the need to find new means of pharmacological protection of the health of military personnel and civilians from the effects of ionizing radiation. Of particular scientific interest in this aspect are natural polyphenols as a promising basis for the development of newdrugs, radiomodifiers. OBJECTIVE: Justification of the prospects of creating radioprotective agents based on compositions of plantpolyphenolic substances (PPS) and polysaccharides. MATERIAL AND METHODS: The experiments were performed on 130 laboratory white rats-male of Wistar line sexually mature weighting 180-240 g. Animals once received a total X-ray dose equivalent to 4.25 Gy. The effects ofquercetin and patulaten to the processes of reparative regeneration under conditions of X-ray irradiation andagainst the background of butadione suppression were investigated. Indicators in the study groups were compared using the Student's t-test for independent samples; the differences were considered statistically significantat p < 0.05. RESULTS: The various biological properties of quercetin, in particular, the ability to bind hydroxyl radicals, is thepotential for developing radioprotective agents based on it. At the first stage of the study, the effect of PPS andtheir compositions with polysaccharides on reparative regeneration was studied against the background of its suppression in intact and irradiated animals. With the oral administration of PPS and their compositions with pectin towhite rats, 30 minutes before the administration of butadion, an increase in the processes of reparative regeneration in the cells of the covering epitheliumof the esophagus was observed. At the same time, quercetin granulescaused the most expressive effect, which increased the statistically significant value of the mitotic index by 78.5 %in relation to the group of animals injected with butadion. At the second stage of the study, the effect of polyphenolic substances and their compositions with pectin on the processes of reparative regeneration in intact and irradiated white rats was studied on a model of linear skin wounds. The prophylactic administration of quercetin granules and the treatment of wounds with 20 % sterile quercetin gel significantly accelerated the healing process.Experimental data indicate that quercetin granules have the ability to stimulate the processes of reparative regeneration, quercetin showed the greatest efficiency with simultaneous use inside and topically. CONCLUSIONS: The research results indicate the promise of developing radioprotective drugs that can stimulatereparative regeneration processes based on compositions of plant polyphenolic substances and polysaccharides invarious qualitative and quantitative ratios.

Impact of Deoxycholic Acid on Oesophageal Adenocarcinoma Invasion: Effect on Matrix Metalloproteinases.

Bile acids (BAs) have been implicated in the development of oesophagitis, Barrett's oesophagus and oesophageal adenocarcinoma (OAC). However, whether BAs promote cancer invasiveness has not been elucidated. We evaluated the role of BAs, in particular deoxycholic acid (DCA), in OAC invasion. Migration and invasiveness in untreated and BA-treated oesophageal SKGT-4 cancer cells were evaluated. Activity and expression of different matrix metalloproteinases (MMPs) were determined by zymography, ELISA, PCR and Western blot. Finally, human OAC tissues were stained for MMP-10 by immunohistochemistry. It was found that SKGT-4 cells incubated with low concentrations of DCA had a significant increase in invasion. In addition, MMP-10 mRNA and protein expression were also increased in the presence of DCA. MMP-10 was found to be highly expressed both in-vitro and in-vivo in neoplastic OAC cells relative to non-neoplastic squamous epithelial cells. Our results show that DCA promotes OAC invasion and MMP-10 overexpression. This study will advance our understanding of the pathophysiological mechanisms involved in human OAC and shows promise for the development of new therapeutic strategies.

Human esophageal myofibroblasts increase squamous epithelial thickness via paracrine mechanisms in an in vitro model of gastroesophageal reflux disease.

The pathogenesis of esophageal injury in gastroesophageal reflux disease (GERD) is incompletely understood. We modeled exposure of human esophageal myofibroblasts (HEMFs) to gastroesophageal reflux by repeated treatment with pH 4.5 and pH 4.5 bile salts and determined the effects on the epithelium in a 3D organotypic-like air-liquid interface model. Total, basal and supra-basal thickness of the epithelium were measured and immunostaining for p63, for basal (CK 14) and supra-basal (CK 4) squamous differentiation markers, and for cell proliferation (PCNA) were performed. Epithelial cell proliferation in response to HEMF conditioned media was also assessed in 2D culture. In the 3D organotypic model, total epithelial thickness increased similarly with pH 4.5 and pH 4.5 bile salt treated versus untreated and bile salt treated HEMF conditioned media. Epithelial p63 immunostaining was increased and multilayered. There was expansion of the CK14+ basal and CK4+ supra-basal layers in the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs versus untreated HEMF conditioned media. PCNA + cells per µm of tissue were unchanged in the basal layer across all treatment conditions while PCNA + cells per total DAPI + cells were decreased. In 2D culture, basal epithelial proliferation decreased with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs compared to conditioned media from untreated HEMF conditioned media. Secreted factors from HEMFs treated with acidic stimuli encountered in GERD increase epithelial thickness compared to secreted factors from untreated HEMFs and expand both basal and supra-basal layers. Our findings demonstrate for the first time paracrine regulation of the squamous epithelium from acid stimulated HEMFs. The effects of secreted factors from acid treated HEMFs on basal cell proliferation in this model and the mechanism mediating the increase in epithelial thickness merit further investigation.

Effects of Lixisenatide Versus Liraglutide (Short- and Long-Acting GLP-1 Receptor Agonists) on Esophageal and Gastric Function in Patients With Type 2 Diabetes.

OBJECTIVE: Short-acting glucagon-like peptide 1 receptor agonists (GLP-1 RAs) decelerate gastric emptying more than long-acting GLP-1 RAs. Delayed gastric emptying is a risk factor for gastroesophageal reflux disease. We aimed to measure esophageal reflux and function as well as gastric emptying and acid secretion during treatment with short-acting (lixisenatide) and long-acting (liraglutide) GLP-1 RAs. RESEARCH DESIGN AND METHODS: A total of 57 subjects with type 2 diabetes were randomized to a 10-week treatment with lixisenatide or liraglutide. Changes from baseline in the number of reflux episodes during 24-h pH registration in the lower esophagus, lower esophagus sphincter pressure, gastric emptying (13C-sodium octanoate acid breath test), and gastric acid secretion (13C-calcium carbonate breath test) were analyzed. RESULTS: Gastric emptying half-time was delayed by 52 min (Δ 95% CI 16, 88) with lixisenatide (P = 0.0065) and by 25 min (3, 46) with liraglutide (P = 0.025). There was no difference in the number of reflux episodes (mean ± SEM 33.7 ± 4.1 vs. 40.1 ± 5.3 for lixisenatide and liraglutide, respectively, P = 0.17) or the extent of gastroesophageal reflux (DeMeester score) (35.1 ± 6.7 vs. 39.7 ± 7.5, P = 0.61), with similar results for the individual GLP-1 RAs. No significant changes from baseline in other parameters of esophageal motility and lower esophageal sphincter function were observed. Gastric acidity decreased significantly by -20.7% (-40.6, -0.8) (P = 0.042) with the GLP-1 RAs. CONCLUSIONS: Lixisenatide exerted a more pronounced influence on gastric emptying after breakfast than liraglutide. Neither lixisenatide nor liraglutide had significant effects on esophageal reflux or motility. Gastric acid secretion appears to be slightly reduced by GLP-1 RAs.

Anticancer effects and possible mechanisms of lycopene intervention on N-methylbenzylnitrosamine induced esophageal cancer in F344 rats based on PPARγ1.

Lycopene, a natural carotenoid, has potential chemopreventive effects in many cancers. This study aimed to examine the effects of lycopene on regulating the inflammation and apoptosis of N-nitrosomethylbenzylamine(NMBzA) induced esophageal cancer in F344 rats. After the rats were fed normal diets containing different concentrations of lycopene for 25 weeks (10, 25, 50 mg/kg·d of lycopene, respectively), the incidence of tumors in the rats treated with lycopene was significantly lower than that in the simple exposed group (P < 0.05). The antioxidant activity of lycopene was exerted by measuring the levels of GSH-PX, SOD and MDA activity by oxidative stress kits. Furthermore, through western blotting analysis lycopene intervention was found to have significantly improved apoptosis cytokines by increasing the protein expression levels of PPARγ and caspase-3, and also significantly reduced inflammatory cytokines by decreasing the protein expression of NF-κB and COX-2 in the esophagus tissue, especially in the 25 mg/kg of lycopene intervention group (all P < 0.05). These results demonstrated that appropriate dose of lycopene intervention could inhibit the carcinogenesis of esophageal in F344 rats through the possible mechanisms of anti-inflammatory and pro-apoptosis.

Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium.

BACKGROUND: The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. METHODS: We established a novel stepwise protocol with transwell culture and an air-liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARß and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus. RESULTS: We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation. CONCLUSION: We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

Antifibrotic Effects of the Thiazolidinediones in Eosinophilic Esophagitis Pathologic Remodeling: A Preclinical Evaluation.

INTRODUCTION: Eosinophilic esophagitis (EoE) is a T-helper 2 (Th2), eosinophilic disease associated with pathologic tissue remodeling that leads to end-organ dysfunction. During early-stage disease, inflammation and subepithelial fibrosis are coupled and reversible, but in late-stage or therapy-resistant disease, there can be uncoupling of these features with progressive esophageal rigidity and strictures contributing to clinical dysphagia and food impactions. No current pharmacotherapeutic interventions directly target esophageal fibrosis. Based on the ability of the thiazolidinediones (TZD) to regulate intestinal and hepatic fibrosis, we tested the antifibrotic effects of the TZDs, rosiglitazone and pioglitazone, in preclinical studies using primary human esophageal fibroblasts. METHODS: Primary fibroblasts isolated from normal or EoE esophagi were treated with transforming growth factor (TGF)-ß1 in the absence or presence of TZDs and, in some experiments, without or with budesonide and analyzed by quantitative real-time PCR and immunoblotting. Immunohistochemical analysis of human esophageal biopsies was performed. RESULTS: EoE esophageal biopsies and esophageal fibroblasts expressed higher levels of the TZD receptor, peroxisome proliferator-activated receptor-γ (PPAR-γ), than normal controls. PPAR-γ was inducible by the Th2 cytokine, interleukin 4 (IL-4). TZD significantly reduced TGF-ß1-induced myofibroblast and fibrotic gene and protein expression preferentially in EoE, but not normal esophageal fibroblasts. In esophageal fibroblasts, TGF-ß1 increased phosphorylated Smad2/3 and p38, but TZDs preferentially inhibited p38 phosphorylation, suggesting signaling pathway-specific effects. The TZDs were more potent than budesonide at decreasing collagen-1α1 expression. DISCUSSION: The TZDs preferentially exert antifibrotic effects in TGF-ß1-activated EoE fibroblasts and provide a preclinical foundation for further investigation of the potential of the TZDs in EoE pathologic remodeling.