Improved gastric residence time of famotidine by raft-forming drug delivery system using DOE.

OBJECTIVE: This study investigated the raft-forming suspension of famotidine as an anti-reflux formulation to improve the oral bioavailability of narrow absorption window drugs by enhancing gastric residence time (GRT) and preventing gastro-esophageal reflux disease (GERD). METHOD: Various combinations of raft-forming agents, such as Tragacanth gum (TG), guar gum (GG), and xanthan gum (XG), were evaluated alongside sodium alginate (SA) to develop an effective raft. Preformulation studies and preliminary screening were conducted to identify the most suitable raft-forming agent, and GG was chosen due to its mucilaginous properties. The formulation was optimized using a 32 full factorial design, with the quantities of GG and SA as independent factors and apparent viscosity and in-vitro drug release (%) as dependent factors. The in vivo floating behavior study was performed for optimized and stabilized formulation. RESULTS: Among the tested batches, F6 was selected as the optimized formulation. It exhibited desirable characteristics such as adequate raft weight for extended floating in gastric fluid, improved apparent viscosity, and a significant percentage of drug release at 12 h. A mathematical model was applied to the in-vitro data to gain insights into the drug release mechanism of the formulation. The stability of the suspension was assessed under accelerated conditions, and it demonstrated satisfactory stability. The formulation remains floating in the Rabbit stomach for more than 12 h. CONCLUSION: It concludes that the developed formulation has enhanced bioavailability in the combination of GG and SA. The floating layer of the raft prevents acid reflux, and the famotidine is retained for an extended period of time in the gastric region, preventing excess acid secretion. The developed formulations are effective for stomach ulcers and GERD, with the effect of reducing acid secretion by H2 receptor antagonists.

Rebound Acid Hypersecretion after Withdrawal of Long-Term Proton Pump Inhibitor (PPI) Treatment-Are PPIs Addictive?

Proton pump inhibitors (PPIs) are widely used in the long-term treatment of gastroesophageal reflux disease (GERD) and other upper gastrointestinal disorders, such as the healing of peptic ulcers and/or prophylactic treatment of peptic ulcers. PPIs are also widely used as symptomatic treatment in patients with functional dyspepsia. One of the adverse effects of the long-term use of PPI is rebound acid hypersecretion (RAHS), which can occur after the withdrawal of PPI therapy due to a compensatory increase in gastric acid production. Mechanisms of the RAHS have been well established. Studies have shown that pentagastrin-stimulated acid secretion after the discontinuation of PPIs increased significantly compared to that before treatment. In healthy volunteers treated with PPIs, the latter induced gastrointestinal symptoms in 40-50% of subjects after the discontinuation of PPI therapy but after stopping the placebo. It is important for practicing physicians to be aware and understand the underlying mechanisms and inform patients about potential RAHS before discontinuing PPIs in order to avoid continuing unnecessary PPI therapy. This is important because RAHS may lead patients to reuptake PPIs as symptoms are incorrectly thought to originate from the recurrence of underlying conditions, such as GERD. Mechanisms of RAHS have been well established; however, clinical implications and the risk factors for RAHS are not fully understood. Further research is needed to facilitate appropriate management of RAHS in the future.

Differentially expressed genes of esophageal tissue in male acute and chronic sleep deprivation mice.

Gastroesophageal reflux disease (GERD) is associated with sleep disturbances. However, mechanisms underlying these interactions remain unclear. Male acute and chronic sleep deprivation (SD) mice were used for this study. Mice in the chronic SD group exhibited anxiety- and depression-like behaviors. We further performed high-throughput genome sequencing and bioinformatics analysis to screen for featured differentially expressed genes (DEGs) in the esophageal tissue. The acute SD group, comprised 25 DEGs including 14 downregulated and 11 upregulated genes. Compared with the acute SD group, more DEGs were present in the chronic SD group, with a total of 169 DEGs, including 88 downregulated and 81 upregulated genes. Some DEGs that were closely related to GERD and associated esophageal diseases were significantly different in the chronic SD group. Quantitative real-time polymerase chain reaction verified the downregulation of Krt4, Krt13, Krt15 and Calml3 and upregulation of Baxl1 and Per3. Notably, these DEGs are involved in biological processes, which might be the pathways of the neuroregulatory mechanisms of DEGs expression.

Saliva Production and Esophageal Motility Influence Esophageal Acid Clearance Related to Post-reflux Swallow-Induced Peristaltic Wave.

BACKGROUND: The post-reflux swallow-induced peristaltic wave (PSPW) brings salivary bicarbonate to neutralize residual distal esophageal mucosal acidification. AIMS: To determine if reduced saliva production and esophageal body hypomotility would compromise PSPW-induced pH recovery in the distal esophagus. METHODS: In this multicenter retrospective cross-sectional study, patients with confirmed Sjogren's syndrome and scleroderma/mixed connective tissue disease (MCTD) who underwent high resolution manometry (HRM) and ambulatory pH-impedance monitoring off antisecretory therapy were retrospectively identified. Patients without these disorders undergoing HRM and pH-impedance monitoring for GERD symptoms were identified from the same time-period. Acid exposure time, numbers of reflux episodes and PSPW, pH recovery with PSPW, and HRM metrics were extracted. Univariate comparisons and multivariable analysis were performed to determine predictors of pH recovery with PSPW. RESULTS: Among Sjogren's syndrome (n = 34), scleroderma/MCTD (n = 14), and comparison patients with reflux symptoms (n = 96), the scleroderma/MCTD group had significantly higher AET, higher prevalence of hypomotility, lower detected reflux episodes, and very low numbers of PSPW (p ≤ 0.004 compared to other groups). There was no difference in pH-impedance metrics between Sjogren's syndrome, and comparison patients (p ≥ 0.481). Proportions with complete pH recovery with PSPW was lower in Sjogren's patients compared to comparison reflux patients (p = 0.009), predominantly in subsets with hypomotility (p < 0.001). On multivariable analysis, diagnosis of Sjogren's syndrome, scleroderma/MCTD or neither (p = 0.014) and esophageal hypomotility (p = 0.024) independently predicted lack of complete pH recovery with PSPW, while higher total reflux episodes trended (p = 0.051). CONCLUSIONS: Saliva production and motor function are both important in PSPW related pH recovery.

Portable and Rapid Fluorescence Turn-On Detection of Total Pepsin in Saliva Based on Strong Electrostatic Interactions.

Salivary pepsin has been proposed as a promising diagnostic marker for gastroesophageal reflux disease (GERD). However, the activity of human pepsin is strongly influenced by pH, and the acidic condition (pH ∼ 2) is optimal, which is a contradiction for the pepsin detection kit based on its catalytic activity. Thus, its accurate quantification in saliva (neutral pH) by readily rapid tools with simplicity and low cost is still challenging. Herein, a convenient fluorescence assay has been developed for the rapid detection of pepsin at neutral pH based on its electrostatic interaction with SYBR Green (SG) rather than the bioactivity. At neutral pH, the positively charged SG fluorophore can be effectively adsorbed by the negatively charged pepsin due to the low isoelectric point (pI) and large molecular size of pepsin. Thus, the molecular rotation of SG is limited, and its fluorescence intensity is significantly increased. The strategy was further confirmed to have the same fluorescence response as that of normally active and inactivated pepsin. Due to the unique pI of pepsin, the fluorescence strategy is highly selective for pepsin compared to other proteins. On the basis of this strategy, a smartphone-based fluorescence capture device integrated with a programmed Python program was fabricated for both color recognition and the accurate detection of pepsin within 3 min. Under the optimal conditions, this turn-on sensor allowed for the on-site analysis of pepsin with a detection limit of 0.2 µg/mL. Moreover, this strategy was successfully used to assess salivary pepsin to aid in the noninvasive diagnosis of GERD.

Gabapentin alleviated the cough hypersensitivity and neurogenic inflammation in a guinea pig model with repeated intra-esophageal acid perfusion.

OBJECTIVE: The anti-tussive effect of gabapentin and its underlying neuromodulatory mechanism were investigated via a modified guinea pig model of gastroesophageal reflux-related cough (GERC). METHODS: Intra-esophageal perfusion with hydrochloric acid (HCl) was performed every other day 12 times to establish the GERC model. High-dose gabapentin (48 mg/kg), low-dose gabapentin (8 mg/kg), or saline was orally administered for 2 weeks after modeling. Cough sensitivity, airway inflammation, lung and esophagus histology, levels of substance P (SP), and neurokinin-1 (NK1)-receptors were monitored. RESULTS: Repeated intra-esophageal acid perfusion aggravated the cough sensitivity in guinea pigs in a time-dependent manner. The number of cough events was significantly increased after 12 times HCl perfusion, and the hypersensitivity period was maintained for 2 weeks. The SP levels in BALF, trachea, lung, distal esophagus, and vagal ganglia were increased in guinea pigs receiving HCl perfusion. The intensity of cough hypersensitivity in the GERC model was significantly correlated with increased SP expression in the airways. Both high and low doses of gabapentin administration could reduce cough hypersensitivity exposed to HCl perfusion, attenuate airway inflammatory damage, and inhibit neurogenic inflammation by reducing SP expression from the airway and vagal ganglia. CONCLUSIONS: Gabapentin can desensitize the cough sensitivity in the GERC model of guinea pig. The anti-tussive effect is associated with the alleviated peripheral neurogenic inflammation as reflected in the decreased level of SP.

MicroRNA dysregulation and therapeutic opportunities in esophageal diseases.

MicroRNAs (miRNAs) are a class of small endogenous RNA molecules between 18 and 25 nucleotides long. The primary function of miRNAs is in the posttranscriptional regulation of mRNA targets through RNA interference culminating in mRNA degradation or translational repression. MiRNAs are fundamental in physiological and pathological processes such as cell proliferation, differentiation, apoptosis, and inflammation. Among this includes the uncovered potential of miRNAs in overall esophageal disease with a focus on the clinicopathologic allergic disease eosinophilic esophagitis (EoE), gastroesophageal reflux disease (GERD), and the tumorigenic continuum from Barrett's esophagus (BE) toward esophageal adenocarcinoma (EAC). Although these pathologies are distinct from one another, they share pathophysiological elements such as an intense inflammatory milieu, esophageal dysfunction, and as presented in this review, an overlap in miRNA expression which contributes to overall esophageal disease. The overlap in the dysregulated miRNA transcriptome of these pathologies highlights the key role miRNAs play in contributing to esophageal disease progression. Owing to this notable dysregulation, there is an attractive utility for miRNAs as diagnostic and prognostic biomarkers in esophageal diseases that already require invasive endoscopies and biopsy retrieval. In this review miRNAs within EoE, GERD, BE, EAC, and esophageal achalasia are discussed, as well as reviewing a core set of miRNAs shared in the disease progression among some of these pathologies, along with the potential utility of targeting miRNAs as therapeutic options in overall esophageal disease.

Differences in salivary secretion and epidermal growth factor concentrations in mild reflux esophagitis and non-erosive reflux disease.

BACKGROUND: Sex differences in salivary secretion have been reported among healthy subjects. In the present study, salivary secretion and salivary epidermal growth factor (EGF) concentrations were investigated in mild reflux esophagitis patients, non-erosive reflux disease (NERD) patients, and healthy controls by matching the sex ratio. METHODS: Thirty-three (male:female = 11:22) patients with NERD, 33 (11:22) with mild reflux esophagitis, and 33 (11:22) healthy controls were recruited for this case-control study. Salivary secretion was assessed as follows: each patient chewed sugar-free gum for 3 min prior to endoscopy, and the amount of saliva secretion, salivary pH, and salivary pH after acid loading as an index of the acid-buffering capacity were measured. Salivary EGF concentrations were measured by ELISA. RESULTS: No significant differences were observed in the amount of saliva secretion, salivary pH, or the acid-buffering capacity between the mild reflux esophagitis and NERD groups. However, the amount of saliva secretion and the acid-buffering capacity in the mild reflux esophagitis group and the amount of saliva secretion, salivary pH, and the acid-buffering capacity in the NERD group were significantly lower than those in the healthy control group. No significant differences were noted in salivary EGF concentrations between the mild reflux esophagitis and NERD groups. CONCLUSION: After matching the sex ratio, the saliva secretion was significantly lower in patients with mild reflux esophagitis and NERD than in healthy controls. However, no significant differences were observed in the amount of saliva secretion or salivary EGF concentrations between both groups.

Dynorphin participates in interaction between depression and non-erosive reflux disease.

BACKGROUND: To explore the relationships between anxiety/depression and NERD, we focused on dynorphin (Dyn), an important member of visceral hypersensitivity, and its related pathways. METHODS: Pearson's correlation analysis on patients with NERD and in vivo experiment on NERD rat model. Part 1: Pearson's correlation analysis among serum levels of Dyn, clinical symptoms and HADS scores of NERD patients were carried on. Part 2: Wistar rats were randomly divided into 2 groups: control group and model group. The data of pH value, immobility time, serum Dyn concentration, NMDAR1 and SP expression were, respectively, derived from automatic pH recorder, tail suspension test, enzyme-linked immunosorbent assay, immunohistochemistry and immunofluorescence. RESULTS: Part 1: Pearson's correlation analysis showed that there was a linear correlation between Clinical Symptom (CS) score and HADS score (HAD-A, HAD-D), and the correlation coefficients were 0.385 and 0.273 respectively; the correlation coefficient between lg (Dyn) and lg (CS score) was r = 0.441, P = 0.002; the correlation coefficient between lg(Dyn) and lg (HAD-D score) was r = 0.447, P = 0.002. Part 2: The pH value of the lower esophagus in the model group was lower than that in the control group (P < 0.01). The tail suspension immobility time of model group was significantly longer than that of control group (P < 0.01). The serum Dyn concentration and the expression level of NMDAR1 in spinal cord and SP in lower esophageal mucosa of model group were significantly higher than those of control group (P < 0.05). CONCLUSION: Increased serum dynorphin level may be a sign of correlation between depression and NERD.

IL-33 Participates in the Development of Esophageal Adenocarcinoma.

Background: The progression from chronic gastroesophageal reflux disease (GERD) to Barrett esophagus (BE) and esophageal adenocarcinoma (EAC) is an inflammatory-driven neoplastic change. Interleukin-33 (IL-33) has identified as a crucial factor in several inflammatory disorders and malignancies. Methods: The high-density tissue microarray of the human EAC was analyzed with IL-33 immunohistochemistry staining (IHC). By anastomosing the jejunum with the esophagus, the rat model of EAC with mixed gastroduodenal reflux was established. The expression of IL-33 was determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blot (WB), IHC and enzyme-linked immunosorbent assay (ELISA). Esophageal adenocarcinoma cells (OE19 and OE33) and human esophageal epithelial cells (HEECs) were used. Results: In the cytoplasm of human EAC tissue, IL-33 expression was substantially greater than in adjacent normal tissue. In rat model, the expression of IL-33 in the EAC group was considerably greater than in the control group, and this expression increased with the upgrade of pathological stage. In in vitro experiment, the mRNA and protein levels of IL-33 were considerably greater in OE19 and OE33 than in HEECs. The stimulation of IL-33 enhanced the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of OE19 and OE33, but soluble ST2 (sST2) inhibited these effects. IL-33 stimulated the release of IL-6 by OE19 and OE33 cells. Conclusion: This study demonstrated the overexpression of IL-33 in the transition from GERD to EAC and that IL-33 promoted carcinogenesis in EAC cells through ST2. IL-33 might be a possible preventive target for EAC.

Visualization of SpoVAEa Protein Dynamics in Dormant Spores of Bacillus cereus and Dynamic Changes in Their Germinosomes and SpoVAEa during Germination.

Bacillus cereus spores, like most Bacillus spores, can survive for years and germinate when their surroundings become suitable, and germination proteins play an important role in the initiation of germination. Because germinated spores lose the extreme resistance of dormant spores, information on the function of germination proteins could be useful in developing new strategies to control B. cereus spores. Prior work has shown that (i) the channel protein SpoVAEa exhibits high-frequency movement in the outer leaflet of the inner membrane (IM) in dormant B. subtilis spores and (ii) the formation of the foci termed germinosomes between two germination proteins, the germinant receptor GerR and the scaffold protein GerD, in developing B. cereus spores is slower than foci formation by GerR and GerD individually. However, the movement dynamics of SpoVAEa in B. cereus spores, and the behavior of the germinosome upon B. cereus spore germination, are not known. In this study, we found that SpoVAEa fluorescent foci in dormant B. cereus spores move on the IM, but slower than in B. subtilis spores, and they likely co-localize transiently with GerD-mScarlet-I in the germinosome. Our results further indicate that (i) the expression of GerR-SGFP2 and SpoVAEa-SGFP2 with GerD-mScarlet-I from a plasmid leads to more heterogeneity and lower efficiency of spore germination in B. cereus, and (ii) germinosome foci observed by Fluorescence resonance energy transfer (FRET) between GerR-SGFP2 and GerD-mScarlet-I can be lost soon after the spore-phase transition. However, this is not always the case, as some GerR-SGFP2 and GerD-mScarlet-I foci continued to exist, co-localize, and even show a weak FRET signal. These data highlight the heterogeneous behavior of spore germination protein complexes and indicate that some complexes may persist beyond the initiation of germination. IMPORTANCE Bacillus cereus is commonly present in soil and infects humans via contaminated food. In this study, we used B. cereus spores to investigate the movement of the spore-specific inner membrane (IM) channel protein SpoVAEa, the interaction between SpoVAEa and the germinosome scaffold protein GerD, and the dynamics of germinosomes with GerR and GerD in spore germination. Our results expand upon observations of interactions between specific B. cereus spore germination proteins, in particular the GerR germinant receptor A, B, and C subunits and GerD, as well as those between SpoVAEa and GerD. The approaches used in this work could also be used to examine the interactions between GerD and SpoVAEa and other germination proteins in spores of other Bacillus species.

Nitric oxide could promote development of Barrett's esophagus by S-nitrosylation-induced inhibition of Rho-ROCK signaling in esophageal fibroblasts.

Barrett's esophagus arises in the process of wound healing in distal esophageal epithelium damaged by gastroesophageal reflux disease. Differentiation of fibroblast into myofibroblasts, a smooth muscle cell-like phenotype and tissue contraction are crucial processes in wound healing. No study has evaluated mechanism by which luminal esophageal nitric oxide (NO) affect Rho-associated coiled coil-forming protein kinase (Rho-ROCK) signaling pathway, a key factor of tissue contraction, in stromal fibroblasts to develop Barrett's esophagus. Using esophageal fibroblasts, we performed collagen-based cell contraction assays and evaluated influence of Rho-ROCK signaling in the exposure to acidic bile salts and NOC-9, which is an NO donor. We found that enhanced cell contraction induced by acidic bile salts was inhibited by NO, accompanied by decrease in phosphorylated myosin light chain expression and stress fiber formation. NO directly S-nitrosylated GTP-RhoA and consequently blocked Rho-ROCK signaling. Moreover, exposure to NO and Y27632, a Rho-ROCK signaling inhibitor, decreased α-SMA expression and increased bone morphogenetic protein-4 (BMP4) expression and secretion. These findings could account for the increased expression of BMP4 in the columnar epithelial cells and stromal fibroblasts in human Barrett's esophagus. NO could impair wound contraction by blocking the Rho-ROCK signaling pathway and promote the development of Barrett's esophagus.NEW & NOTEWORTHY Barrett's esophagus is the condition where esophageal epithelium damaged by gastroesophageal reflux disease (GERD) is abnormally healed via replacing of metaplastic columnar epithelium, but very few studies have conducted focusing wound healing in the development of Barrett's esophagus. Esophageal luminal nitric oxide inhibits Rho-ROCK signaling pathway in esophageal fibroblasts, which leads to delay tissue contraction, a pivotal step in proper wound healing. Moreover, this inhibition increases tissue BMP4 expression. Impaired wound healing could be related to Barrett's esophagus.

Proanthocyanidins mitigate bile acid-induced changes in GSTT2 levels in a panel of racially diverse patient-derived primary esophageal cell cultures.

Persistent and symptomatic reflux of gastric and duodenal contents, known as gastroesophageal reflux disease (GERD), is the strongest risk factor for esophageal adenocarcinoma (EAC). Despite similar rates of GERD and other risk factors across racial groups, EAC progression disproportionately impacts Caucasians. We recently reported that elevated tissue levels of the detoxification enzyme GSTT2 in the esophagi of Blacks compared to Caucasians may contribute protection. Herein, we extend our research to investigate whether cranberry proanthocyanidins (C-PAC) mitigate bile acid-induced damage and GSTT2 levels utilizing a racially diverse panel of patient-derived primary esophageal cultures. We have shown that C-PACs mitigate reflux-induced DNA damage through GSTT2 upregulation in a rat esophageal reflux model, but whether effects are recapitulated in humans or differentially based on race remains unknown. We isolated normal primary esophageal cells from Black and Caucasian patients and assessed GSTT2 protein levels and cellular viability following exposure to a bile acid cocktail with and without C-PAC treatment. Constitutive GSTT2 levels were significantly elevated in Black (2.9-fold) compared to Caucasian patients, as were GSTT2 levels in Black patients with GERD. C-PAC treatment induced GSTT2 levels 1.6-fold in primary normal esophageal cells. GSTT2 induction by C-PAC was greatest in cells with constitutively low GSTT2 expression. Overall, C-PAC mitigated bile-induced reductions of GSTT2 and subsequent loss of cell viability regardless of basal GSTT2 expression or race. These data support that C-PAC may be a safe efficacious agent to promote epithelial fitness through GSTT2 induction and in turn protect against bile acid-induced esophageal injury.

Upregulation of acid sensing ion channels is associated with esophageal hypersensitivity in GERD.

Proton pump inhibitors (PPIs) are the mainstay of therapy for gastroesophageal reflux disease (GERD) but up to 60% of patients have inadequate response to therapy. Acid sensing ion channels (ASICs) play important roles in nociception. This study aimed to investigate whether the increased expression of ASICs results in neuronal hyperexcitability in GERD. Esophageal biopsies were taken from GERD patients and healthy subjects to compare expression of ASIC1 and 3. Next, gene and protein expression of ASIC1 and 3 from esophageal mucosa and dorsal root ganglia (DRG) neurons were measured by qPCR, Western-blot and immunofluorescence in rodent models of reflux esophagitis (RE), non-erosive reflux disease (NERD), and sham operated groups. Excitability of DRG neurons in the GERD and sham groups were also tested by whole-cell patch-clamp recordings. We demonstrated that ASIC1 and 3 expression were significantly increased in patients with RE compared with healthy controls. This correlated positively with symptom severity of heartburn and regurgitation (p < .001). Next, ASIC1 and 3 gene and protein expression in rodent models of RE and NERD were similarly increased in esophageal mucosa as well as T3-T5 DRG neurons compared with sham operation. DRG neurons from RE animals showed hyperexcitability compared with sham group. However, intrathecal injection of ASIC specific inhibitors, PcTx1 and APTEx-2, as well as silencing ASIC1 and 3 genes with specific siRNAs prevented visceral hypersensitivity. Overall, upregulation of ASIC1 and 3 may lead to visceral hypersensitivity in RE and NERD and may be a potential therapeutic target for PPI non-responsive patients.

Associations Between Sleep Position and Nocturnal Gastroesophageal Reflux: A Study Using Concurrent Monitoring of Sleep Position and Esophageal pH and Impedance.

INTRODUCTION: The aim of this study was to investigate the effect of spontaneous sleep positions on the occurrence of nocturnal gastroesophageal reflux. METHODS: In patients referred for ambulatory pH-impedance reflux monitoring, the concurrent sleep position was measured using a sleep position measurement device (measuring left, right, supine, and prone positions). RESULTS: Fifty-seven patients were included. We observed a significantly shorter acid exposure time in the left (median 0.0%, P25-P75, 0.0%-3.0%), compared with the right lateral position (median 1.2%, 0.0%-7.5%, P = 0.022) and the supine position (median 0.6%, 0.00%-8.3%, P = 0.022). The esophageal acid clearance time was significantly shorter in the left lateral decubitus position (median 35 seconds, 16-115 seconds), compared with the supine (median 76 seconds, 22-257 seconds, P = 0.030) and right lateral positions (median 90 seconds, 26-250 seconds, P = 0.002). DISCUSSION: The left lateral decubitus position is associated with significantly shorter nocturnal esophageal acid exposure time and faster esophageal acid clearance compared with the supine and right lateral decubitus positions (see visual abstract).

EZH2-mediated suppression of CLDN1 leads to barrier dysfunction in PPI-refractory gastroesophageal reflux disease.

BACKGROUND: PPI-refractory gastroesophageal reflux disease (RGERD) is characterized as the existence of reflux symptoms resistant to optimized PPI treatment. Alleviated mucosal integrity has been regarded as one of the mechanisms of RGERD. METHODS: RNA sequencing analysis and GSEA were performed. Human biopsy samples, cell lines, and rat models were recruited. Trans-epithelial electrical resistance (TEER) was tested and a FITC-dextran flux assay was performed to detect barrier permeability. Tissue morphology was evaluated using HE staining, while gene expression was measured by qRT-PCR, western blotting, flow cytometry, immunofluorescence, immunohistochemistry, and chromatin immunoprecipitation (ChIP) analysis. RESULTS: The tight junction protein Claudin-1 is significantly weakened in the RGERD epithelium, while levels of EZH2-mediated H3K27me3 were increased. Forced EZH2 expression in epithelial cells led to H3K27me3 accumulation and Claudin-1 suppression, which consequently caused epithelial barrier dysfunction. Notably, studies on esophagogastroduodenal anastomosis (EGDA) rat models showed the attenuation of Claudin-1 level and barrier function could be rescued by an Ezh2 inhibitor GSK126. ChIP analysis followed by qPCR (ChIP-qPCR) revealed H3K27me3 suppressed CLDN1 via accumulating at the TSS area. CONCLUSION: For the first time, we explored the attenuated tight junction of RGERD, demonstrating a potential underlying mechanism that EZH2-mediated H3K27me3 could impair esophageal epithelial barrier function by suppressing the transcription of CLDN1.

Response of Esophageal Epithelium to Acute and Chronic Stress in Rabbits.

We studied electrophysiological changes in rabbit esophageal epithelium following acute (AS) and chronic stress (CS). Esophageal tissue was placed in Ussing chamber and the potential difference U between the luminal and abluminal sides, the short-circuit current Isc, as well as the tissue resistance R were measured. The initial values of these parameters for each sample were determined after the samples were stabilized in Ringer solution. Then, the tissues were exposed for 1 h to normal Ringer solution or Ringer solution with pH 4.0 and pH 1.7 with or without pepsin (0.25 mg/ml). Fluorescein was added to the luminal side of the sample to measure its permeability. In the AS group, U at Ringer solution (pH 1.7)+pepsin was significantly decreased in comparison with the baseline and control values (by 46 and 22%, respectively, p<0.05). R decreased by 74% in comparison with baseline, which little differed from the decrease in control samples exposed to Ringer solution (pH 1.7)+pepsin (by 62%). CS did not change U relative to baseline values, while changes in R were similar to those in the AS group. In the AS group, the permeability of the esophageal tissue perfused with Ringer solution (pH 1.7)+pepsin was significantly higher than in both the control and CS groups. AS, but not CS, made the esophageal epithelium more sensitive to the effects of noxious agents, disrupted barrier properties, and increased permeability. The effects of stress on gastroesophageal reflux disease symptoms can be related to severe exposure to acid and/or pepsin; however, the mechanisms other than epithelial defense should be evaluated.

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.

Cough hypersensitivity in patients with metabolic syndrome: a clinical finding and its possible mechanisms.

PURPOSE: To investigate the changes of cough sensitivity in patients with metabolic syndrome and its possible mechanisms. METHOD: A total of 29 metabolic syndrome (MetS) patients with OSAHS (group-1), 22 MetS patients without OSAHS (group-2), and 25 healthy controls (group-3) were included. All participants underwent a routine physical examination and completed the gastroesophageal reflux disease questionnaire (GerdQ), and the inflammatory mediator profile were determined. The cough threshold for capsaicin, induced sputum cell count and cell classification, and inflammatory mediators in induced sputum supernatants were compared. The correlation between capsaicin cough sensitivity and various indicators in the MetS population was analyzed. RESULTS: The minimum concentration of inhaled capsaicin needed to induce ≥ 5 coughs (C5) was significantly different among three groups (H = 14.393, P = 0.001) and lower for group-1 and group-2 than it for group-3 (P = 0.002, P = 0.005). The percentage of neutrophils in induced sputum and the concentrations of calcitonin gene-related peptide (CGRP), substance P (SP), and interleukin 8 (IL-8) in the sputum supernatant of group-1 and group-2 were significantly higher than those of group-3. Besides, the pepsin concentrations were significantly different among the 3 groups (F = 129.362, P < 0.001), which significantly was highest in group-1 (P < 0.001) and lowest in group-3 (P < 0.001). Triglycerides, AHI, pepsin concentration and BMI were risk factors of increased capsaicin cough sensitivity. CONCLUSION: Increased capsaicin cough sensitivity in MetS patients is closely related to sleep apnea and gastroesophageal reflux. For patients in MetS patients without OSAHS, gastroesophageal reflux is an important factor for increased capsaicin cough sensitivity. Airway inflammation, especially airway neurogenic inflammation, may also play a role in the pathogenesis of increased capsaicin cough sensitivity. Trial registration The protocol was registered in the Chinese Clinical Trials Register ( http://www.chictr.org.cn/ ) (ChiCTR1800014768). Written informed consent was obtained from all participants before enrollment.

Obesity and its effects on the esophageal mucosal barrier.

Obesity is associated with gastroesophageal reflux disease (GERD) and its complications including reflux esophagitis, Barrett's esophagus, and esophageal adenocarcinoma. Traditionally, these associations have been attributed to the mechanical effect of abdominal fat in increasing intra-abdominal pressure, thereby promoting gastroesophageal reflux and causing disruption of antireflux mechanisms at the esophagogastric junction. However, recent studies suggest that visceral adipose tissue (VAT) produces numerous cytokines that can cause esophageal inflammation and impair esophageal mucosal barrier integrity through reflux-independent mechanisms that render the esophageal mucosa especially susceptible to GERD-induced injury. In this report, we review mechanisms of esophageal mucosal defense, the genesis and remodeling of visceral adipose tissue during obesity, and the potential role of substances produced by VAT, especially the VAT that encircles the esophagogastric junction, in the impairment of esophageal mucosal barrier integrity that leads to the development of GERD complications.