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Enteroendocrine cells (EECs) are gut epithelial cells that respond to intestinal contents by secreting hormones, including the incretins glucagon-like peptide 1 (GLP-1) and gastric inhibitory protein (GIP), which regulate multiple physiological processes. Hormone release is controlled through metabolite-sensing proteins. Low expression, interspecies differences, and the existence of multiple EEC subtypes have posed challenges to the study of these sensors. We describe differentiation of stomach EECs to complement existing intestinal organoid protocols. CD200 emerged as a pan-EEC surface marker, allowing deep transcriptomic profiling from primary human tissue along the stomach-intestinal tract. We generated loss-of-function mutations in 22 receptors and subjected organoids to ligand-induced secretion experiments. We delineate the role of individual human EEC sensors in the secretion of hormones, including GLP-1. These represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity, and mucosal immunity.

We describe the histological organisation and mucin content in the digestive tract of the stream catfish Pseudecheneis sulcatus. The aim is to find the modifications of the digestive tract in relation to food resources of its habitat. The oesophageal mucosa consists of stratified squamous epithelium with many mucous-secreting cells. The thick muscularis contains an inner longitudinal and outer circular, striated muscle cells. The stomach is J-shaped and shows 6-7 thick mucosal folds that are separated from the submucosa by an organised muscularis mucosae. The mucosa consists of superficial cells with mucin granules, and deeper simple tubular gastric glands in cardia and fundus, but absent in pyloric region. The glandular epithelium shows oxynticopeptic cells containing zymogen granules and abundant tubulo-vesicular bodies. We provide evidence that the latter arise by budding from smooth endoplasmic reticulum and reach the apical cytoplasm. The anterior intestine shows longer mucosal folds with goblet cells (GC). GC are more in the posterior intestine and highest in the rectum. Myenteric neurons with myelinated and non-myelinated axons innervate the intrinsic musculature from stomach to rectum. Many stem cells are evident in the basal intestinal epithelium. They show darker nuclei and undifferentiated organelles. Mucin histochemistry reveals the predominance of neutral mucin (PAS + positive) from oesophagus to rectum, and neutral and acidic mucin (alcian blue + , pH 2.5) in the posterior intestine to the rectum, with few GC colocalizing both. Ultrastructural features suggest that the species is adapted to omnivory and this is reflected in the predominance of neutral mucin in the digestive tract., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Understanding the diversity of gastrointestinal (GI) immune cells, especially in the muscularis propria, is crucial for understanding their role in the maintenance of enteric neurons and smooth muscle and their contribution to GI motility. Here, we present a detailed protocol for isolating single immune cells from the human gastric muscularis propria. We describe steps for tissue preservation, dissection, and dissociation of the muscularis propria. We then detail procedures for magnetic sorting of CD45 + cells and single-cell RNA sequencing (scRNA-seq) analysis. For complete details on the use and execution of this protocol, please refer to Chikkamenahalli et al. 1 ., Competing Interests: Declaration of interests M.G. has received investigator-initiated grant funding from Takeda Pharmaceuticals and clinical trial funding from Alexza and Donga Pharmaceuticals. He serves as a consultant for Alfasigma, Ardelyx, and Evoke Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background & Aims: Isthmic progenitors, tissue-specific stem cells in the stomach corpus, maintain mucosal homeostasis by balancing between proliferation and differentiation to gastric epithelial lineages. The progenitor cells rapidly adopt an active state in response to mucosal injury. However, it remains unclear how the isthmic progenitor cell niche is controlled during the regeneration of damaged epithelium., Methods: We recapitulated tissue recovery process after acute mucosal injury in the mouse stomach. Bromodeoxyuridine incorporation was used to trace newly generated cells during the injury and recovery phases. To define the epithelial lineage commitment process during recovery, we performed single-cell RNA-sequencing on epithelial cells from the mouse stomachs. We validated the effects of amphiregulin (AREG) on mucosal recovery, using recombinant AREG treatment or AREG-deficient mice., Results: We determined that an epidermal growth factor receptor ligand, AREG, can control progenitor cell lineage commitment. Based on the identification of lineage-committed subpopulations in the corpus epithelium through single-cell RNA-sequencing and bromodeoxyuridine incorporation, we showed that isthmic progenitors mainly transition into short-lived surface cell lineages but are less frequently committed to long-lived parietal cell lineages in homeostasis. However, mucosal regeneration after damage directs the lineage commitment of isthmic progenitors towards parietal cell lineages. During recovery, AREG treatment promoted repopulation with parietal cells, while suppressing surface cell commitment of progenitors. In contrast, transforming growth factor-α did not alter parietal cell regeneration, but did induce expansion of surface cell populations. AREG deficiency impairs parietal cell regeneration but increases surface cell commitment., Conclusions: These data demonstrate that different epidermal growth factor receptor ligands can distinctly regulate isthmic progenitor-driven mucosal regeneration and lineage commitment., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model, Meta1 gastroids, to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells., Methods: We performed single-cell RNA sequencing to characterize Meta1 gastroids, which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation., Results: Meta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids., Conclusions: IL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Restricted gastric motor functions contribute to aging-associated undernutrition, sarcopenia, and frailty. We previously identified a decline in interstitial cells of Cajal (ICC; gastrointestinal pacemaker and neuromodulator cells) and their stem cells (ICC-SC) as a key factor of gastric aging. Altered functionality of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) is central to organismal aging. Here, we investigated the role of EZH2 in the aging-related loss of ICC/ICC-SC., Methods: klotho mice, a model of accelerated aging, were treated with the most clinically advanced EZH2 inhibitor, EPZ6438 (tazemetostat; 160 mg/kg intraperitoneally twice a day for 3 weeks). Gastric ICC were analyzed by Western blotting and immunohistochemistry. ICC and ICC-SC were quantified by flow cytometry. Gastric slow wave activity was assessed by intracellular electrophysiology. Ezh2 was deactivated in ICC by treating Kit creERT2/+ ;Ezh2 fl/fl mice with tamoxifen. TRP53, a key mediator of aging-related ICC loss, was induced with nutlin 3a in gastric muscle organotypic cultures and an ICC-SC line., Results: In klotho mice, EPZ6438 treatment mitigated the decline in the ICC growth factor KIT ligand/stem cell factor and gastric ICC. EPZ6438 also improved gastric slow wave activity and mitigated the reduced food intake and impaired body weight gain characteristic of this strain. Conditional genomic deletion of Ezh2 in Kit-expressing cells also prevented ICC loss. In organotypic cultures and ICC-SC, EZH2 inhibition prevented the aging-like effects of TRP53 stabilization on ICC/ICC-SC., Conclusions: Inhibition of EZH2 with EPZ6438 mitigates aging-related ICC/ICC-SC loss and gastric motor dysfunction, improving slow wave activity and food intake in klotho mice., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The microanatomical features of the oesophageal gastric tract in tetrapod representatives and their function, especially those related to the mucosal layer, have not yet been fully investigated. The mucosal layer cells and their function in the oesophageal gastric tract differ structurally and functionally in tetrapod representatives based on interspecies difference and the type of food and feeding habits. The present study was, therefore, postulated to compare the mucosal microanatomical structure and histochemical biodistribution of different mucin types in oesophageal gastric tract tissues of four tetrapod species. A representative of each tetrapod class was selected, as follows: the Egyptian toad Bufo regularis, the lizard Trachylepis quinquetaeniata, the domestic pigeon Columba livia domestica and the albino mouse Mus musculus for Amphibia, Reptilia, Aves and Mammalia, respectively. Microanatomically, in lower tetrapods (toad and lizard), the mucosal layer of the oesophagus was composed of simple ciliated columnar epithelium with goblet cells, whereas in higher tetrapods (pigeon and mouse) it was composed of stratified squamous epithelium, with non-keratinised epithelium in the pigeon but keratinised epithelium in the mouse. However, the gastric mucosal layer of the stomach in lower tetrapods consists of simple columnar epithelium and gastric glands. Similarly, the mucosa of the pigeon's proventriculus consists of simple columnar epithelium with proventricular glands opened into the lumen, whereas mouse mucosa consists of simple columnar epithelium which folds and forms gastric glands with gastric pits having a variety of cell types. Histochemically, the neutral mucin profile biodistribution in the oesophagus mucosal layer was variable. It was strongly positive in the toad and lizard, but was weak in the pigeon and completely negative in the mouse. In contrast it was strongly positive in the gastric mucosa of the toad, lizard and pigeon, but was weak in the mouse's gastric mucosa. On the other hand, the signals of carboxylated and sulfated mucins were found to be different. They were strong in the mucosa of the lizard oesophagus. In contrast, the carboxylated mucins in the gastric mucosa were positive in all representatives except the mouse. The sulfated mucins were, however, seen localised in the mucosal layer cells of the lizard and pigeon only. The study revealed that the microanatomical structures and functions as well as mucin distribution profiles in the oesophageal gastric tract are in line with interspecies difference and the type of food and feeding habits. However, this may need further investigations including more tetrapod representatives., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The mucosa of the body of the stomach (ie, the gastric corpus) uses 2 overlapping, depth-dependent mechanisms to respond to injury. Superficial injury heals via surface cells with histopathologic changes like foveolar hyperplasia. Deeper, usually chronic, injury/inflammation, most frequently induced by the carcinogenic bacteria Helicobacter pylori, elicits glandular histopathologic alterations, initially manifesting as pyloric (also known as pseudopyloric) metaplasia. In this pyloric metaplasia, corpus glands become antrum (pylorus)-like with loss of acid-secreting parietal cells (atrophic gastritis), expansion of foveolar cells, and reprogramming of digestive enzyme-secreting chief cells into deep antral gland-like mucous cells. After acute parietal cell loss, chief cells can reprogram through an orderly stepwise progression (paligenosis) initiated by interleukin-13-secreting innate lymphoid cells (ILC2s). First, massive lysosomal activation helps mitigate reactive oxygen species and remove damaged organelles. Second, mucus and wound-healing proteins (eg, TFF2) and other transcriptional alterations are induced, at which point the reprogrammed chief cells are recognized as mucus-secreting spasmolytic polypeptide-expressing metaplasia cells. In chronic severe injury, glands with pyloric metaplasia can harbor both actively proliferating spasmolytic polypeptide-expressing metaplasia cells and eventually intestine-like cells. Gastric glands with such lineage confusion (mixed incomplete intestinal metaplasia and proliferative spasmolytic polypeptide-expressing metaplasia) may be at particular risk for progression to dysplasia and cancer. A pyloric-like pattern of metaplasia after injury also occurs in other gastrointestinal organs including esophagus, pancreas, and intestines, and the paligenosis program itself seems broadly conserved across tissues and species. Here we discuss aspects of metaplasia in stomach, incorporating data derived from animal models and work on human cells and tissues in correlation with diagnostic and clinical implications., (Copyright © 2022 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The present study aimed to investigate the protective effects and molecular mechanisms of Dendrobium officinale polysaccharides on gastric mucosal injuries. Following one week of continuous intragastric administration, a gastric mucosal injury model was established using intragastric administration of anhydrous ethanol. The area of gastric ulcer was measured, the contents of interleukin- 6 (IL-6), epidermal growth factor receptor (EGFR), and thyroid transcription factor 1 (TFF-1) in serum were detected by enzyme linked immunosorbent assay (ELISA), and the expressions of EGFR, TFF-1, IL-6, Raf-2, MAP kinase kinase 1 (MEK1), MEK2, and ERK1 in the gastric tissue were determined utilizing qPCR, Western blotting and immunohistochemistry. Simultaneously, Dendrobium officinale polysaccharides and anhydrous ethanol were added to the gastric mucosal cells (GES1) cultured in vitro, and the protective effects of Dendrobium officinale polysaccharides on cell viability was detected using Cell Counting Kit (CCK)-8. The addition of Dendrobium officinale polysaccharides markedly improved the gastric epithelial defect, inflammatory cell infiltration, and redness and swelling stemmed from gastric mucosal injuries and greatly reduced the area of gastric ulcer. The inhibition rates of gastric ulcer were 48.12 ± 2.98, 42.95 ± 1.52, and 27.96 ± 2.05% in the high, medium, and low concentration Dendrobium officinale polysaccharide groups, respectively. Dendrobium officinale polysaccharides could increase the expressions of EGFR and TFF-1 and decrease the expressions of IL-6, Raf-2, MEK1, MEK2, and ERK1. Dendrobium officinale polysaccharides could reduce the level of inflammatory factors and protect gastric mucosa by inhibiting the expression of MAPK pathway genes and proteins.

Helicobacter pylori ( H. pylori ) infection promotes gastric carcinogenesis by increasing oxidative stress, inflammation, and dysregulation of cell survival and proliferation of gastric epithelial cells. Astaxanthin (ASTX), a bioactive carotenoid, exhibits antioxidant and anticancer effects by modulating aberrant signaling pathways that lead to dysregulation of cell death and proliferation. To elucidate the molecular mechanism of H. pylori -induced gastric carcinogenesis and to examine the inhibitory effect of ASTX on H. pylori -induced gastric epithelial cell gene expression changes, we performed comparative RNA-sequencing (RNA-Seq) analysis for H. pylori -infected gastric epithelial cells treated with or without ASTX. RNA-Seq results reveal that differentially expressed genes (DEGs) in H. pylori -infected cells were mainly associated with the Wnt/β-catenin signaling pathway, which is related to cell proliferation. ASTX significantly reversed H. pylori -induced transcriptional alterations of the key mediators involved in β-catenin signaling, notably, porcupine (gene symbol, PORCN) , spermine oxidase ( SMOX ), bone morphogenetic protein (BMP) and activin membrane-bound inhibitor ( BAMBI ), SMAD family member 4 ( SMAD4 ), transforming growth factor-β1 ( TGFB1) , Fos-like 1 ( FOSLI) , and c-myc ( MYC) . We suggest that ASTX may be a potential therapeutic agent that can suppress H. pylori -induced proliferation-associated gene expression changes, in part, by counter-regulating the Wnt/β-catenin signaling pathway.

Objective: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus., Design: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples., Results: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration., Conclusion: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The gastric H + ,K + -ATPase mediates electroneutral exchange of 1H + /1K + per ATP hydrolysed across the membrane. Previous structural analysis of the K + -occluded E2-P transition state of H + ,K + -ATPase showed a single bound K + at cation-binding site II, in marked contrast to the two K + ions occluded at sites I and II of the closely-related Na + ,K + -ATPase which mediates electrogenic 3Na + /2K + translocation across the membrane. The molecular basis of the different K + stoichiometry between these K + -counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H + ,K + -ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K + bound H + ,K + -ATPase, achieved by five mutations, including amino acids directly coordinating K + (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K + -occluded conformation (Tyr799Trp). This quintuple mutant in the K + -occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K + accommodated at their cation-binding site., (© 2021. The Author(s).)

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease., (© 2021 Wiley-VCH GmbH.)

HIV preferentially infects α 4 β 7 + CD4 T cells, forming latent reservoirs that contribute to HIV persistence during antiretroviral therapy. However, the properties of α 4 β 7 + CD4 T cells in blood and mucosal compartments remain understudied. Employing two distinct models of HIV infection, HIV-infected humans and simian-human immunodeficiency virus (SHIV)-infected rhesus macaques, we show that α 4 β 7 + CD4 T cells in blood are enriched for genes regulating cell cycle progression and cellular metabolism. Unlike their circulating counterparts, rectal α 4 β 7 + CD4 T cells exhibited a core tissue-residency gene expression program. These features were conserved across primate species, indicating that the environment influences memory T-cell transcriptional networks. Our findings provide an important molecular foundation for understanding the role of α 4 β 7 in HIV infection., (© 2021 Federation of European Biochemical Societies.)

Background: Helicobacter pylori (H. pylori) is a common pathogen in development of peptic ulcers with pyroptosis. Rabeprazole, a critical component of standard triple therapy, has been widely used as the first-line regimen for H. pylori infectious treatment. The aim of this study to explore the function of Rabeprazole on cell pyroptosis in vitro., Methods: The clinical sample from patients diagnosed with or without H. pylori-infection were collected to analyze by Immunohistochemistry (IHC). Real-time quantitative PCR (qPCR), western blot (WB) and enzyme linked immunosorbent assay (Elisa) were performed to analyze the effect of Rabeprazole on cell pyroptosis, including LDH, IL-1β and IL-18., Results: In this study, we showed that Rabeprazole regulated a phenomenon of cell pyroptosis as confirmed by lactate dehydrogenase (LDH) assay. Further results showed that Rabeprazole inhibited cell pyroptosis in gastric epithelial cells by alleviating GSDMD-executed pyroptosis, leading to decrease IL-1β and IL-18 mature and secretion, which is attributed to NLRP3 inflammasome activation inhibition. Further analysis showed that ASC, NLRP3 and Caspase-1, was significantly repressed in response to Rabeprazole stimulation, resulting in decreasing cleaved-caspase-1 expression. Most important, NLRP3 and GSDMD is significantly increased in gastric tissue of patients with H. pylori infection., Conclusion: These findings revealed a critical role of Rabeprazole in cell pyroptosis in patients with H. pylori infection, suggesting that targeting cell pyroptosis is an alternative strategy in improving H. pylori treatment., (© 2021. The Author(s).)

Energy homeostasis is is determined by food intake and energy expenditure, which are partly regulated by the cross-talk between central and peripheral hormonal signals. Phoenixin (PNX) is a recently discovered pleiotropic neuropeptide with isoforms of 14 (PNX-14) and 20 (PNX-20) amino acids. It is a potent reproductive peptide in vertebrates, regulating the hypothalamo-pituitary-gonadal axis (HPG). It has been identified as a regulator of food intake during light phase when injected intracerebroventricularly in rats. In addition, plasma levels of PNX also increased after food intake in rats, suggesting that it might have possible roles in energy homeostasis. We hypothesized that gut is a source and site of action of PNX in mice. Immunoreactivity for PNX and its putative receptor, super-conserved receptor expressed in brain (SREB3; also known as the G-protein coupled receptor 173/GPR 173) was found in the stomach and intestine of male C57/BL6 J mice, and in MGN3-1 (mouse stomach endocrine) cells and STC-1 (mouse enteroendocrine) cells. In MGN3-1 cells, PNX-20 significantly upregulated ghrelin (10 nM) and ghrelin-O-acyl transferase (GOAT) mRNAs (1000 nM) at 6 h. In STC-1 cells, it significantly suppressed CCK (100 nM) at 2 h. No effects were found on other intestinal hormones tested (glucagon like peptide-1, glucose dependent insulinotropic polypeptide, and peptide YY). Together, these results indicate that PNX-20 is produced in the gut, and it could act directly on gut cells to regulate metabolic hormones., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Gastric cancer is one of the most common and deadly cancers among men and women and is the third leading cause of cancer mortality worldwide. Thus, discovering and developing novel therapeutics for gastric cancer has become a global priority. In this study, we synthesized two novel anthraquinone-based aspirin derivatives, Asp-X 3 and Asp-X 3 -CH 3 , with therapeutic potential for gastric cancer. The structures of the two compounds were determined by 1D, 2D-NMR, and High-Resolution Mass (HRSM). Asp-X 3 and Asp-X 3 -CH 3 could inhibit the growth of gastric cancer cells (SGC7901), yielding IC 50 values 10-fold lower than that of Aspirin. Asp-X 3 and Asp-X 3 -CH 3 were less toxic to gastric mucosal cells, yielding IC 50 values that were about 2-fold higher than the corresponding IC 50 values determined with SGC7901 cells. Asp-X 3 -CH 3 and Asp-X 3 also induced SGC7901 cells to undergo apoptosis, yielding apoptotic rates that were about twice the rate induced by Aspirin. Asp-X 3 -CH 3 did not cause significant loss of COX-1 expression in gastric mucosal cells, whereas Asp-X 3 and Aspirin both caused significant loss of COX-1 expression as demonstrated by Western blot, consistent with their effects on the content of PGE 2 in these cells as determined by ELISA assay. However, both Asp-X 3 -CH 3 and Asp-X 3 exerted a similar effect on the level of COX-2 in gastric cancer cells, causing as much as 90% and 95% reduction in COX-2 expression, respectively. Taken together, the results suggested that Asp-X 3 -CH 3 and Asp-X 3 were potentially better agents than Aspirin for the inhibition of gastric cancer cell growth, but Asp-X 3 -CH 3 was more effective., (Copyright © 2021 Elsevier B.V. All rights reserved.)

There has been increasing evidence that microRNAs (miRNAs) are related to glioma progression, and that genetically engineered mesenchymal stem cells (MSCs) can inhibit the growth of gliomas. However, the underlying mechanism of bone marrow-MSCs (BM--MSCs) and miRs in gastric cancer still remains unclear. Patients with gastric cancer treated in Shijiazhuang First Hospital as well as healthy individuals undergoing physical examinations were recruited to measure the expression of exosomal miR-1228. Receiver operating characteristic (ROC) curves were plotted and the patients were followed up. BM--MSCs from healthy subjects were collected and exosomes were extracted. The MSC cells were transfected with lentiviral vectors carrying miR-1228 and MMP-14 over-expression sequences and scramble sequence, followed by exosome extraction. The exosomes were co-cultured with SGC-7901 and MGC-823 cells to detect cell proliferation, invasion, apoptosis and migration. The correlation between miR-1228 and MMP-14 was determined by dual-luciferase reporter assay. miR-1228 was highly expressed in serum exosomes of patients with gastric cancer with a area under ROC curve (AUC) of 0.865. The exosomes derived from BM-MSCs are expected to be efficient nanocarriers. Up-regulation of miR-1228 can down-regulate the expression of MMP-14 and effectively hinders the development and progression of gastric cancer.

Background and Aim: Muscularis macrophages (MMs) not only mediate the innate immunity, but also functionally interact with cells important for gastrointestinal motility. The aim of this study was to determine the spatial relationship and types of contacts between the MMs and neighboring cells in the muscularis propria of human and mouse stomach, small intestine, and large intestine., Methods: The distribution and morphology of MMs and their contacts with other cells were investigated by immunohistochemistry and transmission electron microscopy., Key Results: Immunohistochemistry showed variable shape and number of MMs according to their location in different portions of the muscle coat. By double labeling, a close association between MMs and neighboring cells, that is, neurons, smooth muscle cells, interstitial cells of Cajal (ICCs), telocytes (TCs)/PDGFRα-positive cells, was seen. Electron microscopy demonstrated that in the muscle layers of both animal species, MMs have similar ultrastructural features and have specialized cell-to-cell contacts with smooth muscle cells and TCs/PDGFRα-positive cells but not with ICCs and enteric neurons., Conclusion & Inferences: This study describes varying patterns of distribution of MMs between different regions of the gut, and reports the presence of distinct and extended cell-to-cell contacts between MMs and smooth muscle cells and between MMs and TCs/PDGFRα-positive cells. In contrast, MMs, although close to ICCs and nerve elements, did not make contact with them. These findings indicate specialized and variable roles for MMs in the modulation of gastrointestinal motility whose significance should be more closely investigated in normal and pathological conditions., (© 2020 John Wiley & Sons Ltd.)

Background & Aims: Inflammation in the gastrointestinal tract may lead to the development of cancer. Dicarbonyl electrophiles, such as isolevuglandins (isoLGs), are generated from lipid peroxidation during the inflammatory response and form covalent adducts with amine-containing macromolecules. Thus, we sought to determine the role of dicarbonyl electrophiles in inflammation-associated carcinogenesis., Methods: The formation of isoLG adducts was analyzed in the gastric tissues of patients infected with Helicobacter pylori from gastritis to precancerous intestinal metaplasia, in human gastric organoids, and in patients with colitis and colitis-associated carcinoma (CAC). The effect on cancer development of a potent scavenger of dicarbonyl electrophiles, 5-ethyl-2-hydroxybenzylamine (EtHOBA), was determined in transgenic FVB/N insulin-gastrin (INS-GAS) mice and Mongolian gerbils as models of H pylori-induced carcinogenesis and in C57BL/6 mice treated with azoxymethane-dextran sulfate sodium as a model of CAC. The effect of EtHOBA on mutations in gastric epithelial cells of H pylori-infected INS-GAS mice was assessed by whole-exome sequencing., Results: We show increased isoLG adducts in gastric epithelial cell nuclei in patients with gastritis and intestinal metaplasia and in human gastric organoids infected with H pylori. EtHOBA inhibited gastric carcinoma in infected INS-GAS mice and gerbils and attenuated isoLG adducts, DNA damage, and somatic mutation frequency. Additionally, isoLG adducts were elevated in tissues from patients with colitis, colitis-associated dysplasia, and CAC as well as in dysplastic tumors of C57BL/6 mice treated with azoxymethane-dextran sulfate sodium. In this model, EtHOBA significantly reduced adduct formation, tumorigenesis, and dysplasia severity., Conclusions: Dicarbonyl electrophiles represent a link between inflammation and somatic genomic alterations and are thus key targets for cancer chemoprevention., (Published by Elsevier Inc.)

Cancer-associated fibroblasts (CAFs) tend to have tumor-promoting capacity, and can provide therapeutic targets. Even without cancer cells, CAF phenotypes are stably maintained, and DNA methylation and H3K27me3 changes have been shown to be involved. Here, we searched for a potential therapeutic target in primary CAFs from gastric cancer and a mechanism for its dysregulation. Expression microarray using eight CAFs and seven non-CAFs (NCAFs) revealed that serum amyloid A1 (SAA1), which encodes an acute phase secreted protein, was second most upregulated in CAFs, following IGF2. Conditioned medium (CM) derived from SAA1-overexpressing NCAFs was shown to increase migration of gastric cancer cells compared with that from control NCAFs, and its tumor-promoting effect was comparable to that of CM from CAFs. In addition, increased migration of cancer cells by CM from CAFs was mostly canceled with CM from CAFs with SAA1 knockdown. Chromatin immunoprecipitation (ChIP)-quantitative PCR showed that CAFs had higher levels of H3K27ac, an active enhancer mark, in the promoter and the two far upstream regions of SAA1 than NCAFs. Also, BET bromodomain inhibitors, JQ1 and mivebresib, decreased SAA1 expression and tumor-promoting effects in CAFs, suggesting SAA1 upregulation by enhancer activation in CAFs. Our present data showed that SAA1 is a candidate therapeutic target from gastric CAFs and indicated that increased enhancer acetylation is important for its overexpression., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The human gastrointestinal mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. In the gastrointestinal tract, the interaction of bacteriophages (phages) and their prokaryotic hosts influences the health of the mammalian host, especially colonization with invasive pathobionts. Antibiotics may be used, but they also kill protective commensals. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, positions it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing mucosal targeting phage to selectively remove invasive pathobiont species from mucosal surfaces. IMPORTANCE Invasive pathobionts or microbes capable of causing disease can reside deep within the mucosal epithelium of our gastrointestinal tract. Targeted effective antibacterial therapies are needed to combat these disease-causing organisms, many of which may be multidrug resistant. Here, we isolated a lytic bacteriophage (phage) that can localize to the epithelial surface by binding heparan sulfated glycans, positioning it near its host, Escherichia coli This targeted therapy can be used to selectively remove invasive pathobionts from the gastrointestinal tract, preventing the development of disease., (Copyright © 2021 Green et al.)

Aromatase, an estrogen synthase, exists in the gastric parietal cells of Wistar rats. The stomach synthesizes large amounts of estrogens and secretes them into the portal vein. We have been particularly studying gastric estrogen synthesis using Wistar rats. However, estrogen synthesis in the stomach of Sprague-Dawley (SD) rats, which are used as frequently as those of the Wistar strain, has not been clarified. We examined steroid synthesis in the stomach of SD rats using immunohistochemistry, in situ hybridization, Western blotting, real-time PCR, and LC-MS/MS. Aromatase also exists in the stomach of SD rats. Its distribution was not found to be different from that of Wistar rats. Results show that H + /K + -ATPase β-subunit and aromatase colocalized in double immunofluorescence staining. Each steroid synthase downstream from progesterone was present in the gastric mucosa. These results suggest that steroid hormones are synthesized in the parietal cells in the same pathway as Wistar rats. Although mRNA expression of steroid synthases were higher in SD, no significant difference was found in the amount of protein and each steroid hormone level in the portal vein. Although differences between strains might exist in steroid hormone synthesis, results show that SD rats are as useful as Wistar rats for gastric estrogen synthesis experimentation.

Background and Aims: Diffuse-type gastric cancer (GC) is currently subdivided into signet-ring cell carcinoma (SRCC) and non-SRCC, referred to as poorly cohesive carcinoma not otherwise specified (PCC-NOS). Although these subtypes are considered to be independent, they often coexist in the same tumors, raising a question of whether they clonally differ or not. To tackle this question, we established an experimental platform for human diffuse GC that enables accurate modeling of histologic subtypes., Methods: Seven patient-derived diffuse GC organoid lines were established, characterized by histopathologic analysis, in situ hybridization, and gene expression analysis. For genetic modeling of diffuse GC, we knocked out CDH1 and/or TP53 in human normal gastric organoids. Green fluorescent protein-labeled GC organoids were xenotransplanted into immune-deficient mice for in vivo assessment., Results: PCC-NOS organoids transformed into SRCC-like structures on removal of Wnt and R-spondin from the culture medium. This morphologic change paralleled downregulation of Wnt-target and gastric stem cell genes, including LGR5, and elevation of differentiation markers, such as KRT20 and MUCs. The association between Wnt target gene expression and histologic subtypes was confirmed in 3 patient-derived GC tissues. In vivo, single clone-derived organoids formed tumors that comprised 2 distinct histologic compartments, each corresponding to SRCC and PCC-NOS. The transition from PCC-NOS to SRCC histology reflected the abundance of surrounding R-spondin-expressing fibroblasts., Conclusions: SRCC and PCC-NOS were clonally identical, and their morphology was regulated by extracellular Wnt and R-spondin expression. Our results decoded how genetic mutations and the tumor environment shape pathohistologic and biologic phenotypes in human diffuse GCs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

An ability to safely harness the powerful regenerative potential of adult stem cells for clinical applications is critically dependent on a comprehensive understanding of the underlying mechanisms regulating their activity. Epithelial organoid cultures accurately recapitulate many features of in vivo stem cell-driven epithelial renewal, providing an excellent ex vivo platform for interrogation of key regulatory mechanisms. Here, we employed a genome-scale clustered, regularly interspaced, short palindromic repeats (CRISPR) knockout (KO) screening assay using mouse gastric epithelial organoids to identify modulators of Wnt-driven stem cell-dependent epithelial renewal in the gastric mucosa. In addition to known Wnt pathway regulators, such as Apc , we found that KO of Alk , Bclaf3 , or Prkra supports the Wnt independent self-renewal of gastric epithelial cells ex vivo. In adult mice, expression of these factors is predominantly restricted to non- Lgr5 -expressing stem cell zones above the gland base, implicating a critical role for these factors in suppressing self-renewal or promoting differentiation of gastric epithelia. Notably, we found that Alk inhibits Wnt signaling by phosphorylating the tyrosine of Gsk3β, while Bclaf3 and Prkra suppress regenerating islet-derived ( Reg ) genes by regulating the expression of epithelial interleukins. Therefore, Alk, Bclaf3, and Prkra may suppress stemness/proliferation and function as novel regulators of gastric epithelial differentiation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background & Aims: Tight junctions form a barrier to the paracellular passage of luminal antigens. Although most tight junction proteins reside within the apical tight junction complex, claudin-18 localizes mainly to the basolateral membrane where its contribution to paracellular ion transport is undefined. Claudin-18 loss in mice results in gastric neoplasia development and tumorigenesis that may or may not be due to tight junction dysfunction. The aim here was to investigate paracellular permeability defects in stomach mucosa from claudin-18 knockout (Cldn18-KO) mice., Methods: Stomach tissue from wild-type, heterozygous, or Cldn18-KO mice were stripped of the external muscle layer and mounted in Ussing chambers. Transepithelial resistance, dextran 4 kDa flux, and potential difference (PD) were calculated from the chambered tissues after identifying differences in tissue histopathology that were used to normalize these measurements. Marker expression for claudins and ion transporters were investigated by transcriptomic and immunostaining analysis., Results: No paracellular permeability defects were evident in stomach mucosa from Cldn18-KO mice. RNAseq identified changes in 4 claudins from Cldn18-KO mice, particularly the up-regulation of claudin-2. Although claudin-2 localized to tight junctions in cells at the base of gastric glands, its presence did not contribute overall to mucosal permeability. Stomach tissue from Cldn18-KO mice also had no PD versus a lumen-negative PD in tissues from wild-type mice. This difference resulted from changes in transcellular Cl - permeability with the down-regulation of Cl - loading and Cl - secreting anion transporters., Conclusions: Our findings suggest that Cldn18-KO has no effect on tight junction permeability in the stomach from adult mice but rather affects anion permeability. The phenotype in these mice may thus be secondary to transcellular anion transporter expression/function in the absence of claudin-18., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Helicobacter pylori chronically infects the gastric mucosa of humans and diseases associated with infection include gastritis, peptic ulceration, and development of gastric cancer. The organism displays a distinct tropism for the gastric mucosa of humans and for the gastric mucin MUC5AC. While the majority of organisms are found in the mucus layer overlying the epithelial cells in the stomach, adherence of the organism to the gastric epithelium is necessary for the development of disease. The interaction of H. pylori with epithelial cells results in subversion of host cell signaling and induction of an inflammatory response. Factors that influence the outcome of infection include host genetics, environmental factors, and the phenotype of the infecting strain. In this chapter, we describe cell culture assays to assess the interaction of H. pylori with epithelial cells, immunofluorescent staining to detect H. pylori in infected human gastric biopsy specimens and the use of flow cytometry to detect mucin binding to H. pylori.

In Cameroon, local plants are traditionally used as remedies for a variety of ailments. In this regard, several papers report health benefits of Cameroonian spices, which include antioxidant and anti-microbial properties, whereas gastric anti-inflammatory activities have never been previously considered. The present study investigates the antioxidant and anti-inflammatory activities of hydro-alcoholic extracts of eleven Cameroonian spices in gastric epithelial cells (AGS and GES-1 cells). The extracts showed antioxidant properties in a cell-free system and reduced H 2 O 2 -induced ROS generation in gastric epithelial cells. After preliminary screening on TNFα-induced NF-κB driven transcription, six extracts from Xylopia parviflora , Xylopia aethiopica , Tetrapleura tetraptera , Dichrostachys glomerata , Aframomum melegueta , and Aframomum citratum were selected for further studies focusing on the anti-inflammatory activity. The extracts reduced the expression of some NF-κB-dependent pro-inflammatory mediators strictly involved in the gastric inflammatory process, such as IL-8, IL-6, and enzymes such as PTGS2 (COX-2), without affecting PTGS1 (COX-1). In conclusion, the selected extracts decreased pro-inflammatory markers by inhibiting the NF-κB signaling in gastric cells, justifying, in part, the traditional use of these spices. Other molecular mechanisms cannot be excluded, and further studies are needed to better clarify their biological activities at the gastric level.

Background: The ability of the gut to detect nutrients is critical to the regulation of gut hormone secretion, food intake, and postprandial blood glucose control. Ingested nutrients are detected by specific gut chemosensors. However, knowledge of these chemosensors has primarily been derived from the intestine, while available information on gastric chemosensors is limited. This study aimed to investigate the nutrient-sensing repertoire of the mouse stomach with particular emphasis on ghrelin cells., Methods: Quantitative RT-PCR was used to determine mRNA levels of nutrient chemosensors (protein: G protein-coupled receptor 93 [GPR93], calcium-sensing receptor [CaSR], metabotropic glutamate receptor type 4 [mGluR4]; fatty acids: CD36, FFAR2&4; sweet/umami taste: T1R3), taste transduction components (TRPM5, GNAT2&3), and ghrelin and ghrelin-processing enzymes (PC1/3, ghrelin O-acyltransferase [GOAT]) in the gastric corpus and antrum of adult male C57BL/6 mice. Immunohistochemistry was performed to assess protein expression of chemosensors (GPR93, T1R3, CD36, and FFAR4) and their co-localization with ghrelin., Key Results: Most nutrient chemosensors had higher mRNA levels in the antrum compared to the corpus, except for CD36, GNAT2, ghrelin, and GOAT. Similar regional distribution was observed at the protein level. At least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36., Conclusions and Inferences: The cellular mechanisms for the detection of nutrients are expressed in a region-specific manner in the mouse stomach and gastric ghrelin cells. These gastric nutrient chemosensors may play a role modulating gastrointestinal responses, such as the inhibition of ghrelin secretion following food intake., (© 2020 John Wiley & Sons Ltd.)

Huntingtin-associated protein 1 (HAP1) is a neuronal cytoplasmic protein that is predominantly expressed in the brain and spinal cord. In addition to the central nervous system, HAP1 is also expressed in the peripheral organs including endocrine system. Different types of enteroendocrine cells (EEC) are present in the digestive organs. To date, the characterization of HAP1-immunoreactive (ir) cells remains unreported there. In the present study, the expression of HAP1 in pyloric stomach in adult male rats and its relationships with different chemical markers for EEC [gastrin, marker of gastrin (G) cells; somatostatin, marker of delta (D) cells; 5-HT, marker of enterochromaffin (EC) cells; histamine, marker of enterochromaffin-like (ECL) cells] were examined employing single- or double-labelled immunohistochemistry and with light-, fluorescence- or electron-microscopy. HAP1-ir cells were abundantly expressed in the glandular mucosa but were very few or none in the surface epithelium. Double-labelled immunofluorescence staining for HAP1 and markers for EECs showed that almost all the G-cells expressed HAP1. In contrast, HAP1 was completely lacking in D-cells, EC-cells or ECL-cells. Our current study is the first to clarify that HAP1 is selectively expressed in G-cells in rat pyloric stomach, which probably reflects HAP1's involvement in regulation of the secretion of gastrin., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

The major circadian clock gene PER2 is closely related to cell proliferation and lipid metabolism in various nonruminant cell types. Objectives of the study were to evaluate circadian clock-related mRNA abundance in cultured goat ruminal epithelial cells (REC), and to determine effects of PER2 on cell proliferation and mRNA abundance of short-chain fatty acid (SCFA) transporters, genes associated with lipid metabolism, cell proliferation, and apoptosis. Ruminal epithelial cells were isolated from weaned Boer goats (n = 3; 2 mo old; ∼10 kg of body weight) by serial trypsin digestion and cultured at 37°C for 24 h. Abundance of CLOCK and PER2 proteins in cells was determined by immunofluorescence. The role of PER2 was assessed through the use of a knockout model with short interfering RNA, and sodium butyrate (15 mM) was used to assess the effect of upregulating PER2. Both CLOCK and PER2 were expressed in REC in vitro. Sodium butyrate stimulation increased mRNA and protein abundance of PER2 and PER3. Furthermore, PER2 gene silencing enhanced cell proliferation and reduced cellular apoptosis in isolated REC. In contrast, PER2 overexpression in response to sodium butyrate led to lower cellular proliferation and ratio of cells in the S phase along with greater ratio of cells in the G 2 /M phase. Those responses were accompanied by downregulated mRNA abundance of CCND1, CCNB1, CDK1, and CDK2. Among the SCFA transporters, PER2 silencing upregulated mRNA abundance of MCT1 and MCT4. However, it downregulated mRNA abundance of PPARA and PPARG. Overexpression of PER2 resulted in lower mRNA abundance of MCT1 and MCT4, and greater PPARA abundance. Overall, data suggest that CLOCK and PER2 might play a role in the control of cell proliferation, SCFA, and lipid metabolism. Further studies should be conducted to evaluate potential mechanistic relationships between circadian clock and SCFA absorption in vivo., (© 2020, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Biofilm growth is a widespread mechanism that protects bacteria against harsh environments, antimicrobials, and immune responses. These types of conditions challenge chronic colonizers such as Helicobacter pylori but it is not fully understood how H. pylori biofilm growth is defined and its impact on H. pylori survival. To provide insights into H. pylori biofilm growth properties, we characterized biofilm formation on abiotic and biotic surfaces, identified genes required for biofilm formation, and defined the biofilm-associated gene expression of the laboratory model H. pylori strain G27. We report that H. pylori G27 forms biofilms with a high biomass and complex flagella-filled 3D structures on both plastic and gastric epithelial cells. Using a screen for biofilm-defective mutants and transcriptomics, we discovered that biofilm cells demonstrated lower transcripts for TCA cycle enzymes but higher ones for flagellar formation, two type four secretion systems, hydrogenase, and acetone metabolism. We confirmed that biofilm formation requires flagella, hydrogenase, and acetone metabolism on both abiotic and biotic surfaces. Altogether, these data suggest that H. pylori is capable of adjusting its phenotype when grown as biofilm, changing its metabolism, and re-shaping flagella, typically locomotion organelles, into adhesive structures.

Background: Chronic gastritis is a common histologic finding in children with functional dyspepsia (FD). While Th17 cells have been implicated in other forms of gastritis, they have not been evaluated in chronic gastritis., Aims: The aim of the current study was to assess Th17 cells in children with FD with and without chronic gastritis., Methods: Densities were determined for Th17 cells, eosinophils, and mast cells, respectively, in both the gastric antrum and the duodenum. Densities were compared between five groups: FD with chronic gastritis (N = 20), FD without chronic gastritis (N = 20), Helicobacter pylori-associated gastritis (N = 10), Crohn's gastritis (N = 10), and normal controls (N = 10). Th17 densities were also compared between patients with and without early satiety., Results: FD with chronic gastritis was associated with higher Th17 cell density as compared to normal controls and comparable to both H. pylori-associated gastritis and Crohn's gastritis. Eosinophil and mast cell densities were higher in FD patients with chronic gastritis as compared to either FD without gastritis or normal controls. Th17 density was higher in patients reporting early satiety but not in those with epigastric pain., Conclusions: FD with chronic gastritis is associated with higher Th17 cell, eosinophil, and mast cell density as compared to FD without chronic gastritis or normal controls. Chronic gastritis demonstrated Th17 cell density similar to that seen in other conditions where Th17 cells are believed to play a pathogenic role. Th17 cells may represent another therapeutic target in these patients.

Rationale: Mesenchymal stem cells (MSCs) have been the focus of many studies because of their abilities to modulate immune responses, angiogenesis, and promote tumor growth and metastasis. Our previous work showed that gastric cancer MSCs (GCMSCs) promoted immune escape by secreting of IL-8, which induced programmed cell death ligand 1 (PD-L1) expression in GC cells. Mounting evidence has revealed that PD-L1 expression is related to intrinsic tumor cell properties. Here, we investigated whether GCMSCs maintained a pool of cancer stem cells (CSCs) through PD-L1 signaling and the specific underlying molecular mechanism. Methods: Stem cell surface markers, aldehyde dehydrogenase (ALDH) activity, migration and sphere formation abilities were tested to evaluate the stemness of GC cells. PD-L1-expressing lentivirus and PD-L1 specific siRNA were used to analyze the effects of PD-L1 on GC cells stemness. Annexin V/PI double staining was used to assess apoptosis of GC cells induced by chemotherapy. Co-Immunoprecipitation (Co-IP) and Mass spectrometry were employed to determine the PD-L1 binding partner in GC cells. PD-L1 Negative and PD-L1 Positive cells were sorted by flow cytometry and used for limiting dilution assays to verify the effect of PD-L1 on tumorigenic ability in GC cells. Results: The results showed that GCMSCs enhanced the CSC-like properties of GC cells through PD-L1, which led to the resistance of GC cells to chemotherapy. PD-L1 associated with CTCF to contribute to the stemness and self-renewal of GC cells. In vivo , PD-L1 Positive GC cells had greater stemness potential and tumorigenicity than PD-L1 Negative GC cells. The results also indicated that GC cells were heterogeneous, and that PD-L1 in GC cells had different reactivity to GCMSCs. Conclusions: Overall, our data indicated that GCMSCs enriched CSC-like cells in GC cells, which gives a new insight into the mechanism of GCMSCs prompting GC progression and provides a potential combined therapeutic target., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Estragole is an aromatic organic compound belonging to the class of phenylpropanoids derived from cinnamic aldehydes and present in essential oils of plant species, such asRavensara anisata (madeira), Ocimum basilicum (manjericão/alfavaca) and Croton zehntneri (canelinha). Pharmacological studies report its anti-inflammatory, antioxidant and vasorelaxant activity., Hypothesis/purpose: This study aimed to evaluate the acute non-clinical toxicity, gastroprotective activity and the related mechanisms of action., Methods: Acute toxicity was assessed according to OECD guide 423 in mice. Ethanol, stress, piroxicam and pylorus ligation-induced gastric ulcer models were used to investigate antiulcer properties. The related mechanisms of action were using the ethanol-gastric lesions protocol., Results: In the acute oral toxicity assay, doses of 300 or 2000 mg/kg of estragole administered orally in Swiss mice did not induce any behavioral changes. However, the dose of 2000 mg/kg showed a decrease in water and feed intake. Lethal dose 50 % (LD50) was set to be equal to or greater than 2500 mg/kg, according to OECD. In all evaluated protocols, estragole (31.25, 62.5, 125 and 250 mg/kg) significantly reduced the area of ​​ulcerative lesion when compared to control groups. To investigate the mechanisms involved in the gastroprotective activity, the antisecretory or neutralizing of gastric secretion, cytoprotectant, antioxidant and immunoregulatory effects were evaluated. Results showed that treatment with estragole (250 mg/kg) reduced (p < 0.05) the volume of the gastric juice. Besides, sulfhydryl groups, nitric oxide, mucus and prostaglandins seems to be involved in the gastroprotective property. Treatment also increased (p < 0.001) levels of reduced glutathione (GSH), interleukin-10 (IL-10) and positive cells marked for glutathione peroxidase (GPx) and cyclooxygenase 2 (COX-2). It also reduced (p < 0.001) malondialdehyde (MDA), myeloperoxidase (MPO), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and inducible nitric oxide synthase (iNOS) (p < 0.05) levels., Conclusion: Thus, it is possible to infer that estragole presents gastroprotective activity related to antisecretory, cytoprotective, antioxidant and immunomodulatory mechanisms., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The stomach is the primary source of the orexigenic and adiposity-promoting hormone, ghrelin. There is emerging evidence on the nutrient-mediated modulation of gastric ghrelin secretion. However, limited information is available on gastric nutrient-sensing mechanisms in high-fat diet (HFD)-induced obesity. This study investigated the impact of HFD-induced obesity on the expression of nutrient chemosensors in mouse stomach, particularly ghrelin cells. Male C57BL/6 mice were fed either a standard laboratory diet (SLD) or HFD for 12 weeks. The expression of ghrelin, enzymes involved in ghrelin production (PC1/3, GOAT) and nutrient chemosensors (CD36, FFAR2&4, GPR93, CaSR, mGluR4 and T1R3) was determined by quantitative RT-PCR in the mouse corpus and antrum. Immunohistochemistry assessed the protein expression of CaSR and ghrelin in the corpus and antrum. Antral mRNA levels of CaSR and PC1/3 were increased in HFD compared to SLD mice, while mRNA levels of all other nutrient chemosensors examined remained unchanged. CaSR immunolabelling was observed in the gastric antrum only. Nearly 80% of antral ghrelin cells expressed CaSR, with a similar cell density and co-expression in SLD and HFD mice. In conclusion, HFD-induced obesity increased CaSR mRNA expression in mouse antrum. However, the high antral co-expression of CaSR and ghrelin was unaltered in HFD compared to SLD mice.

Fumonisin contaminates food and feed extensively throughout the world, causing chronic and acute toxicity in human and animals. Currently, studies on the toxicology of fumonisins mainly focus on fumonisin B1 (FB1). Considering that FB1, fumonisin B2 (FB2) and fumonisin B3 (FB3) could coexist in food and feed, a study regarding a single toxin, FB1, may not completely reflect the toxicity of fumonisin. The gastrointestinal tract is usually exposed to these dietary toxins. In our study, the human gastric epithelial cell line (GES-1) was used as in vitro model to evaluate the toxicity of fumonisin. Firstly, we found that they could cause a decrease in cell viability, and increase in membrane leakage, cell death and the induction of expression of markers for endoplasmic reticulum (ER) stress. Their toxicity potency rank is FB1 > FB2 >> FB3. The results also showed that the synergistic effect appeared in the combinations of FB1 + FB2 and FB1 + FB3. Nevertheless, the combinations of FB2 + FB3 and FB1 + FB2 + FB3 showed a synergistic effect at low concentration and an antagonistic effect at high concentration. We also found that myriocin (ISP-1) could alleviate the cytotoxicity induced by fumonisin in GES-1 cells. Finally, this study may help to determine or optimize the legal limits and risk assessment method of mycotoxins in food and feed and provide a potential method to block the fumonisin toxicity.

The tumor microenvironment favors the growth and expansion of cancer cells. Many cell types are involved in the tumor microenvironment such as inflammatory cells, fibroblasts, nerves, and vascular endothelial cells. These stromal cells contribute to tumor growth by releasing various molecules to either directly activate the growth signaling in cancer cells or remodel surrounding areas. This review introduces recent advances in findings on the interactions within the tumor microenvironment such as in cancer-associated fibroblasts (CAFs), immune cells, and endothelial cells, in particular those established in mouse gastric cancer models. In mice, myofibroblasts in the gastric stroma secrete R-spondin and support normal gastric stem cells. Most CAFs promote tumor growth in a paracrine manner, but CAF population appears to be heterogeneous in terms of their function and origin, and include both tumor-promoting and tumor-restraining populations. Among immune cell populations, tumor-associated macrophages, including M1 and M2 macrophages, and myeloid-derived suppressor cells (MDSCs), are reported to directly or indirectly promote gastric tumorigenesis by secreting soluble factors or modulating immune responses. Endothelial cells or blood vessels not only fuel tumors with nutrients, but also interact with cancer stem cells and immune cells by secreting chemokines or cytokines, and act as a cancer niche. Understanding these interactions within the tumor microenvironment would contribute to unraveling new therapeutic targets., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background & Aims: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis., Methods: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5 + (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB., Results: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5 + cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5 + cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5 + cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation., Conclusions: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Helicobacter pylori ( H. pylori ) infection leads to the massive apoptosis of the gastric epithelial cells, causing gastric ulcers, gastritis, and gastric adenocarcinoma. Autophagy is a cellular recycling process that plays important roles in cell death decisions and can protect cells by preventing apoptosis. Upon the induction of autophagy, the level of the autophagy substrate p62 is reduced and the autophagy-related ratio of microtubule-associated proteins 1A/1B light chain 3B (LC3B)-II/LC3B-I is heightened. AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are involved in the regulation of autophagy. Astaxanthin (AST) is a potent anti-oxidant that plays anti-inflammatory and anti-cancer roles in various cells. In the present study, we examined whether AST inhibits H. pylori -induced apoptosis through AMPK-mediated autophagy in the human gastric epithelial cell line AGS (adenocarcinoma gastric) in vitro. In this study, H. pylori induced apoptosis. Compound C, an AMPK inhibitor, enhanced the H. pylori -induced apoptosis of AGS cells. In contrast, metformin, an AMPK activator, suppressed H. pylori -induced apoptosis, showing that AMPK activation inhibits H. pylori -induced apoptosis. AST inhibited H. pylori -induced apoptosis by increasing the phosphorylation of AMPK and decreasing the phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt) and mTOR in H. pylori -stimulated cells. The number of LC3B puncta in H. pylori -stimulated cells increased with AST. These results suggest that AST suppresses the H. pylori -induced apoptosis of AGS cells by inducing autophagy through the activation of AMPK and the downregulation of its downstream target, mTOR. In conclusion, AST may inhibit gastric diseases associated with H. pylori infection by increasing autophagy through the activation of the AMPK pathway.

Background: microRNAs (miRNAs) play essential roles in the development and progression of gastric cancer (GC). Although aberrant miR-874 expression has been reported in various human cancers, its role in GC remains obscure., Methods: miR-874 expression was assessed by real-time quantitative polymerase chain reaction (RT-qPCR) in 62 matched GC and adjacent normal tissues, as well as in GC cell lines and immortalized human gastric epithelial cells. CCK8 assay, colony formation assay, and flow cytometry were used to assess the role of miR-874 in GC cell proliferation and apoptosis in vitro. Additionally, to determine the effects of miR-874 on GC cell proliferation and apoptosis in vivo, BALB/c nude mice were injected with GC cells transfected with a miR-874 mimic. The role of miR-874 in SPAG9 expression was assessed by luciferase assay, Western blotting, and RT-qPCR., Results: miR-874 was downregulated in GC cell lines and tissues. miR-874 overexpression in GC cells led to inhibition of cell proliferation and induction of apoptosis. Moreover, SPAG9 was identified as a direct miR-874 target, the expression of which was suppressed by miR-874. SPAG9 overexpression markedly promoted GC cell proliferation., Conclusions: miR-874 inhibited cell proliferation and induced apoptosis in GC cells. SPAG9 downregulation was crucial for the tumor-suppressive effects of miR-874. Hence, the miR-874/SPAG9 axis could serve as a novel therapeutic target in GC.

In the present study, the morphological development of the Brycon amazonicus digestive tract is described to provide basic knowledge for nutritional studies and, therefore, increase the survival of this species during larviculture. Samples were collected from hatching up to 25 days of age, measured, processed and observed under a stereomicroscope and light microscopy. Newly hatched larvae presented their digestive tract as a straight tube, dorsal to the yolk sac, lined with a single layer of undifferentiated cells. At 24 h post-hatching (hPH), the buccopharyngeal cavity was open, but the posterior region of the digestive tube remained closed. At 25 hPH, the digestive tube was completely open and could be divided into buccopharyngeal cavity, oesophagus and intestine. At 35 hPH, the intestine presented a dilatation in the proximal region, which had the function of storing food. Differentiation of the stomach started at 83 hPH, and mucous cells were observed in the epithelium. These cells are important in the production of mucus, whose function is to protect the organ against acidity, although the gastric glands began developing only from 171 hPH, when three stomach regions were observed: cardiac, fundic and pyloric. The gastric glands were observed in the cardiac region, indicating that this organ already had digestive functionality. From 243 hPH, the absorption and assimilation of nutrients were already possible but, only from 412 hPH, the digestive tract was completely developed and functional.

We describe the morphological adaptations of the tongue and gastrointestinal tract of the striped sand snake Psammophis sibilans and discuss their functional importance. Using standard histological, histochemical, and scanning electron microscopy techniques, we analyzed 11 adult snakes of both sexes. Our findings showed that the bifurcated non-papillate tongue exhibited chemoreceptive adaptions to squamate foraging behavior. The lingual apex tapered terminally with sensory spines, and the body of the tongue possesses a characteristic central odor-receptor chamber that might serve to trap and retain scent molecules. Furthermore, the intrinsic musculature showed interwoven and well-developed transverse, vertical and longitudinal muscle fibers that control contraction and retraction during probing and flicking. The esophagus displayed highly folded mucosa lined with columnar epithelium with goblet cells. In contrast, the stomach mucosa formed finger-like gastric rugae, encompassing tubular glands with dorsal gastric pits. The intestine is distinct from other vertebrates in lacking the crypts of Lieberkühn in the tunica mucosa and submucosa. The intestine mucosa is mostly arranged in interdigitating villi oriented perpendicular to the luminal surface. We extrapolated subtle variations for both acid and neutral mucopolysaccharides and glycoproteins localization as well as collagen fibers using histochemical analyses. The elaborate histo-morphological and functional adaptation of the tongue and digestive tract plays a pivotal role in foraging and feeding behavior.

The use of anthocyanins are limited by their chemical properties. Recent evidence suggests Cyanidin-3-O-glucoside (C3 G) liposomes via the ethanol injection method exhibit improved stability. In the current study, the characterization and cell absorption of C3 G liposomes were explored via transmission electron microscopy and flow cytometry. The internalization of the C3 G liposomes across the gastric epithelial cell monolayer (GES-1 cells) were investigated. Results showed that the particle size and encapsulation efficiency were 234 ± 9.35 nm and 75.0% ± 0.001, respectively. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content were used to evaluate the antioxidant activity of C3 G liposomes. The C3 G liposomes can obviously increased T-AOC and decreased the MDA content.Collectively, C3 G liposomes protected human GES-1 cells from gastric mucosal injury induced by H 2 O 2 by activating the related antioxidant pathway. Our research could provide a new effective treatment strategy for the absorption of stomach drugs. Abbreviations : C3G: Cyanidin-3-O-glucoside; LP: Liposome; GES-1 cells: Human gastric epithelial cell lines; FBS: Fetal Bovine Serum; PBS: Phosphate-buffered saline; PC: Phosphatidylcholine; CH: Cholesterol; MDA: Malondialdehyde; TEM: Transmission electron microscope; FCM: Flow cytometry; FITC: Fluorescein isothiocyanate; DAPI: 4', 6-diamidino-2phenylidole; FT-IR: Fourier Transform infrared spectroscopy; PFA: Paraformaldehyde.

Background: Nausea is a common symptom in youth with chronic abdominal pain. The aims of the current study were to assess: 1) the frequency of nausea in patients with functional dyspepsia (FD) and irritable bowel syndrome (IBS), respectively, as defined by Rome IV criteria; and, 2) relationships between nausea and mucosal inflammation as defined by antral and duodenal eosinophil and mast cell densities. A secondary aim was to assess relationships between nausea and other gastrointestinal symptoms, non-gastrointestinal somatic symptoms, and psychological dysfunction., Methods: Records from patients with pain associated functional gastrointestinal disorders were retrospectively reviewed for gastrointestinal and somatic symptoms and anxiety, depression, and somatizations scores as assessed by the Behavior Assessment System for Children (BASC-2). In addition, previous gastric and mucosal biopsies were assessed for mast cell and eosinophil densities, respectively., Results: 250 patients, ages 8 to 17 years, were assessed. Nausea was reported by 78% and was equally prevalent in those with FD alone, those with IBS alone, and those with both FD and IBS. Nausea was associated with increased mean (21.4 vs. 17.5) and peak (26.2 vs. 22.9) duodenal mast cell densities as compared those without nausea. Nausea was also associated with a wide variety of individual gastrointestinal symptoms, as well as headaches, fatigue, and dizziness. Lastly, nausea was associated with elevated self-report scores for anxiety (55.2 vs. 50.0), depression (50.2 vs. 46.1), and somatization (70.3 vs. 61.8)., Conclusions: Nausea is common in children and adolescents with pain-associated FGIDs as defined by Rome IV and is not unique to either FD or IBS. Nausea is associated with increased mucosal mast cell density, non-gastrointestinal somatic symptoms, and psychologic dysfunction.

The intake of dietary lipids is known to affect the composition of phospholipids in gastrointestinal cells, thereby influencing passive lipid absorption. However, dietary lipids rich in polyunsaturated fatty acids, such as vegetable oils, are prone to oxidation. Studies investigating the phospholipid-regulating effect of oxidized lipids are lacking. We aimed at identifying the effects of oxidized lipids from moderately (18.8 ± 0.39 meq O 2 /kg oil) and highly (28.2 ± 0.39 meq O 2 /kg oil) oxidized and in vitro digested cold-pressed grape seed oils on phospholipids in human gastric tumor cells (HGT-1). The oils were analyzed for their antioxidant constituents as well as their oxidized triacylglycerol profile by LC-MS/MS before and after a simulated digestion. The HGT-1 cells were treated with polar oil fractions containing epoxidized and hydroperoxidized triacylglycerols for up to six hours. Oxidized triacylglycerols from grape seed oil were shown to decrease during the in vitro digestion up to 40% in moderately and highly oxidized oil. The incubation of HGT-1 cells with oxidized lipids from non-digested oils induced the formation of cellular phospholipids consisting of unsaturated fatty acids, such as phosphocholines PC (18:1/22:6), PC (18:2/0:0), phosphoserine PS (42:8) and phosphoinositol PI (20:4/0:0), by about 40%-60%, whereas the incubation with the in vitro digested oils did not affect the phospholipid metabolism. Hence, the gastric conditions inhibited the phospholipid-regulating effect of oxidized triacylglycerols (oxTAGs), with potential implications in lipid absorption., Competing Interests: The authors declare no conflict of interest.

Background & Aims: Gastric chief cells, a mature cell type that secretes digestive enzymes, have been proposed to be the origin of metaplasia and cancer through dedifferentiation or transdifferentiation. However, studies supporting this claim have had technical limitations, including issues with the specificity of chief cell markers and the toxicity of drugs used. We therefore sought to identify genes expressed specifically in chief cells and establish a model to trace these cells., Methods: We performed transcriptome analysis of Mist1-CreERT-traced cells, with or without chief cell depletion. Gpr30-rtTA mice were generated and crossed to TetO-Cre mice, and lineage tracing was performed after crosses to R26-TdTomato mice. Additional lineage tracing experiments were performed using Mist1-CreERT, Kitl-CreERT, Tff1-Cre, and Tff2-Cre mice crossed to reporter mice. Mice were given high-dose tamoxifen or DMP-777 or were infected with Helicobacter pylori to induce gastric metaplasia. We studied mice that expressed mutant forms of Ras in gastric cells, using TetO-Kras G12D , LSL-Kras G12D , and LSL-Hras G12V mice. We analyzed stomach tissues from GPR30-knockout mice. Mice were given dichloroacetate to inhibit pyruvate dehydrogenase kinase (PDK)-dependent cell competition., Results: We identified GPR30, the G-protein-coupled form of the estrogen receptor, as a cell-specific marker of chief cells in gastric epithelium of mice. Gpr30-rtTA mice crossed to TetO-Cre;R26-TdTomato mice had specific expression of GPR30 in chief cells, with no expression noted in isthmus stem cells or lineage tracing of glands. Expression of mutant Kras in GPR30 + chief cells did not lead to the development of metaplasia or dysplasia but, instead, led to a reduction in labeled numbers of chief cells and a compensatory expansion of neck lineage, which was derived from upper Kitl + clones. Administration of high-dose tamoxifen, DMP-777, or H pylori decreased the number of labeled chief cells. Chief cells were eliminated from epithelia via GPR30- and PDK-dependent cell competition after metaplastic stimuli, whereas loss of GRP30 or inhibition of PDK activity preserved chief cell numbers and attenuated neck lineage cell expansion., Conclusions: In tracing studies of mice, we found that most chief cells are lost during metaplasia and therefore are unlikely to contribute to gastric carcinogenesis. Expansion of cells that coexpress neck and chief lineage markers, known as spasmolytic polypeptide-expressing metaplasia, does not occur via dedifferentiation from chief cells but, rather, through a compensatory response from neck progenitors to replace the eliminated chief cells., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Antimalarials are globally used against plasmodium infections, however, information on the safety of new antimalarial combination therapies on the gastric mucosa is scarce. The aim of this study was to investigate the effects of Artesunate-Amodiaquine and Artemether-Lumefantrine on ulcer induction. Malondialdehyde (MDA), reduced glutathione (GSH) and major histological changes in male Wistar rats following ulcer induction using Indomethacin were investigated. Gastric ulcers were in four groups; Group I was administered Artesunate, group II received Artesunate-Amodiaquine, group III received Artemether-Lumefantrine, and group IV was a positive control (normal saline). Group V was the negative control consisting of healthy rats., Results: Antimalarial combination therapies were associated with a high gastric ulcer index than a single antimalarial agent, Artesunate. In addition, levels of MDA were significantly higher in the combination of therapies while levels of GSH were lower in comparison to Artesunate and the negative control. Microscopically, antimalarial combination therapies were associated with severe inflammation and tissue damage than Artesunate in the gastric mucosa showing that antimalarial combination therapies exert their toxic effects through oxidative stress mechanisms, and this leads to cellular damage. Findings in this study demonstrate a need to revisit information on the pharmacodynamics of major circulating antimalarial agents in developing countries.

Diet is one of the factors contributing to symptom of Helicobacter pylori (H. pylori) infection. Trimethylamine N-oxide (TMAO), a diet-related microbial metabolite, is associated with inflammatory and metabolic diseases. The aim of this study is to investigate the effects of TMAO intake on inflammation and gut microbiota composition in H. pylori-infected mice via 16S rRNA sequencing and biochemical analyses. The in vitro experiments showed that TMAO not only increased the expression of growth- and metabolism-associated genes and the urease activity of H. pylori, but increased the production of virulence factors. Moreover, TMAO intake increased the production of inflammatory markers and reduced the richness and diversity of the gut microbiota in H. pylori-infected mice. Further analysis showed that TMAO increased the relative abundance of Escherichia&#95;Shigella in H. pylori-infected mice, which had positive correlation with the levels of LPS, CRP, and CXCL1. Collectively, our results suggest that TMAO may aggravate H. pylori-induced inflammation by increasing the viability and virulence of H. pylori and may aggravate inflammation in association with the gut microbiota in H. pylori-infected mice. This study may provide a novel insight into the mechanism for the effect of diet-derived metabolites such as TMAO on H. pylori-induced disease development., Competing Interests: Declaration of Competing Interest We declare that we have no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gastric epithelial cells (GES-1) stimulated by Helicobacter pylori (H. pylori) would affect the expression of related genes and induce the immune response of the cells. Abnormal methylation of DNA was one of the main causes. The aim of this study was to investigate phosphoinositol-3-kinase adaptor protein 1(PIK3AP1), which was screened from the chip data as an immune gene candidate to against the inflammatory response of cells caused by H. pylori infection. PIK3AP1 plays a key role in PI3K/AKT signaling pathway. The gene chip analysis and experimental results confirmed that PIK3AP1 expression was downregulated and PIK3AP1 promoter was hypermethylated after H. pylori stimulation in GES-1 cells. Meanwhile, the expression level of PIK3AP1 was significantly upregulated after 5-aza-dc treatment, and its expression was higher after 5-aza-dc and H. pylori co-treatment than that of H. pylori treatment but lower than that of 5-aza-dc treatment. Therefore, hypermethylation was the main reason for the down-regulation of PIK3AP1 after H. pylori stimulation. In addition, the intervention of PIK3AP1 inhibited the expression of downstream gene AKT, and suppressing the expression of the immunoinflammatory gene IL-6 in GES-1 cells. Furthermore, the intervention of PIK3AP1 would promote cell proliferation. In summary, hypermethylation of the PIK3AP1 promoter was accompanied by reduction of the expression level of PIK3AP1 in GES-1 cells by H. pylori stimulation. The expression of PIK3AP1, AKT, and IL-6 genes was positively correlated, Meanwhile, the PIK3AP1 can affect the proliferation of GES-1 cells. These results would be helpful to understand the innate immune response function of PIK3AP1 to pathogenic bacterial infection in the stomach., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)