Human Gastric Epithelial Cells Research Articles

Catha edulis Leaves: Morphological Characterization and Anti-Inflammatory Properties in an In Vitro Model of Gastritis.

Khat leaves, indigenous to eastern Africa, have been chewed for centuries for their stimulant effects, attributed to alkaloids such as cathinone and cathine. Although associated with gastric disorders like gastritis and gastro-oesophageal reflux disease, the underlying molecular mechanisms remain unclear. This study aimed to examine the morpho-anatomy of khat leaves using light microscopy and histochemistry and to assess the effects of leaf extracts and alkaloids on human gastric epithelial cells (GES-1). The study identified specific cells in the palisade-spongy transition zone as storage sites for psychoactive alkaloids. Leaf extracts were prepared by mimicking the chewing process, including a prolonged salivary phase followed by a gastric phase. Cytotoxicity and cell viability were evaluated using LDH and MTT assays, respectively. Additionally, the impact on IL-8 secretion, a key chemokine in gastric inflammation, was analysed under normal and TNF-α-stimulated conditions. The results showed no increase in cytotoxicity up to 250 µg/mL. However, there was a significant decrease in cell metabolism and a reduction in both basal and TNF-α-induced IL-8 secretion, but cathinone and cathine were inactive. These findings suggest that khat may not directly cause the gastric issues reported in the literature, which would rather be attributed to other confounding factors, highlighting the need for further research to clarify its biological impacts.

Construction of Magnolol Nanoparticles for Alleviation of Ethanol-Induced Acute Gastric Injury.

Ethanol (EtOH) has been identified as a potential pathogenic factor in gastric ulcer development primarily due to its association with gastric injury and excessive production of reactive oxygen species. Magnolol (Mag), the principal active compound in Magnolia officinalis extract, is well studied for its notable anti-inflammatory and antioxidant properties. However, its limited solubility, propensity for agglomeration, and low absorption and utilization rates significantly restrict its therapeutic use. This study aims to overcome these challenges by developing a Mag nanoparticle system targeting the treatment and prevention of EtOH-induced gastric ulcers in mice. Utilizing a click chemistry approach, we successfully synthesized this system by reacting thiolated bovine serum albumin (BSA·SH) with Mag. The in vitro analysis revealed effective uptake of the BSA·SH-Mag nanoparticle system by human gastric epithelial cells (GES-1), showcasing its antioxidant and anti-inflammatory capabilities. Additionally, BSA·SH-Mag exhibited gradual disintegration and release in simulated gastric fluid, resulting in a notable reduction of oxidative stress in gastric tissues and mucosal tissue repair and effectively reducing inflammatory expression. Furthermore, BSA·SH-Mag attenuated EtOH-induced gastric inflammation by decreasing the level of NOX4 protein expression and augmenting the level of Nrf2 protein expression. In conclusion, our findings indicate that BSA·SH-Mag represents a promising candidate as an oral therapeutic for gastric ulcer treatment.

Knocking Down HN1 Blocks Helicobacter pylori-Induced Malignant Phenotypes in Gastric Mucosal Cells and Inhibits Gastric Cancer Cell Proliferation, Cytoskeleton Remodeling, and Migration.

Helicobacter pylori (H. pylori) is implicated in the aberrant proliferation and malignant transformation of gastric mucosal cells, heightening the risk of gastric cancer (GC). HN1 is involved in the development of various tumors. However, precise mechanistic underpinnings of HN1 promoting GC progression in H. pylori remain elusive. The study collected 79 tissue samples of GC patients, including 47 with H. pylori-positive GC and 32 H. pylori-negative controls. Using human gastric epithelial cells (GES-1) and human gastric adenocarcinoma cells (HGC-27), the effect of overexpression / knocking down of HN1 and H. pylori infection was evaluated on cell function (proliferation, migration, apoptosis), cytoskeleton, and expression of cell malignant phenotype factors that promote the malignant biological behavior of cancer cells. The expression of HN1 in GC tissues is higher than that in paracancerous tissue and is closely related to infiltration, lymphatic metastasis, distant metastasis, survival, and H. pylori infection. Downregulation of HN1 effectively hinders the ability of H. pylori strains 26695 and SS1 to promote migration of GES-1 and HGC-27 cells, while lowering the expression of key indicators associated with malignant phenotype. Downregulated GSK3B, β-catenin, and Vimentin after knockdown Integrinβ1, but HN1 expression remained largely unchanged, when HN1 and Integrinβ1 were knocked down, GSK3B, β-catenin, and Vimentin expression were considerably reduced. Our research demonstrated the crucial role of HN1 in H. pylori-induced acquisition of a malignant phenotype in GES-1 cells. Knockdown of HN1 blocked the pathogenic mechanism of H. pylori-induced GC and downregulated the expression of GSK3Β, β-catenin and Vimentin via Integrin β1.

Helicobacter pylori glycan biosynthesis modulates host immune cell recognition and response.

The pathogenic bacterium Helicobacter pylori has evolved glycan-mediated mechanisms to evade host immune defenses. This study tests the hypothesis that genetic disruption of H. pylori glycan biosynthesis alters immune recognition and response by human gastric epithelial cells and monocyte-derived dendritic cells. To test this hypothesis, human cell lines were challenged with wildtype H. pylori alongside an array of H. pylori glycosylation mutants. The relative levels of immune response were measured via immature dendritic cell maturation and cytokine secretion. Our findings indicate that disruption of lipopolysaccharide biosynthesis diminishes gastric cytokine production, without disrupting dendritic cell recognition and activation. In contrast, variable immune responses were observed in protein glycosylation mutants which prompted us to test the hypothesis that phase variation plays a role in regulating bacterial cell surface glycosylation and subsequent immune recognition. Lewis antigen presentation does not correlate with extent of immune response, while the extent of lipopolysaccharide O-antigen elaboration does. The outcomes of this study demonstrate that H. pylori glycans modulate the host immune response. This work provides a foundation to pursue immune-based tailoring of bacterial glycans towards modulating immunogenicity of microbial pathogens.

Mitochondrial carrier homolog 2 increases malignant phenotype of human gastric epithelial cells and promotes proliferation, invasion, and migration of gastric cancer cells.

The precise role of mitochondrial carrier homolog 2 (MTCH2) in promoting malignancy in gastric mucosal cells and its involvement in gastric cancer cell metastasis have not been fully elucidated. To determine the role of MTCH2 in gastric cancer. We collected 65 samples of poorly differentiated gastric cancer tissue and adjacent tissues, constructed MTCH2-overexpressing and MTCH2-knockdown cell models, and evaluated the proliferation, migration, and invasion of human gastric epithelial cells (GES-1) and human gastric cancer cells (AGS) cells. The mitochondrial membrane potential (MMP), mitochondrial permeability transformation pore (mPTP) and ATP fluorescence probe were used to detect mitochondrial function. Mitochondrial function and ATP synthase protein levels were detected via Western blotting. The expression of MTCH2 and ATP2A2 in gastric cancer tissues was significantly greater than that in adjacent tissues. Overexpression of MTCH2 promoted colony formation, invasion, migration, MMP expression and ATP production in GES-1 and AGS cells while upregulating ATP2A2 expression and inhibiting cell apoptosis; knockdown of MTCH2 had the opposite effect, promoting overactivation of the mPTP and promoting apoptosis. MTCH2 can increase the malignant phenotype of GES-1 cells and promote the proliferation, invasion, and migration of gastric cancer cells by regulating mitochondrial function, providing a basis for targeted therapy for gastric cancer cells.

Microfluidic device featuring micro-constrained channels for multi-parametric assessment of cellular biomechanics and high-precision mechanical phenotyping of gastric cells

BackgroundCellular biomechanics plays a significant role in the regulation of cellular physiological and pathological processes. In recent years, multiple methods have been developed to evaluate cellular biomechanics, such as atomic force microscopy (AFM), micropipette aspiration, and magnetic tweezers. However, most of these methods only focus on a single parameter and cannot automate the process at a high-efficiency level. A novel microfluidic method is necessary to achieve the simultaneous multi-parametric measurement of cellular biomechanics and high-precision cellular mechanical phenotyping at high throughput. ResultsTo tackle the issue concerning the low-throughput and cellular single-parameter evaluation, we designed and fabricated a microfluidic chip featuring multiple micro-constrained channels structure, providing a simultaneous multi-parametric assessment of cellular biomechanics, including elastic modulus, recovery capability, and deformability. We compared the biomechanical properties of normal human gastric mucosal epithelial cells (GES-1) and human gastric cancer cells (AGS and MKN-45) by the chip. Results demonstrated that the elastic modulus of GES-1, AGS, and MKN-45 cells decreased sequentially, which was the opposite of their invasiveness and metastasis potential, suggesting the inverse correlation between cellular elastic modulus and malignancy. Meanwhile, the recovery capability and deformability of GES-1, AGS, and MKN-45 cells increased sequentially, demonstrating the positive correlation between cellular deformability and malignancy. Furthermore, multiple parameters were used to distinguish gastric cancer cells from normal gastric cells via machine learning. An accuracy of over 94.8% for identifying gastric cancer cells was achieved. SignificanceThis study provides a deep insight into the biophysical mechanism of gastric cancer metastasis at the single-cell level and possesses great potential to function as a valuable tool for single-cell analysis, thereby facilitating high-precision and high-throughput discrimination of cellular phenotypes that are not easily discernible through single-marker analysis.

IL-6 facilitates cross-talk between epithelial cells and tumor- associated macrophages in Helicobacter pylori-linked gastric carcinogenesis

PurposeHelicobacter pylori (H. pylori) is a significant risk factor for development of gastric cancer (GC), one of the deadliest malignancies in the world. However, the mechanism by which H. pylori induces gastric oncogenesis remains unclear. Here, we investigated the function of IL-6 in gastric oncogenesis and macrophage-epithelial cell interactions. MethodsWe analyzed publicly available datasets to investigate the expression of IL-6 and infiltration of M2 macrophages in GC tissues, and determine the inter-cellular communication in the context of IL-6. Human gastric epithelial and macrophage cell lines (GES-1 and THP-1-derived macrophages, respectively) were used in mono- and co-culture experiments to investigate autocrine-and paracrine induction of IL-6 expression in response to H. pylori or IL-6 stimulation. ResultsWe found that IL-6 is highly expressed in GC and modulates survival. M2 macrophage infiltration is predominant in GC and drives an IL-6 mediated communication with gastric epithelium cells. In vitro, IL-6 triggers its own expression in GES-1 and THP-1-derived macrophages cells. In addition, these cell lines are able to upregulate each other's IL-6 levels in an autocrine fashion, which is enhanced by H. pylori stimulation. ConclusionThis study indicates that IL-6 in the tumor microenvironment is essential for intercellular communication. We show that H. pylori enhances an IL-6-driven autocrine and paracrine positive feedback loop between macrophages and gastric epithelial cells, which may contribute to gastric carcinogenesis.

The Effect of Salvia tomentosa Miller Extracts, Rich in Rosmarinic, Salvianolic and Lithospermic Acids, on Bacteria Causing Opportunistic Infections.

Methanolic-aqueous extracts of Salvia tomentosa Miller roots, aerial parts, and inflorescences were examined for their content of polyphenolic derivatives and the antimicrobial and cytotoxic effect. In the polyphenolic-rich profile, rosmarinic, salvianolic, and lithospermic acids along with various derivatives were predominant. A total of twenty phenolic compounds were identified using the UPLC/DAD/qTOF-MS technique. These were caffeic acid, rosmarinic acid derivatives, lithospermic acid derivatives, salvianolic acids B, F, and K derivatives, as well as sagerinic acid, although rosmarinic acid (426-525 mg/100 g of dry weight-D.W.) and salvianolic acid B (83-346.5 mg/100 g D.W.) were significantly predominant in the metabolic profile. Strong antibacterial activity of S. tomentosa extracts was observed against Staphylococcus epidermidis (MIC/MBC = 0.625 mg/mL) and Bacillus cereus (MIC = 0.312-1.25 mg/mL). The extracts showed low cytotoxicity towards the reference murine fibroblasts L929 and strong cytotoxicity to human AGS gastric adenocarcinoma epithelial cells in the MTT reduction assay. The observed cytotoxic effect in cancer cells was strongest for the roots of 2-year-old plant extracts.

Single-cell data revealed CD14-type and FCGR3A-type macrophages and relevant prognostic factors for predicting immunotherapy and prognosis in stomach adenocarcinoma.

Stomach adenocarcinoma (STAD) exhibits profound tumor heterogeneity and represents a great therapeutic challenge. Single-cell sequencing technology is a powerful tool to identify characteristic cell types. Single-cell sequencing data (scRNA-seq) GSE167297 and bulk RNA-seq data from TCGA, GTEx, GSE26901 and GSE15459 database were included in this study. By downscaling and annotating the cellular data in scRNA-seq, critical cell types in tumor progression were identified by AUCell score. Relevant gene modules were then identified by weighted gene co-expression network analysis (WGCNA). A prognostic scoring system was constructed by identifying prognostic factors in STAD by Least absolute shrinkage and selection operator (LASSO) COX model. The prognosis and model performance in the RiskScore groups were measured by Kaplan-Meier (K-M) curves and Receiver operating characteristic (ROC) curves. Nomogram was drawn based on RiskScore and prognosis-related clinical factors. In addition, we evaluated patient's feedback on immunotherapy in the RiskScore groups by TIMER, ESTIMATE and TIDE analysis. Finally, the expression levels of prognostic factors were verified in gastric cancer cell lines (MKN7 and MKN28) and human normal gastric mucosal epithelial cells (GES-1), and the effects of prognostic factors on the viability of gastric cancer cells were examined by the CCK8 assay and cell cycle. scRNA-seq analysis revealed that 11 cell types were identified, and macrophages exhibited relatively higher AUCell scores and specifically expressed CD14 and FCGR3A. High macrophage scores worsened the prognosis of STAD patients. We intersected the specifically expressed genes in macrophages subgroups (670) and macrophage module genes (2,360) obtained from WGCNA analysis. Among 86 common genes, seven prognostic factors (RGS2, GNAI2, ANXA5, MARCKS, CD36, NRP1 and PDE4A) were identified and composed a RiskScore model. Patients in low Risk group showed a better survival advantage. Nomogram also provided a favorable prediction for survival at 1, 3 and 5 years in STAD patients. Besides, we found positive feedback to immunotherapy in patients with low RiskScore. The expression tendency of the seven prognostic factors in MKN7 and MKN28 was consistent with that in the RNA-seq data in addition to comparison of protein expression levels in the public HPA (The Human Protein Atlas) database. Further functional exploration disclosed that MARCKS was an important prognostic factor in regulating cell viability in STAD. This study preliminary uncovered a single cell atlas for STAD patients, and Macrophages relevant gene signature and nomogram displayed favorable immunotherapy and prognostic prediction ability. Collectively, our work provides a new insight into the molecular mechanisms and therapeutic approach for LUAD patients.

Spectroscopic assessment of biomolecular changes in Helicobacter pylori and Epstein–Barr virus co‐infected gastric epithelial cells

AbstractHelicobacter pylori and Epstein–Barr Virus (EBV) are Group 1 carcinogens that can enhance gastric cancer progression. Bioactive substances extracted from plants can be effective therapeutic agents in cancer treatment. For example, Withania somnifera extract‐WSE reduces the Gankyrin oncoprotein, which is upregulated in the presence of H. pylori and EBV. The various biochemical and metabolic changes upon 24 hrs post‐infection followed by W. somnifera extract (WSE) treatment on gastric epithelial cells (AGS) can be studied using spectroscopic techniques. In the biomedical sciences, Raman and NMR spectroscopy have been extensively employed to interpret cellular alterations contributing to the onset of infection and the severity of gastric cancer. More specifically, alterations in cellular biochemical homeostasis are linked to the moieties of cholesterol, collagen, choline, carbohydrate, lipids, tyrosine, and phenylalanine. Further, we have found significantly elevated FWHM for carbohydrates, tumor associated protein, collagen, cholesterol, and cholesterol ester in the co‐infection model. We also looked into the potential correlation between these molecules using molecular network analysis and found several related factors that can be modulated through biomolecular levels. These molecules are crucial in several physiological functions, including cell division, cell proliferation, apoptosis, necrosis, cell migration, and lipid transport. Our study paves the pathway to study H. pylori and EBV co‐infection in human gastric epithelial cells and the therapeutic interventions of WSE in this scenario and highlights specific biomolecular alterations, which can be focused for further mechanistic investigations.

Investigation of Campylobacter concisus gastric epithelial pathogenicity using AGS cells.

Campylobacter concisus is an oral bacterium. Recent studies suggest that C. concisus may be involved in human gastric diseases. The mechanisms, however, by which C. concisus causes human gastric diseases have not been investigated. Here we examined the gastric epithelial pathogenicity of C. concisus using a cell culture model. Six C. concisus strains and the human gastric epithelial cell line AGS cells were used. IL-8 produced by AGS cells after incubation with C. concisus was measured using enzyme-linked immunosorbent assay (ELISA), and AGS cell apoptosis was determined by caspase 3/7 activities. The effects of C. concisus on actin arrangement in AGS cells was determined using fluorescence staining. The effects of C. concisus on global gene expression in AGS cells was determined by transcriptomic analysis and quantitative real-time PCR (qRT-PCR). The role of the upregulated CYP1A1 gene in gastric cancer survival was assessed using the Kaplan-Meier method. C. concisus induced production of IL-8 by AGS cells with strain variation. Significantly increased caspase 3/7 activities were observed in AGS cells incubated with C. concisus strains when compared to AGS cells without bacteria. C. concisus induced actin re-arrangement in AGS cells. C. concisus upregulated 30 genes in AGS cells and the upregulation of CYP1A1 gene was confirmed by qRT-PCR. The Kaplan-Meier analysis showed that upregulation of CYP1A1 gene is associated with worse survival in gastric cancer patients. Our findings suggest that C. concisus may play a role in gastric inflammation and the progression of gastric cancer. Further investigation in clinical studies is warranted.

CircPRDM5 inhibits the proliferation, migration, invasion, and glucose metabolism of gastric cancer cells by reducing GCNT4 expression in a miR-485-3p-dependent manner.

Circular RNA (circRNA) plays a key part in the pathological process of gastric cancer (GC). The study is organized to analyze the function of circPRDM5 in GC cell tumor properties. Expression levels of circPRDM5, miR-485-3p, glucosaminyl (N-acetyl) transferase 4 (GCNT4), ki67, E-cadherin, N-cadherin, and hexokinase 2 (HK2) were analyzed by quantitative real-time polymerase chain reaction (PCR), Western blotting or immunohistochemistry assay. Cell proliferation was assessed by cell colony formation assay and 5-ethynyl-2'-deoxyuridine assay. Cell migration and invasion were investigated by transwell assay. Glycolysis was evaluated by the Seahorse XF Glycolysis Stress Test Kit. Dual-luciferase reporter assay and RNA pull-down assay were performed to identify the associations among circPRDM5, miR-485-3p, and GCNT4. Xenograft mouse model assay was conducted to determine the effects of circPRDM5 on tumor formation in vivo. CircPRDM5 and GCNT4 expression were downregulated, while miR-485-3p expression was upregulated in GC tissues and cells when compared with paracancerous tissues or human gastric epithelial cells. CircPRDM5 overexpression inhibited proliferation, migration, invasion, and glucose metabolism of GC cells; however, circPRDM5 depletion had the opposite effects. CircPRDM5 repressed tumor properties of GC cells in vivo. MiR-485-3p restoration relieved circPRDM5-induced effects in GC cells. GCNT4 overexpression remitted the promoting effects of miR-485-3p mimics on GC cell malignancy. CircPRDM5 acted as a sponge for miR-485-3p, and GCNT4 was identified as a target gene of miR-485-3p. Moreover, circPRDM5 regulated GCNT4 expression by interacting with miR-485-3p.CircPRDM5 acted as a miR-485-3p sponge to inhibit GC progression by increasing GCNT4 expression, proving a potential target for GC therapy.

Ochratoxin A-enhanced glycolysis induces inflammatory responses in human gastric epithelium cells through mTOR/HIF-1α signaling pathway

Ochratoxin A (OTA) is a mycotoxin commonly found in several food commodities worldwide with potential nephrotoxic, hepatotoxic and carcinogenic effects. We previously showed for the first time that OTA treatment enhanced glycolysis in human gastric epithelium (GES-1) cells in vitro. Here, we found that OTA exposure activated inflammatory responses, evidenced by increasing of NF-κB signaling pathway-related protein (p-p65 and p-IκBα) expressions and elevating of inflammatory cytokine (IL-1β and IL-6) mRNA expressions in GES-1 cells. To elucidate the role of glycolysis in inflammatory effects triggered by OTA, we pretreated GES-1 cells with glycolysis inhibitor (2-deoxy-D-glucose, 2-DG) before OTA exposure. The result showed that 2-DG reduced the protein expressions of p-p65 and p-IκBα and alleviated the mRNA expressions of inflammatory cytokines in OTA-treated GES-1 cells. Furthermore, OTA activated the mTOR/HIF-1α pathway by increasing the protein expressions of p-mTOR, p-eIF4E and HIF-1α, and inhibition of mTOR with rapamycin or silencing HIF-1α with siRNA significantly attenuated OTA-enhanced glycolysis by reducing glycolysis related genes and thereby decreasing inflammatory effects of GES-1 cells. These results demonstrate that OTA activates inflammatory responses in GES-1 cells and this is controlled by mTOR/HIF-1α pathway-mediated glycolysis enhancement. Our findings provide a novel mechanistic view into OTA-induced gastric cytotoxicity.

Quercetin ameliorates Helicobacter pylori-induced gastric epithelial cell injury by regulating specificity protein 1/lipocalin 2 axis in gastritis.

Helicobacter pylori (HP) infection is the main cause of most cases of gastritis. Quercetin has been shown to have anti-inflammatory, anti-bacterial, and antiviral activities and has been demonstrated to be involved in HP-induced gastric mucosa injury. Moreover, the secretory protein lipocalin-2 (LCN2) was elevated in HP-infected gastric mucosa. Thus, this work aimed to study the interaction between quercetin and LCN2 in HP-triggered gastric injury during gastritis. Human gastric epithelial cell line GES-1 cells were exposed to HP for functional experiments. Cell viability, apoptosis, and inflammation were evaluated by cell counting kit-8, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Levels of genes and proteins were tested using quantitative reverse transcription polymerase chain reaction and western blotting analyses. The interaction between LCN2 and specificity protein 1 (SP1) was validated using chromatin immunoprecipitation assay and dual-luciferase reporter assay. Thereafter, we found quercetin treatment suppressed HP-induced GES-1 cell apoptotic and inflammatory injury and macrophage M1 polarization. LCN2 was highly expressed in HP-infected gastritis patients and HP-infected GES-1 cells, while quercetin reduced LCN2 expression in HP-infected GES-1 cells; moreover, LCN2 knockdown reversed HP-induced GES-1 cell injury and macrophage M1 polarization, and forced expression of LCN2 abolished the protective effects of quercetin on GES-1 cells under HP infection. Mechanistically, SP1 bound to LCN2 promoter and promoted its transcription. Also, SP1 overexpression counteracted the functions of quercetin on HP-stimulated GES-1 cells. In all, quercetin ameliorated HP-induced gastric epithelial cell apoptotic and inflammatory injuries, and macrophage M1 polarization via the SP1/LCN2 axis.

Licorice flavonoid alleviates gastric ulcers by producing changes in gut microbiota and promoting mucus cell regeneration

Licorice flavonoid (LF) is the main component of Glycyrrhizae Radix et Rhizoma, a “medicine food homology” herbal medicine, which has anti-digestive ulcer activity, but the mechanism in anti-gastric ulcer (GU) remains to be elucidated. In this study, we manifested that LF increased the viability of human gastric mucosal epithelial (GES-1) cells, attenuated ethanol (EtOH)-induced manifestations, reduced histological injury, suppressed inflammation, and restored gastric mucosal barrier in GU rats. After LF therapy, the EtOH-induced gut dysbiosis was partly modulated, and short-chain fatty acids (SCFAs) like butyric acid, propionic acid, and valeric acid were found in higher concentrations. We discovered that the majority of genera that increased in the GU group had a negative correlation with SCFAs in the intestinal tract. In addition, LF-upregulated SCFAs boosted mucus secretion in the gastric epithelium and the expression of mucoprotein (MUC) 5AC and MUC6, particularly the MUC5AC in the gastric foveola. Moreover, LF triggered the EGFR/ERK signal pathway which promoted gastric mucus cell regeneration. Therefore, the findings indicated that LF could inhibit inflammation, promote mucosal barrier repair and angiogenesis, regulate gut microbiota and SCFA metabolism; more importantly, promote epithelial proliferation via activation of the EGFR/ERK pathway, exerting a protective and regenerative effect on the gastric mucosa.

Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization.

Bacterial lipoproteins are post-translationally modified with acyl chains, anchoring these proteins to bacterial membranes. In Gram-negative bacteria, three enzymes complete the modifications. Lgt (which adds two acyl chains) and LspA (which removes the signal peptide) are essential. Lnt (which adds a third acyl chain) is not essential in certain bacteria including Francisella tularensis, Neisseria gonorrhoeae, and Acinetobacter baumannii. Deleting lnt results in mild to severe physiologic changes. We previously showed lnt is not essential for Helicobacter pylori growth in vitro. Here, the physiologic consequences of deleting lnt in H. pylori and the role of Lnt in the host response to H. pylori were examined using in vitro and in vivo models. Comparing wild-type, Δlnt, and complemented mutant H. pylori, no changes in growth rates or sensitivity to acid or antibiotics were observed. Since deleting lnt changes the number of acyl chains on lipoproteins and the number of acyl chains on lipoproteins impacts the innate immune response through Toll-like receptor 2 (TLR2) signaling, primary human gastric epithelial cells were treated with a purified lipoprotein from wild-type or lnt mutant H. pylori. Differential gene expression analysis indicated that lipoprotein from the lnt mutant induced a more robust TLR2 response. In a complementary approach, we infected wild-type and Tlr2-/- mice and found that both the wild-type and complemented mutant strains successfully colonized the animals. However, the lnt mutant strain was unable to colonize either mouse strain. These results show that lnt is essential for H. pylori colonization and identifies lipoprotein synthesis as a target for therapeutic intervention.

Investigating the Molecular Mechanisms Underlying Early Response to Inflammation and Helicobacter pylori Infection in Human Gastric Epithelial Cells.

Helicobacter pylori is a leading cause of chronic gastric inflammation, generally associated with gastritis and adenocarcinoma. Activation of the NF-κB pathway mainly contributes to the inflammatory phenotype observed in H. pylori infection in humans and experimental models. Since the gastric epithelium undergoes rapid turnover, inflammation and pathogenicity of H. pylori result from early phase and chronically activated pathways. In the present study we investigated the early host response to H. pylori in non-tumoral human gastric epithelial cells (GES-1). To dissect the pathogen-specific mechanisms we also examined the response to tumor necrosis factor (TNF), a prototypical cytokine. By analyzing the activation state of NF-κB signaling, cytokine expression and secretion, and the transcriptome, we found that the inflammatory response of GES-1 cells to H. pylori and TNF results from activation of multiple pathways and transcription factors, e.g., NF-κB and CCAAT/enhancer-binding proteins (CEBPs). By comparing the transcriptomic profiles, we found that H. pylori infection induces a less potent inflammatory response than TNF but affects gene transcription to a greater extent by specifically inducing transcription factors such as CEBPβ and numerous zinc finger proteins. Our study provides insights on the cellular pathways modulated by H. pylori in non-tumoral human gastric cells unveiling new potential targets.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

A novel role for Helicobacter pylori cytotoxin-associated gene A in negative regulation of autophagy in human gastric cells

BackgroundAutophagy plays an important role in carcinogenesis and tumor progression in many cancers, including gastric cancer. Cytotoxin-associated gene A (CagA) is a well-known virulent factor in Helicobacter pylori (H. pylori) infection that plays a critical role in gastric inflammation and gastric cancer development. However, its role in autophagy during these processes remains unclear. Therefore, we aimed to clarify the role of CagA in autophagy in CagA-related inflammation.MethodsWe evaluated the autophagic index of AGS cells infected with wild-type cagA-positive H. pylori (Hp-WT) and cagA-knockout H. pylori (Hp-ΔcagA) and rat gastric mucosal (RGM1) cells transfected with CagA genes. To identify the mechanisms underlying the down regulation of autophagy in AGS cells infected with H. pylori, we evaluated protein and mRNA expression levels of autophagy core proteins using western blotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). To determine whether autophagy induced the expression of the pro-inflammatory mediator, cyclooxygenase-2 (COX-2), we evaluated COX-2 expression in AGS cells treated with an autophagy inducer and inhibitor and infected with H. pylori. In addition, we evaluated whether COX-2 protein expression in AGS cells influenced beclin-1 (BECN1) expression with si-RNA transfection when infected with H. pylori.ResultsAutophagic flux assay using chloroquine showed that autophagy in AGS cells was significantly suppressed after H. pylori infection. The autophagic index of AGS cells infected with Hp-WT was decreased significantly when compared with that in AGS cells infected with Hp-ΔcagA. The autophagic index of RGM1 cells transfected with CagA was lower, suggesting that CagA inhibits autophagy. In addition, BECN1 expression levels in AGS cells infected with Hp-WT were reduced compared to those in AGS cells infected with Hp-ΔcagA. Furthermore, COX-2 expression in AGS cells infected with H. pylori was controlled in an autophagy-dependent manner. When AGS cells were transfected with small interfering RNA specific for BECN1 and infected with Hp-WT and Hp-ΔcagA, COX-2 was upregulated significantly in cells infected with Hp-ΔcagA.ConclusionsIn conclusion, the H. pylori CagA protein negatively regulated autophagy by downregulating BECN1. CagA-induced autophagy inhibition may be a causative factor in promoting pro-inflammatory mediator production in human gastric epithelial cells.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.