Gastric Epithelial Cells Research Articles

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.

Helicobacter Pylori and Sociodemographic Characteristics Distribution

Introduction and Aim: Helicobacter pylori is a gram-negative bacterium that affects up to half of the world's population, particularly in poorer nations. It is the leading cause of chronic gastritis, ulcers, gastric lymphoma, and gastric cancer, with more frequent cases in children and adolescents. Transmission can occur through fecal-oral, gastro-oral, oral-oral, or sexual means. The infection is caused by H. pylori's urease activity, invasion of host gastric epithelial cells, and adherence to host cell receptors. Early treatment can enhance gastric protection. Materials and Methods: Between November 2022 and July 2023, 103 samples were collected from patients at Mihrabani Surgical Hospital in Erbil City to identify Helicobacter pylori infection. Blood samples and stool samples were used to test for antibodies. Results: The study found a relatively equal gender representation in the patient and control groups, with 48.48% of men in the patient group and 48.64% in the control group. However, there are significant differences in age distribution, with G3 of age (<35) having the highest prevalence of H.pylori (48.48%). Additionally, 62.12% of patients are rural residents, while 97.29% of the control group are urban residents. Finally, 56.07% of patients are married, indicating a significant difference. Conclusion: The study found equal gender representation in patient and control groups but significant age differences, rural residency, and marital status, with G3 under 35 having the highest H.pylori prevalence.

YTHDF1 mediates N-methyl-N-nitrosourea-induced gastric carcinogenesis by controlling HSPH1 translation.

YT521-B homology (YTH) domain family (YTHDF) proteins serve as readers that directly recognise m6A modifications. In this study, we aim to probe the role of YTHDF1 in environmental carcinogen-induced malignant transformation of gastric cells and gastric cancer (GC) carcinogenesis. We established a long-term low-dose MNU-induced malignant transformation model in gastric epithelial cells. In vivo and in vitro experiments were conducted to validate the malignant phenotype and characterise the roles of YTHDF1 and its downstream genes in malignant transformation cells. Additionally, we explored downstream m6A modification targets of YTHDF1 using RNA-sequencing, RNA immunoprecipitation, and proteomics analyses, and conducted validation experiments in cell experiments and clinical samples. Long-term low-dose exposure of MNU converted normal Gges-1 cells into malignant cells. YTHDF1 mRNA and protein expression are increased in MNU-induced malignant cells (p<0.001). Meanwhile, YTHDF1 knockdown inhibits the malignant potential of MNU-treated cells (p<0.01). YTHDF1 knockdown specifically suppresses HSPH1 protein, but not RNA levels. RIP-qPCR validates HSPH1 is the target of YTHDF1 (p<0.01). HSPH1 knockdown impairs the malignant potential of MNU-induced transformed cells. The increased expression of the key regulatory factor YTHDF1 in MNU-induced gastric carcinogenesis affects malignant transformation and tumorigenesis by regulating the translation of downstream HSPH1. These findings provide new potential targets for preventing and treating environmental chemical-induced gastric carcinogenesis.

Cortactin-dependent control of Par1b-regulated epithelial cell polarity in Helicobacter infection

Cell polarity is crucial for gastric mucosal barrier integrity and mainly regulated by polarity-regulating kinase partitioning-defective 1b (Par1b). During infection, the carcinogen Helicobacter pylori hijacks Par1b via the bacterial oncoprotein CagA leading to loss of cell polarity, but the precise molecular mechanism is not fully clear. Here we discovered a novel function of the actin-binding protein cortactin in regulating Par1b, which forms a complex with cortactin and the tight junction protein zona occludens-1 (ZO-1). We found that serine phosphorylation at S405/418 and the SH3 domain of cortactin are important for its interaction with both Par1b and ZO-1. Cortactin knockout cells displayed disturbed Par1b cellular localization and exhibited morphological abnormalities that largely compromised transepithelial electrical resistance, epithelial cell polarity, and apical microvilli. H. pylori infection promoted cortactin/Par1b/ZO-1 abnormal interactions in the tight junctions in a CagA-dependent manner. Infection of human gastric organoid-derived mucosoids supported these observations. We therefore hypothesize that CagA disrupts gastric epithelial cell polarity by hijacking cortactin, and thus Par1b and ZO-1, suggesting a new signaling pathway for the development of gastric cancer by Helicobacter.

Influence of chlorpyrifos and endosulfan and their metabolites on the virulence of Helicobacter pylori

Organochlorine (OC) and organophosphorus (OP) pesticides such as chlorpyrifos (CPF) and endosulfan (ES) have been associated with a plethora of adverse health effects. Helicobacter pylori (H. pylori) infection can lead to gastrointestinal diseases by regulating several cellular processes. Thus, the current study focuses on the effect of the co-exposure to pesticides and H. pylori on gastric epithelial cells. We have used the in-silico approach to determine the interactive potential of pesticides and their metabolites with H. pylori-associated proteins. Further, various in-vitro methods depict the potential of ES in enhancing the virulence of H. pylori. Our results showed that ES along with H. pylori affects the mitochondrial dynamics, increases the transcript expression of mitochondrial fission genes, and lowers the mitochondrial membrane potential and biomass. They also promote inflammation and lower oxidative stress as predicted by ROS levels. Furthermore, co-exposure induces the multi-nucleated cells in gastric epithelial cells. In addition, ES along with H. pylori infection follows the extrinsic pathway for apoptotic signaling. H. pylori leads to the NF-κB activation which in turn advances the β-catenin expression. The expression was further enhanced in the co-exposure condition and even more prominent in co-exposure with ES-conditioned media. Thus, our study demonstrated that pesticide and their metabolites enhance the pathogenicity of H. pylori infection.

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.

Unraveling the crystal structure of the HpaA adhesin: insights into cell adhesion function and epitope localization of a Helicobacter pylori vaccine candidate.

Helicobacter pylori is a bacterium that can cause chronic gastritis, peptic ulcers, and gastric cancers. The bacterium adheres to the lining of the stomach using proteins called adhesins. One of these proteins, HpaA, is particularly important for H. pylori colonization and is considered a promising vaccine candidate against H. pylori infections. In this work, we determined the atomic structure of HpaA, identifying a characteristic protein fold to the Helicobacter family and delineating specific amino acids that are crucial to support the attachment to the gastric cells. Additionally, we discovered that HpaA can trigger the production of TNF-α, a proinflammatory molecule, in macrophages. These findings provide valuable insights into how H. pylori causes disease and suggest that HpaA has a dual role in both attachment and immune activation. This knowledge could contribute to the development of improved vaccine strategies for preventing H. pylori infections.

A162 DEFINING INNATE IMMUNE CELL MECHANISMS UPON HELICOBACTER PYLORI MEDIATED TUMORIGENESIS

Abstract Aims Hypothesis: H. pylori interacts with eosinophils to inhibit degranulation, alter eosinophil gene expression and promote gastric tumorigenesis Aim 1: Elucidate the mechanisms underlying the interactions between eosinophils and H. pylori. Aim 2: Determine the role of eosinophils during H. pylori mediated gastric tumorigenesis. Methods Histological and immunofluorescent staining. Hemotoxilin and eosin staining is important to analyze gastric gland structure and parietal cell loss. Immunofluorescent staining is used to detect and localize specific antigens present in cells and cell membranes. Gastric organoids: Gastric organoids are an important tool for modelling dissease. Gastric organoids generated from adult stem cells from gastric glands depend on growth factors that mimic tissue regeneration. Bone marrow derived eosinophils: Bone marrow progenitor cells isolated from murine femur and tibia are easily converted into maturer eosinophils with the addition of cytokine interleukin-5. Results Addition of mouse derived eosinophils to gastric organoids significantly increases number of organoids. To understand the relationship between eosinophils and gastric epithelial cell (GEC) growth, I designed a co-culture experiment that involved incubating mature eosinophils with gastric glands in an extracellular matrix. These findings revealed a significant increase in the number of gastric organoids per well when eosinophils were present compared to the gastric organoids without eosinophils. This observation supports the hypothesis that eosinophils may play a crucial role in promoting gastric tumorigenesis. Exposure of mouse derived eosinophils to H. pylori significantly increases gastric organoid proliferation. To explore the impact of stimulating eosinophils prior to incubation with GECs I incubated the eosinophils with H. pylori. As expected, there was a significant increase in the number of organoids per well with the addition of uninfected eosinophils. Interestingly, when H. pylori incubated eosinophils were added to the gastric glands, there was a significant increase in the number of gastric organoids per well compared to uninfected eosinophils. Conclusions My preliminary findings have indicated that eosinophils can promote gastric epithelial stem cell proliferation. These results led me to ask how treatment of eosinophils with H. pylori subsequently impacts gastric organoid growth and gastric stem cell proliferation. Interestingly, I found H. pylori treated eosinophils increase growth and gastric stem cell proliferation. These results support my hypothesis as increased proliferation is considered a hallmark of tumorigenesis. There little known about resident and inflammatory GI eosinophils and the role of eosinophils during tumorigenesis in unknown. A better understanding of the role eosinophils play can uncover novel mechanisms to be targeted for therapeutic intervention. Funding Agencies CIHR

Sustained exposure to Helicobacter pylori induces immune tolerance by desensitizing TLR6.

Helicobacter pylori (H. pylori, Hp) has been designated a class I carcinogen and is closely associated with severe gastric diseases. During colonization in the gastric mucosa, H. pylori develops immune escape by inducing host immune tolerance. The gastric epithelium acts as the first line of defense against H. pylori, with Toll-like receptors (TLRs) in gastric epithelial cells being sensitive to H. pylori components and subsequently activating the innate immune system. However, the mechanism of immune tolerance induced by H. pylori through the TLR signalling pathway has not been fully elucidated. In this research, we detected the expression of TLRs and inflammatory cytokines in GES-1 cells upon sustained exposure to H. pylori or H. pylori lysate from 1 to 30 generations and in Mongolian gerbils infected with H. pylori for 5 to 90 weeks. We found that the levels of TLR6 and inflammatory cytokines first increased and then dropped during the course of H. pylori treatment in vitro and in vivo. The restoration of TLR6 potentiated the expression of IL-1β and IL-8 in GES-1 cells, which recruited neutrophils and reduced the colonization of H. pylori in the gastric mucosa of gerbils. Mechanistically, we found that persistent infection with H. pylori reduces the sensitivity of TLR6 to bacterial components and regulates the expression of inflammatory cytokines in GES-1 cells through TLR6/JNK signaling. The TLR6 agonist obviously alleviated inflammation in vitro and in vivo. Promising results suggest that TLR6 may be a potential candidate immunotherapy drug for H. pylori infection.

Anti-inflammatory effects of extracellular vesicles and cell-free supernatant derived from Lactobacillus crispatus strain RIGLD-1 on Helicobacter pylori-induced inflammatory response in gastric epithelial cells in vitro.

Helicobacter pylori infection is the major risk factor associated with the development of gastric cancer. Currently, administration of standard antibiotic therapy combined with probiotics and postbiotics has gained significant attention in the management of H. pylori infection. In this work, the immunomodulatory effects of Lactobacilluscrispatus-derived extracellular vesicles (EVs) and cell-free supernatant (CFS) were investigated on H. pylori-induced inflammatory response in human gastric adenocarcinoma (AGS) cells. L. crispatus-derived EVs were isolated by ultracentrifugation and physically characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Furthermore, the protein content of L. crispatus-derived EVs was also evaluated by SDS-PAGE. Cell viability of AGS cells exposed to varying concentrations of EVs and CFS was assessed by MTT assay. The mRNA expression of IL-1β, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR. ELISA was used for the measurement of IL-8 production in AGS cells. In addition, EVs (50 μg/mL) and CFS modulated theH. pylori-induced inflammation by downregulating the mRNA expression of IL-1β, IL-6, IL-8, and TNF-α, and upregulating the expression of IL-10, and TGF-ß genes in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with L. crispatus-derived EVs and CFS. In conclusion, our observation suggests for the first time that EVs released by L. crispatus strain RIGLD-1 and its CFS could be recommended as potential therapeutic agents against H. pylori-triggered inflammation.

Helicobacter pylori secretory Proteins-Induced oxidative stress and its role in NLRP3 inflammasome activation

Helicobacter pylori-associated stomach infection is a leading cause of gastric ulcer and related cancer. H. pylori modulates the functions of infiltrated immune cells to survive the killing by reactive oxygen and nitrogen species (ROS and RNS) produced by these cells. Uncontrolled immune responses further produce excess ROS and RNS which lead to mucosal damage. The persistent oxidative stress is a major cause of gastric cancer. H. pylori regulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), nitric oxide synthase 2 (NOS2), and polyamines to control ROS and RNS release through lesser-known mechanisms. ROS and RNS produced by these pathways differentiate macrophages and T cells from protective to inflammatory phenotype. Pathogens-associated molecular patterns (PAMPs) induced ROS activates nuclear oligomerization domain (NOD), leucine rich repeats (LRR) and pyrin domain-containing protein 3 (NLRP3) inflammasome for the release of pro-inflammatory cytokines. This study evaluates the role of H. pylori secreted concentrated proteins (HPSCP) related oxidative stress role in NLRP3 inflammasome activation and macrophage differentiation. To perceive the role of ROS/RNS, THP-1 and AGS cells were treated with 10 μM diphenyleneiodonium (DPI), 50 μM salicyl hydroxamic acid (SHX), 5 μM Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), which are specific inhibitors of NADPH oxidase (NOX), Myeloperoxidase (MPO), and mitochondrial oxidative phosphorylation respectively. Cells were also treated with 10 μM of NOS2 inhibitor l-NMMA and 10 μM of N-acetyl cysteine (NAC), a free radical scavenger·H2O2 (100 μM) treated and untreated cells were used as positive controls and negative control respectively. The expression of gp91phox (NOX2), NOS2, NLRP3, CD86 and CD163 was analyzed through fluorescent microscopy. THP-1 macrophages growth was unaffected whereas the gastric epithelial AGS cells proliferated in response to higher concentration of HPSCP. ROS and myeloperoxidase (MPO) level increased in THP-1 cells and nitric oxide (NO) and lipid peroxidation significantly decreased in AGS cells. gp91phox expression was unchanged, whereas NOS2 and NLRP3 downregulated in response to HPSCP, but increased after inhibition of NO, ROS and MPO in THP-1 cells. HPSCP upregulated the expression of M1 and M2 macrophage markers, CD86 and CD163 respectively, which was decreased after the inhibition of ROS.This study concludes that there are multiple pathways which are generating ROS during H. pylori infection which further regulates other cellular processes. NO is closely associated with MPO and inhibition of NLRP3 inflammasome. The low levels of NO and MPO regulates gastrointestinal tract homeostasis and overcomes the inflammatory response of NLRP3. The ROS also plays crucial role in macrophage polarization hence alter the immune responses duing H. pylori pathogenesis.

MACRO AND NANO ECBALLIUM ELATERIUM IN VITRO ACTIVITY AGAINST MULTIDRUG-RESISTANT SALMONELLA TYPHI WITH GENETIC DETECTION AND SEQUENCING

Multidrug-resistant (MDR) Salmonella typhi is involved in a number of illnesses such as typhoid fever. This research sought to recognize and quantify Salmonella typhi in clinical stool samples by serotyping, with detection and sequencing of blaCMY-2 gene in charge of cephalosporin resistance, DHFR gene responsible for sulfonamide resistance and acrB and rplD genes responsible for macrolide resistance and use of Ecballium elaterium nanoparticles as an all-natural remedy for MDR S. typhi strains. S. typhi was discovered in 76/113 (67.25%) of the collected specimens. 41/76 samples were multidrug-resistant. However, men were likely more prone to infection than women. The findings showed that 95.12% of S. typhi isolates harbored the blaCMY-2 gene, 92.68% harbored the DHFR gene, 48.78% harbored the acrB gene, and 85.36% harbored the rpID gene while 31.70% of them harbored the four genes. Agreement of the four genes sequences showed mutations ranging between deletion, insertion, transversion, and inversion mutations. GC-MS illustrated that the Biochanin B compound was the most popular component, with a percentage of 19.31% in E. elaterium essential oil. 10mg/ml of E. elaterium nanoparticles were found to be the minimal inhibitory concentration (MIC). E. elaterium nanoparticles damaged the cell wall, nuclear material, and cytoplasmic structures of MDR S. typhi. Moreover, they had a more significant cell viability effect on human gastric epithelial cell line (GESI) than chloramphenicol drug. The IC50 of E. elaterium nanoparticles was 1230.05±72.9ug/ml, and the chloramphenicol drug was 73.6924±4.05ug/ml, showing that chloramphenicol was more cytotoxic on GES1 normal cells than E. elaterium nanoparticles. E. elaterium nanoparticles have high effect against MDR S. typhi and more safe than using chloramphenicol drug.

Effect of Lactobacillus gasseri BIO6369 and Lacticaseibacillus rhamnosus BIO5326 on Gastric Carcinogenesis Induced by Helicobacter pylori Infection.

Helicobacter pylori infection-associated gastric adenocarcinoma is influenced by various factors, including the digestive microbiota. Lactic acid bacteria role in digestive carcinogenesis has been discussed, and some Lactobacillaceae family species have been shown to act against H. pylori-induced inflammation and colonization. However, their effects on H. pylori-related carcinogenesis have not yet been studied. Lactobacillaceae family effects on the epithelial-to-mesenchymal transition (EMT), emergence of cells with cancer stem cell (CSC) properties and the pro-inflammatory response of gastric epithelial cells to H. pylori infection were investigated. A co-culture model of AGS gastric epithelial cells infected with a carcinogenic strain of H. pylori associated with 18 different probiotic strains candidates were used. Different EMT indicators and CSC properties were studied, including quantification of the mesenchymal phenotype, tumorsphere formation, EMT marker expression, and tight junction evaluation with immunofluorescence microscopy. The effect of the strains on the pro-inflammatory response to H. pylori was also evaluated by quantifying interleukin-8 (IL-8) production using ELISA. Among the strains tested, Lactobacillus gasseri BIO6369 and Lacticaseibacillus rhamnosus BIO5326 induced a 30.6% and 38.4% reduction in the mesenchymal phenotype, respectively, caused a significant decrease in Snail and Zeb1 EMT marker expression and prevented the loss of tight junctions induced by H. pylori infection. A separate co-culture with a Boyden chamber maintained the effects induced by the two strains. H. pylori-induced IL-8 production was also significantly reduced in the presence of L. gasseri BIO6369 and L. rhamnosus BIO5326. Lactobacillus gasseri BIO6369 and L. rhamnosus BIO5326 strains decreased epithelial-to-mesenchymal transition and inflammation induced by H. pylori infection, suggesting that these species may have a protective effect against H. pylori-induced gastric carcinogenesis.

Punicalagin Restricts Growth, Promotes Apoptosis, and Reduces Invasion in Human Gastric Cancer Cells.

This research investigated the anticancer properties of punicalagin, a prominent bioactive polyphenol extracted from Punica granatum L, in human gastric cancer cell lines. Normal and gastric cancer cells were exposed to different doses of punicalagin for various durations. Punicalagin exhibited cytotoxic effects on gastric cancer cells in a dose- and time-dependent fashion, while sparing normal gastric epithelial cells. It is noteworthy that among the 3 gastric cancer cells, HGC-27 cells were more resistant to punicalagin than 23,132/87 and AGS cells. Furthermore, punicalagin triggered apoptosis in gastric cancer cells, evidenced by a rise in both early and late apoptotic cell percentages. Western blot analysis further revealed that punicalagin elevated the levels of activated caspase-3. Conversely, punicalagin curtailed cell invasion and reduced the expression of MMP-2, MMP-9, Snail, and Slug. From a mechanistic standpoint, Western blotting indicated that punicalagin might inhibit the Erk and NF-κB pathways, leading to apoptosis induction and the inhibition of cell invasion in gastric cancer cells. These results indicate that punicalagin promotes apoptosis and inhibits cell invasion in gastric cancer cells by activating caspase-3 and suppressing MMP-2, MMP-9, Snail, and Slug through the inhibition of the Erk and NF-κB pathways.

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.

Characterization of cytotoxic Citrobacter braakii isolated from human stomach.

Citrobacter braakii (C. braakii) is an anaerobic, gram-negative bacterium that has been isolated from the environment, food, and humans. Infection by C. braakii has been associated with acute mucosal inflammation in the intestine, respiratory tract, and urinary tract. However, the pathogenesis of C. braakii in the gastric mucosa has not yet been clarified. In this study, the bacterium was detected in 35.5% (61/172) of patients with chronic gastritis (CG) and was closely associated with the severity of mucosal inflammation. Citrobacter braakii P1 isolated from a patient with CG exhibited urease activity and acid resistance. It contained multiple secretion systems, including a complete type I secretion system (T1SS), T5aSS and T6SS. We then predicted the potential pilus-related adhesins. Citrobacter braakii P1 diffusely adhered to AGS cells and significantly increased lactate dehydrogenase (LDH) release; the adhesion rate and LDH release were much lower in HEp-2 cells. Strain P1 also induced markedly increased mRNA and protein expression of IL-8 and TNF-α in AGS cells, and the fold increase was much higher than that in HEp-2 cells. Our results demonstrate proinflammatory and cytotoxic role of C. braakii in gastric epithelial cells, indicating the bacterium is potentially involved in inducing gastric mucosa inflammation.

The modulatory effect of Lactobacillus gasseri ATCC 33323 on autophagy induced by extracellular vesicles of Helicobacter pylori in gastric epithelial cells in vitro

Helicobacter pylori has been recognized as a true pathogen, which is associated with various gastroduodenal diseases, and gastric adenocarcinoma. The crosstalk between H. pylori virulence factors and host autophagy remains challenging. H. pylori can produce extracellular vesicles (EVs) that contribute to gastric inflammation and malignancy. Some probiotic strains have been documented to modulate cell autophagy process. This study was aimed to investigate the modulatory effect of cell-free supernatant (CFS) obtained from Lactobacillus gasseri ATCC 33323 on autophagy induced by H. pylori-derived EVs. EVs were isolated from two clinical H. pylori strains (BY-1 and OC824), and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The viability of AGS cells was assessed after exposure to different concentrations of H. pylori EVs, and L. gasseri CFS. Based on MTT assay and Annexin V-FITC/PI staining, 50 μg/ml of H. pylori EVs and 10 % v/v of L. gasseri CFS were used for further cell treatment experiments. Autophagy was examined using acridin orange (AO) staining, RT-qPCR analysis for autophagy mediators (LC3B, ATG5, ATG12, ATG16L1, BECN1, MTOR, and NOD1), and western blotting for LC3B expression. H. pylori EVs were detected to range in size from 50 to 200 nm. EVs of both H. pylori strains and L. gasseri CFS showed no significant effect on cell viability as compared to untreated cells. H. pylori EVs promoted the development of acidic vesicular organelles and the expression of autophagy-related genes (LC3B, ATG5, ATG12, ATG16L1, BECN1, and NOD1), and decreased the expression of MTOR in AGS cells at 12 and 24 h time periods. In addition, the production of LC3B was increased following 12 h of treatment in AGS cells. In contrast, L. gasseri CFS effectively inhibited EVs-induced autophagy, as evidenced by reduced acidic vesicular organelle formation and modulation of autophagy markers. Our study indicated that L. gasseri CFS can effectively suppress H. pylori EV-induced autophagy in AGS cells. Further investigations are required to decipher the mechanism of action L. gasseri CFS and its metabolites on autophagy inhibition induced by H. pylori.

LOX-1 acts as an N6-methyladenosine-regulated receptor for Helicobacter pylori by binding to the bacterial catalase.

The role of N6-methyladenosine (m6A) modification of host mRNA during bacterial infection is unclear. Here, we show that Helicobacter pylori infection upregulates host m6A methylases and increases m6A levels in gastric epithelial cells. Reducing m6A methylase activity via hemizygotic deletion of methylase-encoding gene Mettl3 in mice, or via small interfering RNAs targeting m6A methylases, enhances H. pylori colonization. We identify LOX-1 mRNA as a key m6A-regulated target during H. pylori infection. m6A modification destabilizes LOX-1 mRNA and reduces LOX-1 protein levels. LOX-1 acts as a membrane receptor for H. pylori catalase and contributes to bacterial adhesion. Pharmacological inhibition of LOX-1, or genetic ablation of Lox-1, reduces H. pylori colonization. Moreover, deletion of the bacterial catalase gene decreases adhesion of H. pylori to human gastric sections. Our results indicate that m6A modification of host LOX-1 mRNA contributes to protection against H. pylori infection by downregulating LOX-1 and thus reducing H. pylori adhesion.

Autophagy-Related Gene ATG7 Polymorphism Could Potentially Serve as a Biomarker of the Progression of Atrophic Gastritis.

Cytotoxin-associated gene A (CagA) is an oncoprotein that H. pylori injects into the host's gastric epithelial cells and that induces proinflammatory cytokines, such as interleukin (IL)-18 and IL-1β. As a result, it leads to atrophic gastritis (AG), a precancerous lesion of gastric cancer. On the other hand, host cells degrade CagA using autophagy systems. However, few studies exist about the single nucleotide polymorphisms (SNPs) in MAP1LC3A, MAP1LC3B, ATG4A, ATG4B, ATG4C, ATG7, and ATG13, which belong to the autophagy-related genes concerning AG. This study aimed to detect biomarkers associated with AG. Herein, H. pylori-positive subjects (n = 200) were divided into the AG (n = 94) and non-AG (n = 106) groups. Thirty tag SNPs were selected from the above seven candidate genes. The SNP frequency between the two groups was analyzed. The frequency of the C/T or T/T genotype at rs4683787 of ATG7 was significantly lower in the AG group than in the non-AG group (p = 0.034, odds ratio = 0.535). Based on multivariate analysis, the C/C genotype of rs4684787 and age were independently associated with gastric mucosal atrophy. This finding helps stratify the patients needing timely endoscopic screening or early eradication of H. pylori.