Human Gastric Epithelial Cells Research Articles

Ochratoxin A induces cytotoxicity through ROS-mediated endoplasmic reticulum stress pathway in human gastric epithelium cells

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species that greatly threatens human health. We previously showed that OTA induced cycle arrest, apoptosis and autophagy in human gastric epithelium cells (GES-1). However, the mechanism underlying these effects is still unclear. Here, we showed that OTA exposure increased the expression of endoplasmic reticulum (ER) stress indicators (GRP78, PERK, ATF6, eIF2α, and CHOP), suggesting the activation of the unfolded protein response pathway. 4-phenylbutyric acid (4-PBA), an ER stress-specific inhibitor, attenuated OTA-induced loss of cell viability and apoptosis in GES-1 cells. It also attenuated the G2 phase arrest and autophagy induced by OTA, as evidenced by upregulated G2 phase-related proteins (Cdc2, Cdc25C, and cyclinB1) and downregulated autophagy markers (LC3B and Beclin-1). Moreover, OTA was found to increase ROS generation, and the inhibition of ROS formation by N-acetylcysteine (NAC), an ROS inhibitor, attenuated OTA-induced ER stress and subsequent apoptosis, cell cycle arrest, and autophagy. Collectively, these results suggest that the ROS-mediated ER stress pathway contributes to the OTA toxin-induced cytotoxicity in GES-1 cells. This study offers new insights into the molecular mechanisms underlying OTA toxicity in gastric cells.

Ornithine Decarboxylase in Gastric Epithelial Cells Promotes the Immunopathogenesis of Helicobacter pylori Infection.

Colonization by Helicobacter pylori is associated with gastric diseases, ranging from superficial gastritis to more severe pathologies, including intestinal metaplasia and adenocarcinoma. The interplay of the host response and the pathogen affect the outcome of disease. One major component of the mucosal response to H. pylori is the activation of a strong but inefficient immune response that fails to control the infection and frequently causes tissue damage. We have shown that polyamines can regulate H. pylori-induced inflammation. Chemical inhibition of ornithine decarboxylase (ODC), which generates the polyamine putrescine from l-ornithine, reduces gastritis in mice and adenocarcinoma incidence in gerbils infected with H. pylori However, we have also demonstrated that Odc deletion in myeloid cells enhances M1 macrophage activation and gastritis. Here we used a genetic approach to assess the specific role of gastric epithelial ODC during H. pylori infection. Specific deletion of the gene encoding for ODC in gastric epithelial cells reduces gastritis, attenuates epithelial proliferation, alters the metabolome, and downregulates the expression of immune mediators induced by H. pylori Inhibition of ODC activity or ODC knockdown in human gastric epithelial cells dampens H. pylori-induced NF-κB activation, CXCL8 mRNA expression, and IL-8 production. Chronic inflammation is a major risk factor for the progression to more severe pathologies associated with H. pylori infection, and we now show that epithelial ODC plays an important role in mediating this inflammatory response.

α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the gastric mucosa and triggers various stomach diseases. H. pylori induces reactive oxygen species (ROS) production and DNA damage. The heterodimeric Ku70/Ku80 protein plays an essential role in the repair of DNA double-strand breaks (DSB). Oxidative stress stimulate apoptosis and DNA damage that can be repaired by Ku70/80. However, excessive reactive oxygen species (ROS) can cause Ku protein degradation, resulting in DNA fragmentation and apoptosis. α-lipoic acid (α-LA), which is found in organ meats such as liver and heart, spinach, broccoli, and potatoes, quenches free radicals, chelates metal ions, and reduces intracellular DNA damage induced by oxidative stress. Here, we investigated whether H. pylori decreases Ku70/80 and induces apoptosis, and whether α-LA inhibits changes induced by H. pylori. We analyzed ROS, DNA damage markers (γ-H2AX, DNA fragmentation), levels of Ku70/80, Ku–DNA binding activity, Ku80 ubiquitination, apoptosis indices (Bcl-2, Bax, apoptosis-inducing factor (AIF), and caspase-3), and viability in a human gastric epithelial adenocarcinoma cell line (AGS). H. pylori increased ROS, DNA damage markers, Ku80 ubiquitination, and consequently induced apoptosis. It also decreased nuclear Ku70/80 levels and Ku–DNA-binding activity; increased Bax expression, caspase-3 cleavage, and truncated AIF; but decreased Bcl-2 expression. These H. pylori-induced alterations were inhibited by α-LA. The antioxidant N-acetylcysteine and proteasome inhibitor MG-132 suppressed H. pylori-induced cell death and decreased nuclear Ku70/80 levels. The results show that oxidative stress induced Ku70/80 degradation via the ubiquitin–proteasome system, leading to its nuclear loss and apoptosis in H. pylori-infected cells. In conclusion, α-LA inhibited apoptosis induced by H. pylori by reducing ROS levels and suppressing the loss of Ku70/80 proteins in AGS cells.

PSMB2 knockdown suppressed proteasome activity and cell proliferation, promoted apoptosis, and blocked NRF1 activation in gastric cancer cells.

Proteasome 20S Subunit Beta 2 (PSMB2) has been suggested to play several roles in cancer. However, the role of PSMB2 and its underlying mechanisms in gastric cancer have not been studied. In this study, qRT-PCR was employed to detect the expression of genes that encode for 26s proteasome subunit proteins. PSMB2 expression and its prognostic ability were assessed by collecting patient tissue samples and reviewing the TCGA and Kaplan-Meier Plotter databases. Immunofluorescence and western blotting experiments were performed to evaluate the expression of PSMB2 in human gastric cancer cells and normal gastric epithelial cells. Subsequently, PSMB2 was knocked down in HGC-27 and SNU-1 cells and overexpressed in N-87 and AGS cells. Proteasome activity assays, 5-Ethynyl-2'-deoxyuridine staining, and TUNEL assays were used to assess proteasome activity, cell proliferation, and apoptosis. Tumor xenograft assays were conducted to evaluate PSMB2 function in vivo. Our results showed that a total of 8 genes encoding for the 26s proteasome subunit protein were highly expressed in a variety of gastric cancer cells. Next, PSMB2 was selected as the focus of subsequent studies which showed that PSMB2 was highly expressed in samples of gastric cancer tissue. Furthermore, a review of the TCGA database revealed that a high level of PSMB2 expression was associated with a poor clinical prognosis. Our results indicated that PSMB2 overexpression promoted proteasome activity, cell proliferation, and suppressed the apoptosis of gastric cancer cells, while those effects were reversed by treatment with a proteasome inhibitor (MG132). In contrast, PSMB2 knockdown produced the opposite effects and also blocked NRF1 activation. Moreover, PSMB2 knockdown inhibited tumor growth in vivo, decreased PSMB2 expression and cell proliferation, and promoted apoptosis in tumor tissues. Our findings revealed the role played by PSMB2 in gastric cancer and suggest PSMB2 as a new target molecule for use in diagnosing and treating gastric cancer.

KLF4 Mutation Shapes Pathologic Characteristics of Foveolar-Type Gastric Adenoma in Helicobacter pylori–Naive Patients

Along with a recent remarkable decrease in Helicobacter pylori-infected individuals, reports of gastricneoplasms such as sporadic foveolar-type gastric adenoma (FGA) in H. pylori-naive patients have been increasing. This tumor, with its raspberry-like appearance, is common in H. pylori-naive gastric mucosa. The current study investigated the genomic features of sporadic FGA. Fresh-frozen sporadic FGA tissue samples from H. pylori-naive patients were subjected to whole genome analysis using a next-generation sequencer. Proliferation ability and apoptotic profiles of human gastric epithelial cells, along with plasmid transfection of candidate variants, were examined. A mean of 6.65×108 total reads were obtained for each sample. Common genetic abnormalities in well-known proliferation driver genes of conventional gastric dysplasia/cancer were not found. However, a common single-nucleotide variation (SNV) was noted within the DNA-binding domain of the tumor suppressor gene KLF4. This novel SNV was located in the zinc finger 2region. Additional experiments showed that it significantly suppressed proliferation of gastric epithelial cells compared with wild-type KLF4 plasmid-transfected cells, although suppression was reduced in early apoptotic phase-related genes. A novel SNV in the KLF4 zinc finger 2 region was commonly found in sporadic FGA tissue samples, which may explain the slow-growing properties of this neoplasm.

High Expression of UPK3A Promotes the Progression of Gastric Cancer Cells by Inactivating p53 Pathway.

Background Gastric cancer is a common gastrointestinal tract cancer and is a considerable health burden worldwide. TCGA analysis found Uroplakin 3A (UPK3A) was upregulated in gastric cancer tissues. Our study was designed to investigate the underlying mechanism of Uroplakin 3A (UPK3A) in gastric cancer. Methods Data from TCGA database were used to assess the expression, and Kaplan-Meier plotter analysis was used to assess the prognosis value of UPK3A. Furthermore, there are effects of UPK3A silencing on the activity, proliferation, migration, and invasion of human gastric cancer cells (SNU-216 and HGC-27) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing, and Transwell assays. In addition, the expression of UPK3A, p53, KLF4, ZMAT3, MDM2, and SP1 was detected by qRT-PCR and Western blot assay. Results UPK3A was markedly upregulated in gastric cancer tissues compared to that in normal tissues, and patients with high UPK3A level showed poor prognosis. UPK3A was highly expressed in human gastric cancer cell lines compared to that in a normal human gastric epithelial cell line. Silencing of UPK3A inhibited the proliferation, migration, and invasion of gastric cancer cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that UPK3A was involved in the p53 signaling pathway. UPK3A suppressed the activation of p53 signaling pathway, and treatment with Pifithrin-α (an inhibitor of the p53 signaling pathway) or silencing of p53 significantly reversed the effect of UPK3A silencing on the expression of p53, KLF4, ZMAT3, MDM2, and SP1. Conclusion Our findings showed that UPK3A promotes the progression of gastric cancer by regulating the p53 signaling pathway and could be a potential therapeutic target for gastric cancer.

Abstract 5293: Rationally developing antibody drug conjugates targeting genomically stable gastric cancer

Abstract Background: Gastric cancer (GC) ranks the fifth most common cancer and the fourth most frequent cause of cancer-related deaths. Genomically stable gastric cancers (GSGCs) represent 20% of all GCs and GSGC patients have the worst prognosis among all GC subtypes. Although two HER2-targeted antibody-drug conjugates (ADCs), RC48 and DS8201, have shown promising clinical efficacies, most GSGC patients do not benefit from these ADCs due to the infrequent and heterogeneous HER2 expression. Therefore, there remains a significant and unmet medical need for developing novel ADCs targeting GSGC tumors. Methods: In this presented study, a quantitative and unbiased approach was first used to identify potential targets for GSGC in a panel of human GSGC cell lines in reference with two normal human gastric epithelial cells. Cell membrane localizations of potential targets on GSGC cell lines were determined by immunofluorescent (IF) staining. To correlate it with clinically-relevant GSGC tumor samples, immunohistochemical (IHC) staining of potential targets were performed in human diffuse GC tumor tissues along with normal gastric tissues. Next, GSGC cellular internalization capability of antibodies against potential targets was evaluated. Then, an optimized target was selected for developing GSGC-targeted ADCs. A series of ADC formulations with different linkers and payloads were constructed and characterized. Their anti-tumor activities against GSGC cell lines were subsequently screened in order to identify the optimized ADC formulation. Eventually, the anti-tumor activity and biosafety profile of the optimized ADC formulation were determined in suppressing tumor growth, progression and metastasis by using established orthotopic and peritoneal metastasis models of GSGC. Results: We first identified CD54 as a potential molecular target for human GSGCs using comparative flow cytometric analysis. We next evaluated its potential as an ADC target for GSGC-targeted therapy by determining its overexpression levels, cell membrane localizations, and cell internalization activity in GSGC cells. Furthermore, we constructed and characterized a panel of CD54-targeted ADCs and identified an optimized ADC formulation by quantitatively determining their half maximum inhibitory concentrations (IC50s) and validated its anti-tumor efficacy against GSGC tumors. Meanwhile, there was no evidence to indicate histopathological damages to normal organs in the optimized ADC treated group. Conclusions: In this study, we identified a molecular target for GSGC using an unbiased and quantitative screening assay and explored its potential application in developing GSGC-targeted ADCs. In order to maximize its efficacy, we optimized the ADC formulations against GSGC cells using different ADC linkers and payloads. Our study provides a promising targeted therapeutic candidate for the clinical treatment of GSGC. Citation Format: Rui Xu, Li Yuan, Xiao-Qing Guan, Shi-Li Yao, Bing Zhu, Tong Yang, Chun Liu, Peng Guo, Jiang-Jiang Qin, Xiang-Dong Cheng. Rationally developing antibody drug conjugates targeting genomically stable gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5293.

Impact of Amarogentin on Gastric Carcinoma Cell Multiplication, Apoptosis and Migration via circKIF4A/miR-152-3p

Objective The active ingredients extracted from natural plants have anti-GC actions and can slow down gastric carcinoma (GC) progression. To investigate the impact of Amarogentin (AG) on GC cell multiplication, apoptosis and migration and the possible mechanisms. Methods qRT-PCR quantification of circKIF4A and miR-152-3p in GC tissues and normal counterparts as well as HGC-27 (human GC cell strain) and GES-1 (human gastric mucosal epithelial cell strain) was performed. HGC-27 cells were intervened by AG of various concentrations. si-NC, si-circKIF4A were further transfected into HGC-27 cells. Besides, pcDNA and pcDNA-circKIF4A were transfected into HGC-27 cells, after which 60 mmol/L AG was added for intervention. Cell multiplication, clone formation, as well as apoptosis and migration measurements were made by MTT, plate clone formation, flow cytometry and Transwell assays, respectively; Double luciferase reporter assay was performed for targeting relationship identification between circKIF4A and miR-152-3p; Western blots were carried out to measure Bax and Bcl-2 protein levels. Results circKIF4A increased (P <0.05) and miR-152-3p decreased (P <0.05) in GC tissues and cell strains. Concentration-dependently, AG intervention contributed to enhanced cell multiplication inhibitory rate, apoptosis rate, miR-152-3p expression and Bax protein level (P <0.05), together with declined number of cell clones formed, migrating cells, circKIF4A expression and Bcl-2 protein level (P <0.05). After transfection of si-circKIF4A, cell multiplication inhibition rate, apoptosis rate and Bax protein level enhanced (P <0.05), while cell clones formed and migrating cells as well as Bcl-2 protein level reduced (P <0.05). miR-152-3p can be controlled by circKIF4A; pcDNA-circKIF4A transfection antagonized AG's effects on HGC-27 cell multiplication, clone formation, apoptosis and migration. Conclusion AG can decrease GC multiplication, clone formation and migration and induce apoptosis via modulating circKIF4A/miR-152-3p expression.

Astaxanthin Inhibits Helicobacter pylori-Induced MMPs Expression and Cell Invasion by Suppressing PI3K-AKT-mTOR Pathways in AGS Cells

ObjectivesMatrix metalloproteinases (MMPs) are members of the metzincin group of proteases which can degrade various components of extracellular matrix components (ECM). ECM degradation by MMPs not only enhances tumor invasion but also affects tumor cell behavior and leads to cancer progression. Resent reports demonstrated that PI3K-AKT-mTOR signaling pathways are involved in expression of MMPs. Especially when it comes to gastric cancer, H. pylori infection promotes the invasion and metastasis of tumor cells by reactive oxygen species (ROS). Astaxanthin, a xanthophyll carotenoid, has been well known for its strong antioxidant and anticancer properties. Thus, the present study was aimed to investigate the inhibitory effects and mechanisms of astaxanthin on H. pylori-induced MMPs expression and cell invasion in human gastric epithelial cells.MethodsHuman gastric epithelial cell lines, AGS were used. AGS cells were pre-treated with astaxanthin for 3 hours prior to H. pylori (cag A positive NCTC 11,637 strains) infection at bacterium/cell ratio of 50:1 and cultured for 24 hours in the presence of H. pylori. mRNA expression and protein levels of MMP-7 and 10 was measured by real time PCR and Western blot analysis, respectively. Intracellular ROS levels were determined using DCF-DA. Invasive cell phenotype was measured using invasion assay.ResultsAstaxanthin inhibited expression of MMP-7&10. Intracellular ROS production and cell invasion induced by H. pylori was inhibited by astaxanthin. In addition, H. pylori-induced activation of PI3K-AKT-mTOR was inhibited by astaxanthin.ConclusionsAstaxanthin exerts an inhibitory effect on H. pylori-induced MMPs expression and cell invasion by suppression of ROS production and PI3K-AKT-mTOR signaling pathway in gastric epithelial cells. Thus astaxanthin may hold promise for clinical cancer therapy.Funding SourcesNo funding sources.

Natural Compound 2-Chloro-1,3-dimethoxy-5-methylbenzene, Isolated from Hericium Erinaceus, Inhibits Fungal Growth by Disrupting Membranes and Triggering Apoptosis.

In this study, 2-chloro-1,3-dimethoxy-5-methylbenzene (CDM), a natural product with anti-Candida albicans activity, was discovered from the Hericium erinaceus mycelium. The minimum inhibitory concentration of CDM was 62.5 μg/mL. Moreover, structural analogues of CDM obtained from chemical synthesis were applied to explore the structure-activity relationship (SAR) of CDM against C. albicans. It was found that methoxy groups, halogen atoms (except fluorine atoms), and methoxy-meta-position methyl groups in the structure of CDM were the key active groups. Furthermore, we investigated the anti-C. albicans mechanism of CDM. Experiments suggested that CDM destroyed the cell membrane of C. albicans, including the cytoplasmic membrane and mitochondrial membrane, and caused the accumulation of reactive oxygen species and mitochondrial dysfunction, which ultimately led to apoptosis of C. albicans. In addition, CDM had no toxicity on human normal gastric mucosal epithelial cells exposed to a concentration of 125 μg/mL. Experiments showed that CDM reduced the damage of C. albicans to the visceral tissue of infected mice and improved the survival rate of mice. Our research provides a scientific basis for the discovery of effective and safe anti-C. albicans drugs from H. erinaceus.

Gossypol from Gossypium spp. Inhibits Helicobacter pylori Clinical Strains and Urease Enzyme Activity: Bioactivity and Safety Assessments

This study investigates the inhibitory activities of gossypol, a natural polyphenolic compound from Gossypium spp., against Helicobacter pylori (HP) clinical strains and a urease enzyme that plays a key role in the pathogenesis of HP. Gossypol was detected to exhibit a bacteriostatic action against all the HP strains tested with minimum inhibitory concentration (MIC) values ranging from 3.51 to 4.14 µg/mL. The activity of HP urease (HPU) was efficiently impeded by gossypol with a 50% inhibitory concentration (IC50) value of 3.3 µM using an Electrospray Ionization–Mass Spectrometry (ESI-MS)-based method. The in vitro cytotoxicity assay showed no significant cytotoxic properties of gossypol against human gastric epithelial cells. Additionally, molecular docking studies were performed to assess the binding mode and the molecular interactions of gossypol with HPU with a binding affinity value of −8.1 kcal/mol compared with an HPU–acetohydroxamic acid (a standard urease inhibitor) docking complex (–6.1 kcal/mol). The overall results reveal that gossypol might help fight against HP infection by two mechanisms of action: inhibition of the growth of HP and inhibition of urease.

Tauroursodeoxycholic Acid Inhibits Nuclear Factor Kappa B Signaling in Gastric Epithelial Cells and Ameliorates Gastric Mucosal Damage in Mice.

Previous studies have reported the protective effects of tauroursodeoxycholic acid (TUDCA) on gastric epithelial cells in some animal models, but the precise mechanisms are unclear. This study examined the effects of TUDCA on NF-κB signaling in gastric epithelial cells. Moreover, the protective effects of TUDCA in experimental gastritis models induced by ethanol and NSAID were evaluated and compared with ursodeoxycholic acid (UDCA). After a pretreatment with TUDCA or UDCA, human gastric epithelial MKN-45 cells were stimulated with tumor necrosis factor (TNF)-α to activate NF-κB signaling. A real-time PCR (RT-PCR) for human interleukin (IL)-1 mRNA was performed. An electrophoretic mobility shift assay (EMSA) and immunoblot analyses were carried out. In murine models, after a pretreatment with TUDCA or UDCA, ethanol and indomethacin were administered via oral gavage. Macroscopic and microscopic assessments were performed to evaluate the preventive effects of TUDCA and UDCA on murine gastritis. A pretreatment with TUDCA downregulated the IL-1α mRNA levels in MKN-45 cells stimulated with TNF-α, as assessed by RT-PCR. As determined using EMSA, a pretreatment with TUDCA reduced the TNF-α-induced NF-κB DNA binding activity. A pretreatment with TUDCA inhibited IκBα phosphorylation induced by TNF-α, as assessed by immunoblot analysis. TUDCA attenuated the ethanol-induced and NSAID-induced gastritis in murine models, as determined macroscopically and microscopically. TUDCA inhibited NF-κB signaling in gastric epithelial cells and ameliorated ethanol- and NSAID-induced gastritis in murine models. These results support the potential of TUDCA for the prevention of gastritis in humans.

Biodegradation of Fumonisins by the Consecutive Action of a Fusion Enzyme.

Fumonisins (FBs) are toxic mycotoxins that commonly exist in food and feed. FBs can induce many aspects of toxicity, leading to adverse effects on human and animal health; therefore, investigating methods to reduce fumonisin contamination is necessary. In our study, we generated a recombinant fusion enzyme called FUMDI by linking the carboxylesterase gene (fumD) and the aminotransferase gene (fumI) by overlapping polymerase chain reaction (PCR). The fusion enzyme FUMDI was successfully, secretively expressed in the host Pichia pastoris (P. pastoris) GS115, and its expression was optimized. Our results demonstrated that the fusion enzyme FUMDI had high biodegradation activity of fumonisin B1 (FB1) and other common FBs, such as fumonisin B2 (FB2) and fumonisin B3 (FB3), and almost completely degraded 5 μg/mL of each toxin within 24 h. We also found that FUMDI enzyme and its reaction products had no negative effect on cell viability and did not induce cell apoptosis, oxidative stress, or endoplasmic reticulum (ER) stress in a human gastric epithelial cell line (GES-1). The results indicated that these FBs degradation products cannot have adverse effects in a cell model. In conclusion, a safe and efficient fumonisin-degrading enzyme was discovered, which could be a new a technical method for hazard control of FBs in the future.

Exosomal CagA from Helicobacter pylori aggravates intestinal epithelium barrier dysfunction in chronic colitis by facilitating Claudin-2 expression

BackgroundThe chronic infection with Helicobacter pylori (H. pylori), especially cytotoxin-associated gene A-positive (CagA+) strains, has been associated with various extragastric disorders. Evaluating the potential impacts of virulence factor CagA on intestine may provide a better understanding of H. pylori pathogenesis such as colitis. The intestinal mucosal barrier is essential for maintaining its integrity and functions. However, how persistent CagA+H. pylori colonization influences barrier disruption and thereby affects chronic colitis is not fully understood.ResultsChronic colitis models of CagA+H. pylori-colonized mice treated with 2% Dextran sulphate sodium (DSS) were established to assess the disease activity and pertinent expression of tight junction proteins closely related to mucosal integrity. The aggravating effect of CagA+H. pylori infection on DSS-induced chronic colitis was confirmed in mouse models. In addition, augmented Claudin-2 expression was detected in CagA+H. pylori infection conditions and selected for mechanistic analysis. Next, GES-1 human gastric epithelial cells were cultured with CagA+H. pylori or a recombinant CagA protein, and exosomes isolated from conditioned media were then identified. We assessed the Claudin-2 levels after exposure to CagA+ exosomes, CagA− exosomes, and IFN-γ incubation, revealing that CagA+H. pylori compromised the colonic mucosal barrier and facilitated IFN-γ-induced intestinal epithelial destruction through CagA-containing exosome-mediated mechanisms. Specifically, CagA upregulated Claudin-2 expression at the transcriptional level via a CDX2-dependent mechanism to slow the restoration of wounded mucosa in colitis in vitro.ConclusionsThese data suggest that exosomes containing CagA facilitate CDX2-dependent Claudin-2 maintenance. The exosome-dependent mechanisms of CagA+H. pylori infection are indispensable for damaging the mucosal barrier integrity in chronic colitis, which may provide a new idea for inflammatory bowel disease (IBD) treatment.

Immunomodulatory Efficacy-Mediated Anti-HCV and Anti-HBV Potential of Kefir Grains; Unveiling the In Vitro Antibacterial, Antifungal, and Wound Healing Activities.

The utilization of fermented foods with health-promoting properties is becoming more popular around the world. Consequently, kefir, a fermented milk beverage made from kefir grains, was shown in numerous studies to be a probiotic product providing significant health benefits. Herein, we assessed the antibacterial and antifungal potential of kefir against a variety of pathogenic bacteria and fungi. This study also showed the effectiveness of kefir in healing wounds in human gastric epithelial cells (GES-1) by (80.78%) compared with control (55.75%) within 48 h. The quantitative polymerase chain reaction (qPCR) results of kefir-treated HCV- or HBV- infected cells found that 200 µg/mL of kefir can eliminate 92.36% of HCV and 75.71% of HBV relative to the untreated infected cells, whereas 800 µg/mL (the highest concentration) completely eradicated HCV and HBV. Moreover, the estimated IC50 values of kefir, at which HCV and HBV were eradicated by 50%, were 63.84 ± 5.81 µg/mL and 224.02 ± 14.36 µg/mL, correspondingly. Kefir can significantly suppress the elevation of TNF-α and upregulate IL-10 and INF-γ in both treated HCV- and HBV-infected cells. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analysis of kefir revealed the presence of numerous active metabolites which mainly contribute to the antimicrobial, antiviral, and immunomodulatory activities. This study demonstrated, for the first time, the anti-HBV efficacy of kefir while also illustrating the immunomodulatory impact in the treated HBV-infected cells. Accordingly, kefir represents a potent antiviral agent against both viral hepatitis C and B, as well as having antimicrobial and wound healing potential.

Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells.

BACKGROUNDGastric injury is the most common digestive system disease worldwide and involves inflammation, which can lead to gastric ulcer or gastric cancer (GC). Matrix metallopeptidase-9 [MMP-9 (gelatinase-B)] plays an important role in inflammation and GC progression. Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities. However, the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIMTo assess whether tumor necrosis factor-α (TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODSThe normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the anti-inflammatory effects of quercetin. The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line. Cell migration was measured using Transwell assay. The expression of proto-oncogene tyrosine-protein kinase Src (c-Src), phospho (p)-c-Src, extracellular-signal-regulated kinase 2 (ERK2), p-ERK1/2, c-Fos, p-c-Fos, nuclear factor kappa B (NF-κB/p65), and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity. p65 expression was detected by immunofluorescence. MMP-9 mRNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction (qRT–PCR) and gelatin zymography, respectively.RESULTSqRT-PCR and gelatin zymography showed that TNF-α induced MMP-9 mRNA and protein expression in a dose- and time-dependent manner. These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-α antagonist (TNFR inhibitor) in a dose- and time-dependent manner. Quercetin and TNF-α antagonists decreased the TNF-α-induced phosphorylation of c-Src, ERK1/2, c-Fos, and p65 in a dose- and time-dependent manner. Quercetin, TNF-α antagonist, PP1, U0126, and tanshinone IIA (TSIIA) reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase (Luc) activity in a dose- and time-dependent manner. Pretreatment with quercetin, TNF-α antagonist, PP1, U0126, or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose- and time-dependent manner. TNF-α significantly increased GES-1 cell migration, and these results were reduced by pretreatment with quercetin or a TNF-α antagonist.CONCLUSIONQuercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.

Blueberry anthocyanin extracts protect against Helicobacter pylori-induced peptic epithelium injuries both in vitro and in vivo: the key role of MAPK/NF-κB pathway.

Anthocyanins are well-characterized by anti-oxidative and anti-inflammatory potentials. Peptic ulcers contribute to the development of severe gastric disorders. In the current study, the effects of blueberry anthocyanin extracts (BE) on the Helicobacter pylori lipopolysaccharide (LPS)-induced peptic epithelium injures were assessed and the associated mechanism driving the effects was explored by focusing on MAPK/NF-κB pathway. Peptic injures were induced in a mouse model using LPS plus ligation method and then the mice were treated with BE. Then changes in gastric histology, inflammatory response, and MAPK/NF-κB axis were detected. To reveal the role of MAPK/NF-κB axis in the effects of BE, human gastric epithelial cells (HGECs) were further subjected to co-treatment of BE, LPS, and MAPK activator. The assays of mouse model showed that BE attenuated gastric epithelial injuries by improving epithelial structure and suppressing gastric inflammatory response, which was associated with the inhibition of MAPK/NF-κB axis. In in vitro assays, BE suppressed viability and production of cytokines, and induced apoptosis in LPS-treated HGECs. The re-activation of MAPK pathway counteracted the effects of BE by re-inducing cell viability and suppressing cell apoptosis. The protective effects of BE against LPS-induced injuries in mouse stomach depended on the inhibition of both MAPK pathway and the downstream NF-κB signaling.

Differential H. pylori-Induced MAPK Responses Regulate Lewis Antigen Expression and Colonization Density on Gastric Epithelial Cells Between Children and Adults.

Helicobacter pylori causes gastrointestinal diseases, the manifestations of diseases are more serious in adults than in children. Lewis antigen expressions on the gastric epithelium serves as receptors targeted by H. pylori. Moreover, the MAPK signaling pathway involves glycoprotein synthesis of Lewis antigens. We aimed to investigate whether differences in H. pylori-induced MAPK responses mediate gastric Lewis antigens expression and colonization density differently in children and adults. We used human stomach fetal epithelium (HSFE) and SV40-immortalized human normal gastric epithelial (GES-1) cell lines to mimic primary gastric epithelium of children and adults, respectively. H. pylori colonization intensity and Lewis antigens were significantly higher in GES-1 than in HSFE cells, whereas IL-8 and IL-6 levels were significantly higher in HSFE than in GES-1 cells after infection. c-Jun N-terminal kinase (JNK) siRNA and inhibitor (SP600125) experiments showed that Lewis antigen expression and H. pylori colonization were reduced in GES-1 cells but increased in HSFE cells. Furthermore, p-p38 intensity was significantly higher in the superficial epithelium of the children than in the adults with/without H. pylori infection. The overexpression of p38 in GES-1 cells downregulated H. pylori-induced JNK activity mimicking H. pylori infection in children. In conclusion, a higher p38 expression in gastric epithelium counteracting JNK activity in children may contribute to lower Lewis antigen expression and colonization density than in adults after H. pylori infection.

Iridoid glucosides from the leaves of Vitex negundo var. cannabifolia

• Five new and nine known iridoid glucosides were obtained from the leaves of Vitex negundo var. cannabifolia . • The absolute configurations of sugar moieties were determined using a HPLC-UV-based method. • Six isolates increased the cell viability of GES-1 cell insulted by TCA. Five new iridoid glucosides, cannabifolisides A–E ( 1 – 5 ), together with nine analogues ( 6 – 14 ), were isolated from the leaves of Vitex negundo var. cannabifolia. Their structures were elucidated by spectroscopic data (NMR, UV, IR, and MS) analyses and comparison of their spectroscopic and physical data with the literature values. The protective effect of isolated compounds on human gastric epithelial cells were evaluated by measuring the enhanced cell viability of GES-1 cell insulted by TCA. Among these, compounds 4 , 6 , 8 , 10 , 12 , and 14 remarkably increased the cell viability in a concentration-depend manner.

Total astragalosides decrease apoptosis and pyroptosis by inhibiting enterovirus 71 replication in gastric epithelial cells.

Enterovirus 71 (EV71) is one of the primary pathogens involved in severe hand, foot and mouth disease in children. EV71 infection causes various types of programmed cell death. However, there are currently no clinically approved specific antiviral drugs for control of EV71 infection. Astragalus membranaceus (AM), a Traditional Chinese medicine, has been used in antiviral therapy in China. The aim of the present study was to determine whether total astragalosides (ASTs), bioactive components of AM, protect against EV. DAPI nuclear staining was used to observe morphological changes of the nucleus and the protective effect of ASTs, which revealed that the nucleus shrank following EV71 infection, while ASTs reversed it. Cell Counting Kit-8 assay found that human normal gastric epithelial cell (GES-1 cell) viability decreased following EV71 infection, while lactate dehydrogenase (LDH) assay showed that EV71 infection induced GES-1 cell damage. Western blotting was used to measure the expression levels of apoptosis and pyroptosis marker protein to determine whether EV71 infection induced apoptosis and pyroptosis in GES-1 cells. Reverse transcription-quantitative PCR was used to determine the anti-EV71 effect of ASTs. The results showed that ASTs protected GES-1 cells from EV71-induced cell apoptosis and pyroptosis. Furthermore, the present data demonstrated that the protective effect of ASTs was exerted by suppressing EV71 replication and release. These findings suggested that ASTs may represent a potential antiviral agent for the treatment of EV71 infection.