Mouse Gastric Tissue Research Articles

Abstract 5583: Helicobacter pylori induces the wee1, promoting immune suppressive in human gastric adenocarcinoma

Abstract Background: Gastric cancer (GC) is the fourth most common cause of cancer-related death worldwide. GC has a dismal 5-year survival rate of about 32%. Helicobacter pylori (H. pylori) infection is the strongest risk factor for gastric carcinogenesis. 75-90% of gastric cancer patients exhibit positive H. pylori serology. WEE1 is a nuclear kinase that regulates cell cycle progression by inhibiting cyclin-dependent kinases. Methods & Results: Analysis of non-cancer normal stomach tissue (n=360) and human GC samples (n=1221) identified that WEE1 mRNA was significantly overexpressed in GC. Using immunohistochemistry (IHC) staining in 41 normal and 44 GC human samples, the data indicated that WEE1 protein was significantly overexpressed in GC. Western blot and immunofluorescence (IF) staining confirmed that H. pylori infection induces WEE1 overexpression in GC cells with an unexpected cytosolic localization of WEE1 in GC cells. Tff1 knockout GC mouse model (Tff1-/-) was infected with mouse-adapted H. pylori strain PMSS1. Western blot and immunohistochemistry data demonstrated that H. pylori infection induced WEE1 expression in Tff1 -/- HGD/cancer mouse gastric tissues compared to control Tff1 -/- mouse stomach. To study how H. pylori infection induces WEE1 in GC, analysis from miRTAR, Target Scan, and miRDB demonstrated that WEE1 is predicted to be targeted by miR-497-5p. Using qRT-PCR analysis, we assessed 30 normal and 30 GC human tissue samples, revealing a pronounced downregulation of miR-497-5p in GC specimens. Western blot analysis confirmed that the restoration of miR-497-5p in GC cells markedly reduced WEE1 protein expression. Interestingly, H. pylori infection further decreased miR-497-5p levels in GC cells, upregulating WEE1 protein expression. Since the miR-497-5P host gene promoter region contains CpG islands, we confirmed that treatment with 5 Azacytidine (demethylating agent) or DNMT1 knockdown induced expression of miR-497-5p with decreased WEE1 level in GC cells. Furthermore, our preliminary results indicate that aberrant WEE1 overexpression in GC demonstrates a significant positive correlation with immune-suppression gene signatures, underscoring its predictive value for unfavorable patient survival outcomes in GC. Using the TFF1 knockout mouse GC model, we found that WEE1 inhibition notably decreases the number and activation of CD4+ and CD8+ T cells in neoplastic gastric tissues. These findings also indicate a crucial relationship between WEE1 and the immunosuppressive microenvironment in GC, establishing WEE1 as a promising therapeutic target to enhance therapeutic efficacy and overcome immunotherapy resistance in GC. Conclusions: Our findings demonstrated a novel mechanism in which H. pylori activates the WEE1 in GC, promoting an immune suppressive microenvironment. WEE1 inhibition with immunotherapy could be a promising therapeutic method for GC patients. Citation Format: Md Obaidul Islam, Krishnapriya Thangaretnam, Heng Lu, Dunfa Peng, Nadeem Sidiq Bhat, Mohammed Soutto, Wael El-Rifai, Zheng Chen. Helicobacter pylori induces the wee1, promoting immune suppressive in human gastric adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5583.

Effects of Helicobacter pylori and Moluodan on the Wnt/β-catenin signaling pathway in mice with precancerous gastric cancer lesions.

Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer (GC), the Wnt/β-Catenin signaling pathway is closely linked to tumourigenesis. GC has a high mortality rate and treatment cost, and there are no drugs to prevent the progression of gastric precancerous lesions to GC. Therefore, it is necessary to find a novel drug that is inexpensive and preventive to against GC. To explore the effects of H. pylori and Moluodan on the Wnt/β-Catenin signaling pathway and precancerous lesions of GC (PLGC). Mice were divided into the control, N-methyl-N-nitrosourea (MNU), H. pylori + MNU, and Moluodan groups. We first created an H. pylori infection model in the H. pylori + MNU and Moluodan groups. A PLGC model was created in the remaining three groups except for the control group. Moluodan was fed to mice in the Moloudan group ad libitum. The general condition of mice were observed during the whole experiment period. Gastric tissues of mice were grossly and microscopically examined. Through quantitative real-time PCR (qRT-PCR) and Western blotting analysis, the expression of relevant genes were detected. Mice in the H. pylori + MNU group showed the worst performance in general condition, gastric tissue visual and microscopic observation, followed by the MNU group, Moluodan group and the control group. QRT-PCR and Western blotting analysis were used to detect the expression of relevant genes, the results showed that the H. pylori + MNU group had the highest expression, followed by the MNU group, Moluodan group and the control group. H. pylori can activate the Wnt/β-catenin signaling pathway, thereby facilitating the development and progression of PLGC. Moluodan suppressed the activation of the Wnt/β-catenin signaling pathway, thereby decreasing the progression of PLGC.

MiR-520f-3p blocks MNNG-induced gastric precancerous lesions via the KLF7/NFκB pathway

Studying the regulatory mechanism of gastric disease progression to gastric cancer (GC) is essential. miR-520f expression is down-regulated in GC and inhibits the proliferation of gastric cancer cells, suggesting that it is associated with the development of GC, but whether it plays a role in the gastric precancerous lesion (GPL) is unclear. This study aimed to investigate the effect of miR-520f-3p in the N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced GPL model and to elucidate the role of its downstream target gene Kruppel-like factor 7 (KLF7) in it. The experimental results showed that miR-520f-3p expression was down-regulated in the MNNG-induced GES-1 cell model, and overexpression of miR-520f-3p reversed the effects of MNNG on cell migration, invasion and epithelial-mesenchymal transition (EMT) -related protein expression. Meanwhile, overexpression of KLF7 attenuated the effect of miR-520f-3p on GPL. In a mouse GPL model, it was observed that MNNG elicited inflammation and EMT processes in mouse gastric tissues through the KLF7/ Nuclear Factor Kappa B (NFκB) pathway, and silencing KLF7 alleviated MNNG-induced gastric epithelial cell injury and gastric atrophy symptoms. These results provide a new perspective for understanding the development of GPL, and the development of new therapies targeting miR-520f-3p and KLF7 may provide new ideas for the prevention and treatment of gastric cancer.

Gintonin Alleviates HCl/Ethanol- and Indomethacin-Induced Gastric Ulcers in Mice.

Gintonin, newly extracted from ginseng, is a glycoprotein that acts as an exogenous lysophosphatidic acid (LPA) receptor ligand. This study aimed to demonstrate the in vivo preventive effects of gintonin on gastric damage. ICR mice were randomly assigned to five groups: a normal group (received saline, 0.1 mL/10 g, p.o.); a control group (administered 0.3 M HCl/ethanol, 0.1 mL/10 g, p.o.) or indomethacin (30 mg/kg, p.o.); gintonin at two different doses (50 mg/kg or 100 mg/kg, p.o.) with either 0.3 M HCl/ethanol or indomethacin; and a positive control (Ranitidine, 40 mg/kg, p.o.). After gastric ulcer induction, the gastric tissue was examined to calculate the ulcer index. The expression of gastric damage markers, such as tumor necrosis factor (TNF)-α, cyclooxygenase 2 (COX-2), and LPA2 and LPA5 receptors, were measured by Western blotting. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) levels were measured by enzyme-linked immunosorbent assay. The platelet endothelial cell adhesion molecule (PECAM-1), Evans blue, and occludin levels in gastric tissues were measured using immunofluorescence analysis. Both HCl/ethanol- and indomethacin-induced gastric ulcers showed increased TNF-α, IL-6, Evans blue permeation, and PECAM-1, and decreased COX-2, PGE2, occludin, and LPA5 receptor expression levels. However, oral administration of gintonin alleviated the gastric ulcer index induced by HCl/ethanol and indomethacin in a dose-dependent manner. Gintonin suppressed TNF-α and IL-6 expression, but increased COX-2 expression and PGE2 levels in mouse gastric tissues. Gintonin intake also increased LPA5 receptor expression in mouse gastric tissues. These results indicate that gintonin can play a role in gastric protection against gastric damage induced by HCl/ethanol or indomethacin.

일회용 종이컵에서 발생하는 나노플라스틱의 생체 내 잔존량 측정

In this study, an experiment was conducted to determine whether nanoplastics extracted from disposable paper cups that are easily accessible and widely used in daily life remain in vivo. In order to extract nanoplastics, water boiled at 95-100℃ was added to a disposable paper cup, and the extracted nanoplastics were observed with a digital microscope. To check whether nanoplastics remained in the body, 1mg/mL of nanoplastic was administered to mice, urine and feces were collected to measure the amount of nanoplastics discharged, and the gastric tissue of mice was collected to confirm the amount of nanoplastics remaining on the stomach. As a result, it was confirmed that a significant amount of nanoplastic in the disposable paper cup was extracted at high water temperature, and the amount of nanoplastic was discharged out of the body, but some remained in the body.

Protective Effects of Cudrania tricuspidata Against Helicobacter pylori-Induced Inflammation in C57BL/6 Mice.

Helicobacter pylori modulates the host inflammatory response, resulting in chronic gastritis, which contributes to gastric cancer pathogenesis. We verified the effect of Cudrania tricuspidata on H. pylori infection by inhibiting H. pylori-induced inflammatory activity. Five-week-old C57BL/6 mice (n = 8) were administered C. tricuspidata leaf extract (10 or 20 mg/kg per day) for 6 weeks. An invasive test (campylobacter-like organism [CLO]) and noninvasive tests (stool antigen test [SAT] and H. pylori antibody enzyme-linked immunosorbent assay) were performed to confirm the eradication of H. pylori. To evaluate the anti-inflammatory effect of C. tricuspidata, pro-inflammatory cytokines levels and inflammation scores were measured in mouse gastric tissue. C. tricuspidata significantly decreased the CLO score and H. pylori immunoglobulin G antibody optical density levels at both 10 and 20 mg/kg per day doses (P < .05). C. tricuspidata decreased the H. pylori antibody levels in a concentration-dependent manner, increased negative responses to SAT by up to 37.5%, and inhibited the pro-inflammatory cytokines interleukin (IL; IL-1β, IL-6, 1L-8, and tumor necrosis factor alpha). C. tricuspidata also relieved gastric erosions and ulcers and significantly reduced the inflammation score (P < .05). We measured rutin in C. tricuspidata extract as a standard for high-performance liquid chromatography. C. tricuspidata leaf extract showed anti-H. pylori activity through the inhibition of inflammation. Our findings suggest that C. tricuspidata leaf extract is potentially an effective functional food material against H. pylori.

Anti-CD40L therapy prevents the formation of precursor lesions to gastric B-cell MALT lymphoma in a mouse model.

Mucosa-associated lymphoid tissue (MALT) lymphoma is a B-cell tumour that develops over many decades in the stomachs of individuals with chronic Helicobacter pylori infection. We developed a new mouse model of human gastric MALT lymphoma in which mice with a myeloid-specific deletion of the innate immune molecule, Nlrc5, develop precursor B-cell lesions to MALT lymphoma at only 3months post-Helicobacter infection versus 9-24 months in existing models. The gastric B-cell lesions in the Nlrc5 knockout mice had the histopathological features of the human disease, notably lymphoepithelial-like lesions, centrocyte-like cells, and were infiltrated by dendritic cells (DCs), macrophages, and T-cells (CD4+ , CD8+ andFoxp3+ ). Mouse and human gastric tissues contained immune cells expressing immune checkpoint receptor programmed death 1 (PD-1) and its ligand PD-L1, indicating an immunosuppressive tissue microenvironment. We next determined whether CD40L, overexpressed in a range of B-cell malignancies, may be a potential drug target for the treatment of gastric MALT lymphoma. Importantly, we showed that the administration of anti-CD40L antibody either coincident with or after establishment of Helicobacter infection prevented gastric B-cell lesions in mice, when compared with the control antibody treatment. Mice administered the CD40L antibody also had significantly reduced numbers of gastric DCs, CD8+ and Foxp3+ T-cells, as well as decreased gastric expression of B-cell lymphoma genes. These findings validate the potential of CD40L as a therapeutic target in the treatment of human gastric B-cell MALT lymphoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation in Helicobacter pylori-associated gastritis by regulating ROS and autophagy

BackgroundThe NLRP3 inflammasome activation is the molecular basis of Helicobacter pylori (Hp)-associated gastritis. Tripartite motif (TRIM) 31 is involved in diverse pathological events. However, whether TRIM31 plays a role in the activation of NLRP3 inflammasome in Hp infection is not clarified.MethodsA mouse model of chronic Hp infection was established, and the gastric tissues were subjected to the polymerase chain reaction, western blotting, histopathological analysis, and RNA sequencing. The mitochondrial membrane potential and ROS in the human gastric epithelium GES-1 cells with or without Hp infection were measured by flow cytometry. GES-1 cells with or without TRIM31 knockdown were transfected with mCherry-EGFP-LC3 adenovirus. After rapamycin and bafilomycin A1 stimulation, autophagy flux in the above primed GES-1 cells was assessed by laser confocal microscope. Lysosomal acidification and expression levels of cathepsin B and cathepsin D in GES-1 cells with Hp infection were measured.ResultsNLRP3 inflammasome was activated in the gastric tissues of mice with chronic Hp infection in vivo and the GES-1 cells with Hp infection in vitro. TRIM31 was downregulated in Hp infection. TRIM31 negatively regulated the NLRP3 inflammasome activation. Enhanced ROS, impaired autophagy flux, and decreased expression of lysosomal cathepsin B and cathepsin D were observed in TRIM31-deficient GES-1 cells with Hp infection. In turn, inhibition of ROS led to the decreased expression of NLRP3 inflammasome.ConclusionsTogether, our data identified that TRIM31 negatively regulated the activation of NLRP3 inflammasome in Hp-associated gastritis by affecting ROS and autophagy of gastric epithelial cells.BaPkEPhQjKRgDMqnjfdJ-VVideo abstract

Posttranslational Modifications of Rev-Erbα Protein and Abnormal Inflammatory Response in Gastric Cancer.

We reported that Rev-erbα, a transcriptional repressor, is reduced in human gastric cancer and that it inhibits glycolysis in cultured gastric cancer cells. However, it is unclear whether Rev-erbα undergoes posttranslational modifications in gastric cancer. Here, we determined levels of Rev-erbα and its posttranslational modifications including phosphorylation, SUMOylation, and ubiquitination in N-methyl-N-nitrosourea (MNU)/Helicobacter pylori (H. pylori)-induced gastric cancer in mice and in cultured human gastric cancer cells. Administration of MNU plus H. pylori infection successfully induced gastric tumor in C57BL/6J mice. MNU/H. pylori decreased the levels of Rev-erbα in gastric tumor tissues of mice accompanied by an increase in the level of lactic acid. Rev-erbα protein SUMOylation and ubiquitination modifications were significantly increased, whereas phosphorylation was unchanged, in gastric cancer cells line BGC-823 and MNU/H. pylori-induced mouse gastric cancer tissues. Using human gastric cancer tissues, we found that Rev-erbα was specifically reduced in mucosal epithelial cells in gastric tissue. Cytokine levels were increased in MNU/H. pylori-exposed mice compared with control mice. Similarly, the levels of IL-6 IL-10, TNF-α, and VEGF were higher in the BGC-823 cell line compared with GES-1 cells. IL-6 and IL-1 incubation did not affect Rev-erbα levels in BGC-823 cells. Furthermore, Rev-erbα was recruited on the promoters of these cytokine genes, which suppressed their expression. Conclusively, Rev-erbα SUMOylation and subsequent ubiquitination may contribute to its protein reduction, which leads to increased glycolysis and abnormal inflammatory responses during the development of gastric cancer. Targeting Rev-erbα and its SUMOylation represents promising approaches for prevention and management of gastric cancer.

Induction of Fibroblast Growth Factor Receptor 4 by Helicobacter pylori via Signal Transducer and Activator of Transcription 3 With a Feedforward Activation Loop Involving SRC Signaling in Gastric Cancer

Helicobacter pylori (H pylori) infection is the main risk factor for gastric cancer. The role of fibroblast growth factor receptors (FGRFs) in H pylori-mediated gastric tumorigenesis remains largely unknown. This study investigated the molecular and mechanistic links between H pylori, inflammation, and FGFR4 in gastric cancer. Cell lines, human and mouse gastric tissue samples, and gastric organoids models were implemented. Infection with H pylori was performed using invitro and invivo models. Western blot, real-time quantitative reverse-transcription polymerase chain reaction, flow cytometry, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and luciferase reporter assays were used for molecular, mechanistic, and functional studies. Analysis of FGFR family members using The Cancer Genome Atlas data, followed by validation, indicated that FGFR4 messenger (m)RNA was the most significantly overexpressed member in human gastric cancer tissue samples (P < .001). We also detected high levels of Fgfr4 mRNA and protein in gastric dysplasia and adenocarcinoma lesions in mouse models. Infection with J166, 7.13, and PMSS1 cytotoxin-associated gene A (CagA)+ H pylori strains induced FGFR4 mRNA and protein expression in invitro and invivo models. This was associated with a concordant activation of signal transducer and activator of transcription 3 (STAT3). Analysis of the FGFR4 promoter suggested several putative binding sites for STAT3. Using chromatin immunoprecipitation assay and an FGFR-promoter luciferase reporter containing putative STAT3 binding sites and their mutants, we confirmed a direct functional binding of STAT3 on the FGFR4 promoter. Mechanistically, we also discovered a feedforward activation loop between FGFR4 and STAT3 where the fibroblast growth factor 19-FGFR4 axis played an essential role in activating STAT3 in a steroid receptor coactivator-dependent manner. Functionally, we found that FGFR4 protected against H pylori-induced DNA damage and cell death. Our findings demonstrated a link between infection, inflammation, and FGFR4 activation, where a feedforward activation loop between FGFR4 and STAT3 is established via steroid receptor coactivator in response to H pylori infection. Given the relevance of FGFR4 to the etiology and biology of gastric cancer, we propose FGFR4 as a druggable molecular vulnerability that can be tested in patients with gastric cancer.

MiR-1298-5p level downregulation induced by Helicobacter pylori infection inhibits autophagy and promotes gastric cancer development by targeting MAP2K6

Helicobacter pylori infection is a leading cause of gastric cancer (GC). However, the underlying mechanisms have not yet been fully elucidated. We aimed to identify microRNAs (miRNAs) regulated by H. pylori infection and their underlying mechanisms in gastric carcinogenesis. Using a mouse model, it was established that H. pylori infection inhibited autophagy in the gastric mucosa. Importantly, H. pylori infection decreased miR-1298-5p levels in human and mouse gastric tissues and human gastric cell lines. Furthermore, the downregulation of miR-1298-5p levels remarkably inhibited autophagy, ultimately increasing the intracellular H. pylori load, which was detected using a gentamicin protection assay. A series of in vitro assays showed that the downregulation of miR-1298-5p expression promoted GC cell proliferation, migration, and invasion. Mechanistically, using bioinformatics prediction, miRNA pull-down assays, and luciferase reporter assays, mitogen-activated protein kinase kinase 6 (MAP2K6) was found to be the direct target of miR-1298-5p, through which miR-1298-5p regulated autophagy and GC cell viability and motility. Moreover, MAP2K6/p38 mitogen-activated protein kinase (MAPK) axis was determined to be the downstream pathway of miR-1298-5p. These findings revealed that H. pylori infection was found to inhibit autophagy and promote tumor growth by regulating miR-1298-5p expression and the miR-1298-5p/MAP2K6/p38 MAPK axis might be a new avenue for the clinical management of H. pylori infection and H. pylori-associated GC.

Enhanced ASGR2 by microplastic exposure leads to resistance to therapy in gastric cancer.

Background: Microplastics (MPs) are a new global environmental threat. Previously, we showed the biodistribution of MPs using [64Cu] polystyrene (PS) and PET in mice. Here, we aimed to identify whether PS exposure has malignant effects on the stomach and induces resistance to therapy.Methods: BALB/c nude mice were fed 1.72 × 104 particles/mL of MP. We investigated PS accumulation in the stomach using radioisotope-labeled and fluorescent-conjugated PS. Further, we evaluated whether PS exposure induced cancer stemness and multidrug resistance, and whether it affected tumor development, tumor growth, and survival rate in vivo using a 4-week PS-exposed NCI-N87 mouse model. Using RNA-Seq analysis, we analyzed whether PS exposure induced gene expression changes in gastric tissues of mice.Results: PET imaging results showed that a single dose of [64Cu]-PS remained for 24 h in the mice stomach. The 4-week daily repetitive dose of fluorescent conjugated PS was deposited in the gastric tissues of mice. When PS was exposed, a 2.9-fold increase in migration rate was observed for NCI-N87 cells. Immunocytochemistry results showed decreased E-cadherin and increased N-cadherin expression, and flow cytometry, qPCR, and western blot analysis indicated a 1.9-fold increase in N-cadherin expression after PS exposure. Further, PS-induced multidrug resistance to bortezomib, paclitaxel, gefitinib, lapatinib, and trastuzumab was observed in the NCI-N87 mouse model due to upregulated CD44 expression. RNA-seq results identified increased asialoglycoprotein receptor 2 (ASGR2) expression after PS exposure, and ASGR2 knockdown decreased cell proliferation, migration, invasion, and drug resistance.Conclusion: We demonstrated that ASGR2 enhanced cancer hallmarks on PS exposure and induced resistance to chemo- and monoclonal antibody-therapy. Our preclinical findings may provide an incentive for further epidemiological studies on the role of MP exposure and its association with gastric cancer.

The role of l-cysteine/Hydrogen sulfide pathway on β3-Adrenoceptor- induced relaxation in mouse gastric fundus

In this study, we investigated the possible role of the l-cysteine/hydrogen sulfide pathway in β3-adrenoceptors-mediated relaxation in isolated mouse gastric fundus tissue. l-cysteine (endogenous H2S; 10−6-10−2 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10−6-10−3 M), selective β3-adrenoceptors agonist BRL 37344 (10−9-10−4 M) and non-selective β-adrenoceptor agonist isoprenaline (10−9-10−4 M) produced concentration-dependent relaxation in mouse gastric fundus. The non-selective β-adrenoceptors antagonist propranolol (10−6 M) inhibited the relaxant response to isoprenaline but not to BRL 37344. On the other hand, the selective β3-adrenoceptors antagonist SR 59230A (10−5 M) inhibited the relaxant responses to BRL 37344. In addition, cystathionine-gamma-lyase (CSE) inhibitor D,L-propargylglycine (PAG, 10−2 M), cystathionine-beta-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10−2 M), and the combination of these inhibitors significantly reduced the relaxant responses induced by l-cysteine and BRL 37344. Pre-incubation of gastric fundal strips with propranolol (10−6 M) and SR 59230A (10−5 M) did not affect relaxations to l-cysteine and NaHS. Also, the existence of CSE, CBS, 3-mercaptopurivate sulfur transferase (3-MST) enzymes and β3-adrenoceptors were detected in gastric fundal tissue. Furthermore, basal H2S release was detected in the measurements. H2S level increased in the presence of l-cysteine, NaHS, and BRL 37344. The increase in H2S level by l-cysteine and BRL 37344 decreased significantly with PAG and AOAA enzyme inhibitors. These results suggest that endogenous H2S is synthesized from l-cysteine at least by CBS and CSE enzymes. Also, β3-adrenoceptors are found in the mouse stomach fundus and mediate BRL 37344-induced relaxations, and l-cysteine/hydrogen sulfide pathway plays a partial role in β3-adrenoceptors-mediated relaxation in mouse gastric fundus tissue.

Mast1 mediates radiation-induced gastric injury via the P38 MAPK pathway

Radiation-induced gastric injury is a serious adverse effect and reduces the efficacy of radiotherapy treatment. However, the mechanisms underlying radiation-induced stomach injury remain unclear. Here, mouse stomach and gastric epithelial cells were irradiated with different doses of X-ray radiation. The results showed that radiation induced gastric injury in vivo and in vitro. Differentially expressed functional mRNAs in irradiation-induced gastric tissues were screened from the Gene Expression Omnibus (GEO) database. We found that the expression of microtubule-associated serine/threonine kinase 1 (Mast1) was downregulated in mouse gastric tissues and gastric epithelial cells after irradiation. Furthermore, functional assays showed that knockdown of Mast1 inhibited growth and promoted apoptosis in gastric epithelial cells, while overexpression of Mast1 protected gastric epithelial cells from radiation damage. Mechanistically, Mast1 negatively regulated radiation-induced injury in gastric epithelial cells by inhibiting the activation of P38. The apoptosis caused by knockdown of Mast1 in gastric epithelial cells could be partially reversed by the P38 inhibitor SB203580. Moreover, data from several gastric cancer cell lines and online databases revealed that Mast1 was not involved in the development of gastric cancer. Collectively, our findings demonstrated that Mast1 is essential for radiation-induced gastric injury, providing a promising prognostic and therapeutic target.

Loss of ARID1A activates mTOR signaling and SOX9 in gastric adenocarcinoma—rationale for targeting ARID1A deficiency

BackgroundGastric adenocarcinoma (GAC) is a lethal disease with limited therapeutic options. Genetic alterations in chromatin remodelling gene AT-rich interactive domain 1A (ARID1A) and mTOR pathway activation occur frequently in GAC....

Loss of NFKB1 Results in Expression of Tumor Necrosis Factor and Activation of Signal Transducer and Activator of Transcription 1 to Promote Gastric Tumorigenesis in Mice

Activity of nuclear factor κB transcription factors and signaling via signal transducer and activator of transcription (STAT) are frequently altered in gastric cancer cells. Mice lacking NFKB1 (Nfkb1-/- mice) develop invasive gastric cancer, and their gastric tissues have increased levels of cytokines, such as interleukin (IL) 6, IL22, IL11, and tumor necrosis factor (TNF), as well as increased activation of STAT1. We investigated whether these cytokines were required for STAT1 activation in gastric tissues of mice and critical for gastric tumorigenesis. We crossed Nfkb1-/- mice with Il6-/-, Il22-/-, Il11Rα-/-, and Tnf-/- mice. Stomach tissues from compound mutant mice were analyzed by histology, immunoblotting, and RNA sequencing. Lymphoid, myeloid, and epithelial cells were isolated from stomachs, and the levels of cytokines were determined by flow cytometric analysis. Nfkb1-/- mice developed gastritis, oxyntic atrophy, gastric dysplasia, and invasive tumors, whereas Nfkb1-/-Stat1-/- mice did not, even when followed for as long as 2 years. The levels of Il6, Il11, Il22, and Tnf messenger RNA were increased in the body and antrum of the stomachs from Nfkb1-/- mice, from 3-6 months of age. However, Nfkb1-/-Il6-/-, Nfkb1-/-Il22-/-, and Nfkb1-/-Il11Rα-/- mice still developed gastric tumors, although the absence of IL11 receptor (IL11R) significantly reduced development of invasive gastric tumors. Stomachs from Nfkb1-/-Tnf-/- mice exhibited significantly less gastritis and oxyntic atrophy and fewer tumors than Nfkb1-/- mice. This correlated with reduced activation of STAT1 and STAT3 and fewer numbers of T cells and B cells infiltrating the gastric body. Loss of STAT1 or TNF significantly reduced expression of PD-L1 on epithelial and myeloid (CD11b+) cells in the gastric mucosa of Nfkb1-/- mice-indeed, to the levels observed on the corresponding cells from wild-type mice. In studies of gastric tumor development in knockout mice, we found that loss of NFKB1 causes increased expression of TNF in the stomach and thereby drives activation of STAT1, resulting in an inflammatory immune response and the development of gastric cancer. IL11R appears to be required for the progression of gastric tumors to the invasive stage. These findings suggest that inhibitors of TNF, and possibly also inhibitors of IL11/IL11Rα, might be useful in the treatment of gastric cancer.

Inhibitory Effect of Flavonoid Extract of Lotus Leaf on Alcohol-Induced Gastric Injury by Antioxidant Capacity in Mice

The aim of this study was to investigate the antioxidant effects of flavonoid extract of lotus leaf (FELL) in vitro and in mice with gastric injury and elucidate the inhibitory effect of FELL on alcohol-induced gastric injury by antioxidant capacity. Gastric juice volume and acid value were measured in the established alcohol-induced acute gastric injury model in mice. Hematoxylin and eosin staining demonstrated the morphological changes in the gastric mucosa. FELL improved the alcohol-induced gastric lesions. Serological testing results showed that FELL significantly increased the levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and nitric oxide (NO) and significantly reduced the levels of MDA and MPO in mice with gastric injury. Besides, FELL significantly reduced the levels of interleukin 6 (IL-6), interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ). The quantitative polymerase chain reaction (qPCR) and Western blot results proved that FELL upregulated the mRNA and protein expressions of SOD1, SOD2, CAT, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), neuronal nitric oxide synthase (nNOS), and endothelial nitric oxide synthase (eNOS) and downregulated the expression of inducible nitric oxide synthase (iNOS) in the gastric tissue of mice with gastric injury. Five compounds, including kaempferitrin, hyperoside, astragalin, phloridzin, and quercetin, were detected in FELL using high-performance liquid chromatography. Thus, FELL has an inhibitory effect on experimental alcohol-induced gastric injury because of the antioxidant capacity of the five compounds. Its effect increases as the FELL concentration increases, which is close to that of ranitidine. FELL, which is an active substance, provides a good gastroprotective effect.

Innate Immune Molecule NLRC5 Protects Mice From Helicobacter-induced Formation of Gastric Lymphoid Tissue

Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5møKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P < .01), and correlated with gastritis severity (P < .05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P < .05). After 3 months of infection with H felis, Nlrc5mø-KO mice developed gastric hyperplasia (P < .0001), splenomegaly (P < .0001), and increased serum antibody titers (P < .01), whereas control mice did not. Nlrc5mø-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P < .0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein that promoted B-cell hyperproliferation in Nlrc5mø-KO mice. NLRC5 is a negative regulator of gastric inflammation and mucosal lymphoid formation in response to Helicobacter infection. Aberrant NLRC5 signaling in macrophages can promote B-cell lymphomagenesis during chronic Helicobacter infection.

Preventive Effect of Liupao Tea Polyphenols on HCl/Ethanol-Induced Gastric Injury in Mice

Liupao tea is a traditional Chinese tea drink. The preventive effect of crude polyphenols in Liupao tea on HCl/ethanol-induced gastric injury was investigated in this study. After a model of gastric injury in mice was established, mouse serum and tissues were analyzed by biochemical and molecular biological methods. The results showed that Liupao tea polyphenols (LTPs) could effectively reduce the area of gastric mucosal lesions, decrease the volume of gastric juice, and increase the pH of gastric juice in mice with gastric injury. Observations of the pathology revealed that LTPs could alleviate cell necrosis and gastric mucosal injury in mice with gastric injury. The SOD activity and GSH level were decreased in mice after gastric injury, while the level of MDA was increased. LTPs could inhibit the changes caused by gastric injury and make the SOD activity, GSH, and MDA levels close to the normal levels. In addition, LTPs could upregulate the mRNA expression of Cu/Zn-SOD, Mn-SOD, CAT, nNOS, and eNOS and downregulate the expression of iNOS in the gastric tissue of mice with gastric injury. Therefore, LTPs can effectively prevent HCl/ethanol-induced gastric injury. HPLC analysis showed that LTP contains six bioactive substances of gallic acid, catechin, caffeine, epicatechin, epigallocatechin gallate, and epicatechin gallate, so the effect of LTP might mainly come from these six components. The effect of a high concentration of LTP is similar to that of ranitidine. LTPs represent a kind of active substance with a protective effect on gastric tissue.