Early prevention of pathological changes underlying gastric cancer (GC) development is a critical strategy, offering the most effective opportunity to limit malignant progression and improve patient outcomes. We have previously demonstrated that Helicobacter pylori (Hp) (cagA+vacA+) contributes to GC development by activating gastric fibroblasts toward CAF-like phenotype, eliciting aggressive, cancer stem cells (CSCs)-related malignant transformation of LGR5+ normal epithelial cells. A key mediator of these processes appears to be the NF-κB/STAT3 axis. Therefore, our aim was to investigate the protective role of hydrogen sulfide (H2S) as a potential novel strategy for counteracting Hp-induced fibroblast reprogramming. Human fibroblasts were infected with Hp (cagA+vacA+) for 120 h. The fast-releasing H2S donor NaHS (50, 100, 200 and 400 µM) was added every 24 h. Activation markers, corresponding signaling pathways, H2S release and activities of H2S-metabolizing enzymes were determined. NaHS reduced Hp-induced fibroblast activation and their pro-inflammatory, pro-tumorigenic markers, which was associated with the inhibition of NF-κB/STAT3 axis and Twist expression. Additionally, it modulated sulfur metabolism while preserving sulfur-enzyme homeostasis. NaHS limited Hp adhesion (high doses), reduced reinfection-induced activation and increased sensitivity of Hp to metronidazole. These findings suggest that H2S signaling may represent a modulatory factor of NF-κB/STAT3-driven inflammatory responses during Hp infection and warrant further investigation.

Adiponectin is adipokine exhibiting beneficial metabolic action through lipid and carbohydrate metabolism stimulation, as well as anti-inflammatory action. We have determined the role of adiponectin in gastroprotection against the formation of acute gastric lesions induced by ischemia-reperfusion (I/R). Gastric lesions evoked by I/R are a serious clinical entity; however, the participation of reactive oxygen species (ROS) and lipid peroxidation products and the involvement of nitric oxide (NO), neuropeptides released from sensory afferent nerves, and the hormone gastrin in the potential gastroprotective action of adiponectin remains unknown. Therefore, we determined the interplay between capsaicin-sensitive afferent nerves, the NO/NOS system, lipid peroxidation products, and the expression of pro-inflammatory and antioxidative factors in the gastroprotective action of adiponectin against gastric I/R. injury. Wistar rats was administered with adiponectin in graded doses (1-40 μg/kg i.v.) with or without: (a) blockade of nitric oxide (NO) activity by L-nitro-L-arginine (L-NNA) and (b) deactivation of sensory nerves by capsaicin (125 mg/kg s.c. 10 days before experiment conduction). They were then exposed to 30 min of ischemia by clamping of the celiac artery followed by 3 h of reperfusion after clamp release. After 3 h, the rats were euthanized with pentobarbital and their gastric blood flow (GBF) was determined by laser Doppler flowmetry, their blood was withdrawn to assess plasma gastrin levels, and the area of gastric lesions was measured by planimetry. Gastric biopsy samples were excised to determine gastric mucosal levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). In separate groups of animals with chronic gastric fistula, the effect of adiponectin on gastric acid secretion was determined. Adiponectin dose-dependently reduced the gastric lesions induced by I/R and this effect was accompanied by an increase in GBF. Blockade of NO-synthase with L-NNA (20 mg/kg i.p.) reversed the protective effect and the rise in GBF induced by this adipokine, and both these effects were restored when L-arginine was added to L-NNA. Capsaicin denervation also impeded the beneficial action of adiponectin in rats, but these effects were in part restored when exogenous CGRP was combined with adiponectin. Adiponectin dose-dependently decreased gastric acid secretion, the expression of mRNA for pro-inflammatory cytokines, and MDA plus 4-HNE content, while significantly increasing SOD, GSH and plasma gastrin increments. We conclude that adiponectin exerts gastroprotection against I/R-induced gastric lesions, through mechanisms involving NO and neuropeptides such as CGRP being released from sensory nerves, a decrease in lipid peroxidation (MDA+4-HNE), an increase of antioxidative factors (SOD, GSH), and the inhibition of gastric acid secretion.

Intestinal alkaline phosphatase (IAP) is an enzyme that plays a protective role in the gut. This study investigated the effect of IAP treatment on experimental colitis in mice subjected to forced exercise on a high-fat diet. C57BL/6 mice with TNBS colitis were fed a high-fat diet and subjected to forced treadmill exercise with or without IAP treatment. Disease activity, oxidative stress, inflammatory cytokines, and gut microbiota were assessed. Forced exercise exacerbated colitis in obese mice, as evidenced by increased disease activity index (DAI), oxidative stress markers, and proinflammatory adipokines and cytokines. IAP treatment significantly reduced these effects and promoted the expression of barrier proteins in the colonic mucosa. Additionally, IAP treatment altered the gut microbiota composition, favoring beneficial Verrucomicrobiota and reducing pathogenic Clostridia and Odoribacter. IAP treatment ameliorates the worsening effect of forced exercise on murine colitis by attenuating oxidative stress, downregulating proinflammatory biomarkers, and modulating the gut microbiota. IAP warrants further investigation as a potential therapeutic strategy for ulcerative colitis.

The gut mucosal barrier plays a key role in the physiology of gastrointestinal (GI) tract, preventing under homeostatic conditions, the epithelial cells of the gastric mucosa from hydrochloric acid and intestinal mucosa from alkaline secretion, food toxins and pathogenic microbiota. Previous studies have documented that blockade of both isoforms of cyclooxygenase (COX): constitutive (COX-1) and inducible (COX-2), as well NO synthase in the stomach exacerbated the gastric damage induced by various ulcerogens, however, such as effects of non-selective and selective inhibition of COX-1, COX-2 and NOS enzymes on colonic damage have been little studied. The supplementation of NO by intragastric (i.g.) treatment with NO-releasing compound NO-aspirin (NO-ASA) or substrate for NO synthase L-arginine ameliorated the damage of upper GI-tract, but whether similar effect can be observed in colonic mucosa associated with the experimental colitis, and if above mentioned compounds can be effective in aggravation or protection of experimental colitis remains less recognized. In this study rats with experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzosulphonic acid (TNBS) were daily treated for 7 days with: 1) vehicle (i.g.), 2) ASA 40 mg/kg (i.g.), 3) rofecoxib 10 mg/kg (i.g.), 4) resveratrol 10 mg/kg (i.g.), 5) NO-ASA 40 mg/kg (i.g.), 6) L-arginine 200 mg/kg (i.g.) with or without of L-NNA 20 mg/kg (i.p.). The macroscopic and microscopic area of colonic damage was determined planimetrically, the colonic blood flow (CBF) was assessed by Laser flowmetry, and the oxidative stress biomarkers malondialdehyde and 4-hydroxynonenal (MDA+4-HNE), the antioxidative factors superoxide dismutase (SOD) and glutathione (GSH), as well as proinflammatory cytokines in the colonic mucosa (tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1β)) were measured. We have documented that administration of TNBS produced gross and microscopic colonic damage and significantly decreased CBF (p<0.05). Treatment with ASA significantly increased the area of colonic damage (p<0.05), an effect accompanied by a significant decrease in the CBF, the significant increment of MDA+4-HNE, and the attenuation of the antioxidative properties in colonic mucosa, documented by a significant decrease of SOD activity and GSH concentration, and elevation of the colonic tissue levels of TNF-α and IL-1β comparing to control Veh-treated TNBS rats. Administration of rofecoxib or resveratrol also significantly increased the colonic damage and significantly decreased the CBF, causing an increase in MDA+4-HNE and mucosal content of TNF-α and IL-1α and a significant decrease of the SOD activity and GSH content (p<0.05), however, these changes were significantly less pronounced as compared with ASA. On the contrary, the treatment with NO-ASA, or L-arginine, significantly diminished the area of colonic lesions, the MDA+4-HNE concentration, attenuated the TNF-α and IL-1β levels, while increasing the CBF, SOD activity and GSH content (p<0.05). The concomitant treatment of L-NNA with rofecoxib or resveratrol reversed an increase in area of colonic damage and accompanying changes in CBF, colonic mucosa TNF-α and IL-1β levels, the MDA+4-HNE concentration, and SOD activity and GSH content comparing to those observed in TNBS rats treated with these COX-inhibitors alone (p<0.05). In contrast, co-treatment with L-NNA and NO-ASA or L-arginine failed to significantly affect the decrease of colonic lesions accompanied by the rise in CBF, the attenuation of MDA+4-HNE concentration, TNF-α and IL-1β levels, SOD activity and GSH content exerted by NO-ASA- or L-arginine treatment of the respective control TNBS-rats without L-NNA administration. These observations suggest that 1) the increase of NO availability either from NO-releasing donors such as NO-ASA or NO precursors such as L-arginine, can inhibit the inflammatory and microvasculature alterations, as well as increase in lipid peroxidation due to the enhanced efficacy of these compounds to increase the antioxidative properties of colonic mucosa, 2) unlike ASA which exacerbated the severity of colitis, the treatment with rofecoxib, the specific 'safer' COX-2 inhibitor or resveratrol, the polyphenolic compound known to act as the dual COX-1 and COX-2 inhibitor, can attenuate the colonic damage during course of TNBS colitis possibly via anti-inflammatory and antioxidative properties, and 3) the blockade of endogenous NO activity by L-NNA which also exacerbated the severity of mucosal damage in colitis, can abolish the sparing effect of rofecoxib and resveratrol indicating the NO bioavailability plays an important role in enhanced efficacy of both specific and dual COX inhibitors to ameliorate the experimental colitis.

Intestinal inflammation in inflammatory bowel disease (IBD) is closely linked to nutrition. This study aimed to evaluate associations between nutritional, inflammatory, and intestinal barrier parameters in patients with IBD. We assessed nutritional status, fecal short-chain fatty acid profile, serum cytokine levels, and mRNA expression of enzymes and tight junction proteins in intestinal biopsies obtained from 35 patients, including 11 patients with inactive IBD, 18 patients with active IBD, and six controls. Patients with active IBD were characterized by hypoalbuminemia, fluctuations in body weight, and restriction of fiber-containing foods. In addition, they had significantly reduced levels of isovaleric acid and tended to have lower levels of butyric, acetic, and propionic acids. Patients with active IBD had higher mRNA expression of peroxisome proliferator-activated receptor γ and inducible nitric oxide synthase, and lower mRNA expression of claudin-2 and zonula occludens-1, compared with patients with inactive IBD. Moreover, patients with a body mass index (BMI) of ≥25 kg/m2 had higher median tumor necrosis factor-α levels that those with a lower BMI. We comprehensively evaluated inflammatory parameters in relation to IBD activity and nutritional status. The discrepancies between proinflammatory and anti-inflammatory parameters depending on IBD activity may be related to nutritional factors, including diet and abnormal body weight.

Despite of the improvement in gastric cancer (GC) therapies patients still suffer from cancer recurrence and metastasis. Recently, the high ratio of these events combined with increased chemoresistance has been related to the asymptomatic Helicobacter pylori (Hp) infections. The limited efficiency of GC treatment strategies is also increasingly attributed to the activity of tumor stroma with the key role of cancer-associated fibroblasts (CAFs). In order to investigate the influence of Hp infection within stromal gastric tissue on cancer initiation and progression, we have exposed normal gastric epithelial cells to long-term influence of Hp-activated gastric fibroblast secretome. We have referred obtained results to this secretome influence on cancer cell lines. The invasive properties of cells were checked by time-lapse video microscopy and basement membrane assays. The expression of invasion-related factors was checked by RT-PCR, Western Blot, immunofluorescence and Elisa. Hp-activated gastric fibroblast secretome induced EMT type 3-related shifts of RGM1 cell phenotype; in particular it augmented their motility, cytoskeletal plasticity and invasiveness. These effects were accompanied by Snail1/Twist activation, the up-regulation of cytokeratin19/FAP/TNC/Integrin-β1 and MMPs, and by the induction of cMethigh/pEGFRhigh phenotype. Mechanistic studies suggest that this microevolution next to TGFβ relies also on c-Met/EGFR signaling interplay and engages HGF-Integrin-Ras-dependent Twist activation leading to MMP and TNC upregulation with subsequent positive auto- and paracrine feedback loops intensifying this process. Similar shifts were detected in cancer cells exposed to this secretome. Collectively, we show that the secretome of Hp-infected fibroblasts induces reprogramming/microevolution of epithelial and cancer cells towards type 3 EMT-related invasive phenotype in a manner reciprocally reliant next to TGFβ on cMet/Integrin-β1/p-EGFR-dependent axis. Apparently, the phenotypical plasticity of Hp-activated fibroblast reprogrammed gastric epithelial cells determines their susceptibility to the pro-invasive signaling, which results in re-organization of gastric niches and provides the cues for GC promotion/progression.

Although the natural niche for H. pylori (Hp) is the human stomach, for widespread infection to occur this microorganism may need to survive in the external environment. Molecular techniques such as polymerase (PCR) have revealed the presence of Hp DNA in water, indicating that this environment could act as a reservoir for this bacterium. The aim of this study was to analyse the occurrence of Hp in tap water from Cracow and to examine the relationship between 26 parameters and the presence of Hp DNA due to the lack of such information related to this issue in Poland. Additional aim of this study was to determine whether the correlation between Hp DNA detection and seasonal changes of water quality in 379 water samples collected from various water treatment plants (WTPs), could be found. Water samples were subjected to PCR for glmM and cagA genes. Ionic and organic composition of microelements were determined in accordance to Polish and ISO standards. The data obtained from tests show that 212 (55.96%) objects were Hp DNA (glmM) positive and among them 145 (68.40%) waters samples revealed expression of cagA. Linear Discriminant Analysis and Principal Component Analysis were used and provided that the selected variables (p < 0.05): colour, pH, conductivity at 25°C, chlorides, nitrites, nitrates, phosphates, chlorates, chlorites, sulphates, free chlorine, sodium, magnesium, potassium, calcium, organic carbon, trichloromethane, bromodichloromethane, dibromochloroethane, total iron, ammonium ion, and ƩTMHs distinguished the water samples that contain Hp DNA and do not contain Hp DNA. We conclude that the ionic and organic composition of microelements in water might influence the presence of Hp. Thus, determination of the selected microelements may indirectly indicate or sometimes predict the presence of Hp in drinking water.

Intestinal alkaline phosphatase (IAP) is an essential mucosal defense factor involved in the process of maintenance of gut homeostasis. We determined the effect of moderate exercise (voluntary wheel running) with or without treatment with IAP on the course of experimental murine 2,4,6-trinitrobenzenesulfonic acid (TNBS) colitis by assessing disease activity index (DAI), colonic blood flow (CBF), plasma myokine irisin levels and the colonic and adipose tissue expression of proinflammatory cytokines, markers of oxidative stress (SOD2, GPx) and adipokines in mice fed a standard diet (SD) or high-fat diet (HFD). Macroscopic and microscopic colitis in sedentary SD mice was accompanied by a significant decrease in CBF, and a significant increase in the colonic expression of tumor necrosis factor-alpha (TNF-α), IL-6, IL-1β and leptin mRNAs and decrease in the mRNA expression of adiponectin. These effects were aggravated in sedentary HFD mice but reduced in exercising animals, potentiated by concomitant treatment with IAP, especially in obese mice. Exercising HFD mice demonstrated a substantial increase in the mRNA for adiponectin and a decrease in mRNA leptin expression in intestinal mucosa and mesenteric fat as compared to sedentary animals. The expression of SOD2 and GPx mRNAs was significantly decreased in adipose tissue in HFD mice, but these effects were reversed in exercising mice with IAP administration. Our study shows for the first time that the combination of voluntary exercise and oral IAP treatment synergistically favored healing of intestinal inflammation, strengthened the antioxidant defense and ameliorated the course of experimental colitis; thus, IAP may represent a novel adjuvant therapy to alleviate inflammatory bowel disease (IBD) in humans.

Gut-brain axis plays a central role in the regulation of stress related diseases such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD). It is increasingly recognized that stress modulates gut microbiota community structure and activity and represents an important causal factor in dysbiosis. This study was designed to determine the effect of daily treatment with synbiotic (Syngut) containing inulin, Lactobacillus acidophilus, Bifidobacterium lactis W51, Lactobacillus plantarum W21 and Lactococcus lactis applied i.g. at a dose of 50 mg/kg i.g. on the colonic damage and colonic mucosal blood flow in rats with experimentally induced TNBS-colitis that were additionally exposed or not to acute stress (episodes of cold restraint stress every other day before colitis induction). Control rats received daily treatment with vehicle (saline, i.g.) or mesalazine (50 mg/kg-d i.g.), the standard drug recommended in therapy of IBD. At the termination of TNBS colitis, the histologic evaluation of colonic mucosa, mucosal malonyldialdehyde (MDA) level and plasma concentrations of proinflammatory cytokines (TNF-α, IL-1β) and adipokine adiponectin were assessed. the samples of colonic mucosa not involving colonic lesions and surrounding the flared mucosa were excised for the determination of mRNA expression for proinflammatory biomarkers TNF-α, IL-1β, IL-10 and COX-2 as well as antioxidazing factors SOD-1 and SOD-2. Finally, the gut microbial profiles were analyzed by 16S rRNA sequencing at phylum, family and genus level. Episodes of cold stress significantly aggravated the course of TNBS colitis, and significantly increased the release of proinflammatory cytokines as well as the significant increase in the MDA concentration has been observed as compared with non-stressed TNBS rats. These changes were followed by the significant fall in the CBF and plasma adiponectin levels and by the overexpression of mRNA of proinflammatory biomarkers. Synbiotic treatment with Syngut significantly reduced the area of colonic lesions observed macroscopically and microscopically in rats with TNBS colitis with or without exposure to cold stress, significantly increased the CBF, normalized plasma adiponectin levels and significantly attenuated the release and colonic expression of proinflammatory cytokines and biomarkers. the analysis of the gut microbiota showed a significant reduction of microbial diversity (Shannon index) in rats with TNBS colitis with or without exposure to stress. The therapy with Syngut failed to significantly affect the alpha diversity. At the phylum level, the significant rise in Proteobacteria has been observed in stressed rats with TNBS colitis and this effects was attenuated by treatment with Syngut. At family level, TNBS colitis alone or in combination with stress led to a significant decrease of SCFA producing bacterial taxa such as Ruminococaceae and Lachnospiraceae and Syngut counteracted this effect. We conclude that: 1) cold stress exacerbates the gastrointestinal inflammation in experimental colitis; 2) the synbiotic therapy with Syngut ameliorates the gut inflammation in rats with TNBS colitis combined with cold stress; 3) the beneficial effect of Syngut is accompanied by increase of anti-inflammatory taxa such as Ruminococaceae and Lachnospiraceae, and 4) the modulation of gut microbiota with Syngut alleviates stress-related intestinal inflammation suggesting a potential usefulness of synbiotic therapy in intestinal disorders accompanied by stress in patients with IBD.

Helicobacter pylori (Hp)-induced inflammatory reaction leads to a persistent disturbance of gastric mucosa and chronic gastritis evidenced by deregulation of tissue self-renewal and local fibrosis with the crucial role of epithelial–mesenchymal transition (EMT) in this process. As we reported before, Hp activated gastric fibroblasts into cells possessing cancer-associated fibroblast properties (CAFs), which secreted factors responsible for EMT process initiation in normal gastric epithelial RGM1 cells. Here, we showed that the long-term incubation of RGM1 cells in the presence of Hp-activated gastric fibroblast (Hp-AGF) secretome induced their shift towards plastic LGR5+/Oct4high/Sox-2high/c-Mychigh/Klf4low phenotype (l.t.EMT+RGM1 cells), while Hp-non-infected gastric fibroblast (GF) secretome prompted a permanent epithelial–myofibroblast transition (EMyoT) of RGM1 cells favoring LGR−/Oct4high/Sox2low/c-Myclow/Klf4high phenotype (l.t.EMT−RGM1 cells). TGFβ1 rich secretome from Hp-reprogrammed fibroblasts prompted phenotypic plasticity and EMT of gastric epithelium, inducing pro-neoplastic expansion of post-EMT cells in the presence of low TGFβR1 and TGFβR2 activity. In turn, TGFβR1 activity along with GF-induced TGFβR2 activation in l.t.EMT−RGM1 cells prompted their stromal phenotype. Collectively, our data show that infected and non-infected gastric fibroblast secretome induces alternative differentiation programs in gastric epithelium at least partially dependent on TGFβ signaling. Hp infection-activated fibroblasts can switch gastric epithelium microevolution towards cancer stem cell-related differentiation program that can potentially initiate gastric neoplasm.

Gastric cancer (GC) originating from the lining of the stomach is one of the most frequent malignancies in humans. The most efficient method giving hope of full recovery from GC is gastric resection combined with adjuvant chemotherapy and radiotherapy. However, over 50% of patients after treatment suffer from recurrence and peritoneal metastasis. The bacteria Helicobacter pylori (Hp) is nowadays considered as the major pathogen capable of colonizing gastric mucosa. This bug causes deregulation of multiple signaling pathways including the activation of nuclear factor kappaB (NFκB) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) responsible for inflammation and development of carcinogenic cascade. The pathomechanism of these changes remains little understood, but the Hp virulence factors affecting mainly gastric epithelium have been postulated. Nevertheless, the changes associated with inflammation progressing to cancer are not only limited to epithelial cells. The cells surrounding the tumor, mainly activated fibroblasts (CAFs - cancer-associated fibroblasts) create molecular microenvironment promoting tumorigenesis and cancer invasion. The downstream targets of STAT3, epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) are expressed in activated fibroblasts providing them with additional properties. Thus, our aim was to determine if the Hp strain expressing CagA and VacA cytotoxins may result in the activation/differentiation of rat gastric fibroblasts resulting in NFκB and STAT3 signaling, which could lead to EMT-TFs expression and secretome responsible for inflammatory and EMT inducing microenvironment. In this study, gastric samples were harvested from 8-week-old Spraque-Dowley rats and the primary and secondary fibroblast cultures were established. The 70% confluent secondary fibroblast cultures were infected with 1 x 109 of live Hp expressing cytotoxins CagA VacA per dish and incubated in humidified atmosphere for 3, 24, 48 and 72 hours, before the conditioned media or the cells were used for endpoint experiments. As the control, fibroblast culture in DMEM with 10% FBS and antibiotics, free from Hp infection was used. The expression of mRNA for 18S (control), toll-like receptors: TLR2 and TLR4, STAT3, NFκB p65/Rel A, inhibitor of NF-κB (Iκβ), Snail and Twist was determined by RT-PCR. The protein expression of Snail and Twist was assessed by Western blot technique. The fibroblast supernatant was collected at 72 hours from non-infected and Hp (cagA+ vacA+)-infected culture and the concentrations of interleukin 8 (IL-8), hepatocyte growth factor (HGF) and stromal derived factor-1 (SDF-1) were measured by ELISA. In fibroblasts infected with Hp (cagA+ vacA+), the significant increase of mRNA expression for both, TLR2 and TLR4, as well as STAT3, NFκB/RelA subunit was observed already after 3 hours of fibroblasts infection with Hp strain compared with control non-infected fibroblasts. Simultaneously, the significant decrease of Iκβ mRNA has been noticed starting from 48 hours after the Hp infection of fibroblasts was carried out. The strong increase in the expression of Snail1 and Twist mRNA was recorded already at 3 hours in Hp-infected fibroblasts comparing to control non-infected fibroblasts and this increase persisted at 24 and 48 hours being the most pronounced at 72 hours post incubation with Hp. The expression of Snail1 protein was observed after 3 hours post Hp infection and this increase persisted throughout entire time periods upon Hp infection. In contrast, no detectable level of Twist protein expression was observed up to 72 hours post-infection neither in control conditions nor in fibroblasts co-infected with Hp. These changes in fibroblasts were accompanied by a significant increase in the release of HGF, SDF-1 and IL-8 determined in cell supernatants collected from Hp-infected fibroblasts. These data indicate that the activation/differentiation of rat gastric fibroblasts can occur directly by Hp releasing CagA and indirectly through TLR2 and TLR4 and these effects can be mediated by transcription factors NFκB and STAT3 signaling leading to rapid Snail1 protein expression. We conclude that NFκB and STAT3 signaling together with Snail1 protein expression may activate the secretome responsible for fibroblasts inflammatory and EMT-inducing microenvironment likely serving as prerequisite for GC development.