Helicobacter Pylori Antigens, Acetylsalicylic Acid, Ldl and 7-ketocholesterol — Their Potential Role in Destabilizing the Gastric Epithelial Cell Barrier. an in Vitro Model of Kato Iii Cells*

Colonization of gastric tissue in humans by H. pylori Gram-negative bacteria initiates gastric and duodenal ulcers and even gastric cancers. Infections promote inflammation and damage to gastric epithelium which might be followed by the impairment of its barrier function. The role of H. pylori components in these processes has not been specified. H. pylori cytotoxicity may potentially increase in the milieu of anti-inflammatory drugs including acetylsalicylic acid (ASA). The lipid transport-associated molecule such as low density lipoprotein (LDL), which is a classic risk factor of coronary heart disease (CHD) and 7-ketocholesterol (7-kCh) a product of cholesterol oxidation, which may occur during the oxidative stress in LDL could also be considered as pro-inflamma-tory. The aim of this study was to evaluate the cytotox-icity of H. pylori antigens, ASA, LDL and 7-kCh towards Kato III gastric epithelial cells, on the basis of the cell ability to reduce tetrazolium salt (MTT) and morphology of cell nuclei assessed by 4',6-diamidino-2-phenylindole (DAPI) staining. Kato III cells were stimulated for 24 h, at 37 o C and 5% CO 2 , with H. pylori antigens: cytotoxin associated gene A (CagA) protein, the urease A subunit (UreA), lipopolysaccharide (LPS) and ASA, LDL or 7-kCh. H. pylori LPS, ASA, LDL and 7-kCh, but not H. pylori gly-cine acid extract (GE), demonstrated cytotoxicity against Kato III cells, which was related to a diminished percentage of MTT reducing cells and to an increased cell population with the signs of DNA damage. The results suggest that damage to gastric epithelial cells can be induced independently by H. pylori antigens, ASA and en-dogenous lipid transport-associated molecules. During H. pylori infection in vivo, especially in CHD patients, syn-ergistic or antagonistic interactions between these factors might possibly influence the disease course. Further study is necessary to explain these potential effects.


INTRODUCTION
Helicobacter pylori Gram-negative bacteria have a high affinity to gastric epithelium.In humans, chronic colonization of the gastric tissue by H. pylori may be associated with both, hypochlorhydria and hyperchlorhydria, depending on the distribution of inflammation, and thus initiates gastric and duodenal ulcers and even gastric cancers (Neuman & Crabtree 2004;Derakhshan et al., 2006).H. pylori infections promote an excessive inflammatory response which is deleterious to gastric epithelial cells (Israel & Peek, 2001;Chmiela & Michetti, 2006).Changes occurring in the gastric epithelium colonized by H. pylori may promote the penetration of H. pylori antigens into the basal membrane, where they interact with extracellular matrix proteins (ECM) (Valkonen et al., 1994).This may be followed by an impairment of the gastric barrier function.H. pylori bacteria also interact with and activate the infiltrating immune cells via Pathogen Recognition Receptors (PRR) (Dubreuil et al., 2002;Wessler & Backert, 2008).The role of individual H. pylori compounds in these processes has not been fully specified.Urease is a major H. pylori virulence factor, which increases gastric pH and may be involved in degradation of intracellular tight junctions (Posselt et al., 2013).Cytotoxin associated gene A protein (CagA) is the most extensively studied virulence factor of H. pylori.The cagA is located at the cag pathogenicity island (PAI) encoding a type IV secretion system (T4SS), through which CagA is delivered into host cells.A higher risk of gastric ulcers and stomach cancer is related to infections caused by CagA producing, rather than CagA non-producing, H. pylori strains (Yamaoka, 2010).H. pylori CagA positive strains have functional vacA, which encodes a vacuolating cytotoxin A (VacA).In addition to vacuolation, VacA can induce multiple cellular activities, including membrane channel formation, cytochrome c release from mitochondria leading to apoptosis, and binding to cell-membrane receptors, which is followed by the initiation of proinflammatory response (Yamaoka, 2012).In addition, VacA can modulate antigen presentation by B lymphocytes and inhibit T-cell activation as well as proliferation (Gebert et al., 2003).During natural infection in vivo this toxin may possibly modulate the influence of CagA on gastric epithelial barrier.
H. pylori lipopolysaccharide (LPS) is an important proinflammatory compound.However, its lipid A has probably evolved in the mode which promotes persistence of the infection by regulating the expression of adhesins and modulating the secretion of cytokines by host immune cells as well as their phagocytic, proliferative and cytotoxic activities (Taylor et al., 2006;Grebowska et al., 2008;Grebowska et al., 2010;Rudnicka et al., 2015).
H. pylori cytotoxicity may potentially increase in the milieu of anti-inflammatory drugs such as acetylsalicylic acid (ASA), which is widely used to reduce fever, pains and inflammation.In addition, ASA is preventively used as an anticoagulant in patients with coronary heart disease (CHD) (Baigent et al., 2009).The use of aspirin increases a risk of adverse reactions in the gastrointestinal tract such as erosions and ulcers due to irritation, impaired blood flow, accumulation of neutrophils and inhibition of prostaglandin synthesis (Perini et al., 2004;Vella, 2005).However, literature about interactions between H. pylori infections and a low dose of ASA is scarce and controversial (Sosters et al., 2014, Buzas 2014).
The role of lipid transport-associated molecules, including low density lipoprotein (LDL), which is a classic risk factor for CHD in the development of gastric disorders is a new approach.It has been shown that in the gastric epithelium, there might be present specific structures called lipid islands.The precise mechanism of their formation is not clear.It is hypothesized that the lipid islands might be formed by the accumulation of cholesterol, LDL and oxidized LDL (ox-LDL) in the gastric epithelium damaged due to inflammatory or degenerative processes (Kaiserling et al., 1996).It has been suggested that rather than being eliminated via reverse cholesterol transport machinery using LDL receptor, ox-LDL is recognized and captured by scavenger receptors abundant in peripheral tissues.In vascular cells, internalization of ox-LDL has been shown to trigger signaling events resulting in overproduction of reactive oxygen species, inflammation and proliferation (Khaidakov & Mehta, 2012).Various epithelial cells have been also shown to express ox-LDL specific scavenger receptors such as CD36 molecule (Wadsack et al., 2003;Susztak et al., 2005).CD36 is a transmembrane protein of class B scavenger receptor family and it is involved in multiple biological processes: angiogenesis, atherosclerosis, inflammation, lipid metabolism (Febbraio et al., 2001).
Gastric damage is observed in patients with chronic diseases (gastritis, peptic ulcer disease, and also after partial gastrectomy) (Chandan et al., 2004).It has been shown that infection and inflammation induce LDL oxidation and it is known that oxidative modification of lipoproteins plays a major role in atherosclerosis (Memon et al., 2000).The association between H. pylori infection and the serum lipid, lipoproteins, apolipoprotein A1 and apolipoprotein B in patients with gastritis has also been indicated (Memon et al., 2000;Kucukazman et al., 2008;Ansari et al., 2010;Kim HL et al., 2011).By the ability to create an atherogenic lipid profile and insulin resistance, H. pylori has been considered as an infectious agent promoting atherosclerosis, type 2 diabetes mellitus and a metabolic syndrome.However, the epidemiologic link between H. pylori infection and these syndromes is inconsistent and controversial (Buzas, 2014).While H. pylori does not enter blood circulation, these extragastric manifestations are probably mediated by the cytokines and acute phase proteins produced by the inflammatory mucosa (Lamb & Chen, 2013).However, it could not be ruled out that gastric epithelial damage due to H. pylori infection may be enhanced by the effect of drugs and some endogenous factors occurring in circulation.Their effects on the gastric epithelium may manifest in the situation of erosions and ulcers or as a consequence of activation of immune cells infiltrating the gastric mucosa.
Considering various effects of infectious agents that chronically colonize the gastric epithelium, as well as pharmacological agents and lipid transport-associated molecules on the gastric barrier function, in this study we evaluated the cytotoxic effects of selected and well defined H. pylori antigens, as well as ASA, LDL and 7-ketocholesterol (7-kCh) using an in vitro model of Kato III gastric epithelial cells.The ability of the cells to reduce tetrazolium salt (MTT) and morphology of cell nuclei assessed by 4',6-diamidino-2-fenyloindol (DAPI) staining were selected as cytotoxicity markers.We expect that this study will demonstrate the properties of individual compounds used as cell stimulators and will enable to determine the conditions to explore their putative synergistic or antagonistic effects on the integrity and function of gastric epithelial cells.Simultaneous evaluation of these effects is a new approach, which will help to better understand the relationship between H. pylori infection and CHD.

H. pylori culture conditions.
A reference Helicobacter pylori strain CCUG 17874 (Culture Collection, University of Gothenburg, Gothenburg, Sweden), VacA and CagA positive, was used in this study.H. pylori bacteria were stored at -80°C in Tris-buffered saline (TBS) containing 10% glycerol.Before being used in the experiments, H. pylori bacteria were grown for 5 days on modified Helicobacter agar (Becton Dickinson, Heidelberg, Germany) under microaerophilic conditions (Gas Pak, Becton Dickinson, Heidelberg, Germany), at 37°C.Bacteria were harvested by scraping from agar plates, suspended in 0.85% sodium chloride (NaCl), pelleted by centrifugation (4 000 × g, for 15 min), and then washed twice under the same conditions.Bacterial pellet was used for the preparation of antigens.
Bacterial stimulators.Surface H. pylori antigens present in a glycine acid extract (GE) were extracted from the reference H. pylori strain CCUG 17874 using 0.2 M glycine buffer, pH 2.2, as previously described (Logan & Trust 1983, Rechciński et al., 1997), with evaluation of protein composition by SDS-PAGE and a Western blot -Immuno blot (Milenia ® Blot H. pylori, DPC Biermann, GmbH, Bad Nanheim, Germany) performed with the reference serum samples from patients infected with H. pylori (Czkwianianc et al., 1997).Major proteins in GE recognized by sera from H. pylori positive patients were 120 kDa, 80 kDa, between 66-42 kDa and 29-26 kDa.The protein content in GE was 600 µg/ml (NanoDrop 2000c Spectrophotometer, ThermoScientific, Wlatman, WY, USA).The GE preparation contained < 0.001 EU ml of LPS as shown by the chromogenic Limulus amebocyte lysate test (Lonza, Braine-Alleud, Belgium).The H. acynonychis UreA subunit was amplified by a polymerase chain reaction (PCR) as previously described (Hinc The influence of H. pylori, lipid transport-associated molecules and ASA on gastric epithelial barrier et al., 2010), and used as a homologue of H. pylori UreA protein.Recombinant CagA protein (rCagA) was from IRIS, Siena, Italy.LPS from the reference strain of H. pylori CCUG 17874 (courtesy of AP.Moran) was prepared by hot phenol-water extraction after pre-treatment of the bacterial biomass with a protease.Then, the crude LPS preparation was purified by RNAse, DNAse and proteinase K treatment and by ultracentrifugation, as previously described (Moran et al., 1992).The E. coli LPS derived from the O55:B5 strain (Sigma, St. Louis, MI, USA) was used as control.
Culture and stimulation conditions of Kato III cells.Human gastric epithelial cells Kato III, ATCC ® HTB TM (American Type Cell Culture Collection, Rockville, MD, USA) were used in this study.The cells were cultured in RPMI-1640 medium containing 10% fetal calf serum, l-glutamine and standard antibiotics, penicillin/streptomycin, at a concentration of 1 × 10 6 cells ml in tissue culture flasks up to 90% of confluence.The cells were detached from the tissue culture flasks by 0.25% trypsin solution in ethylenediaminetetraacetic acid (EDTA), 10 min, 37°C, and then washed twice with a culture medium by centrifugation (10 min, 300 × g) and evaluated for cell viability by trypan blue staining.For experiments, a cell suspension containing 1×10 6 cells/ml was distributed into the wells of 96-well culture plates (100 µl/well) and attached for 24 h, at 37°C and 5% CO 2 .The cells were stimulated for 24 h with H. pylori stimulators used at a concentration adjusted in the previous cellular experiments: GE (10 µg/ml), UreA (10 µg/ ml), CagA (1 µg/ml), H. pylori LPS (1 ng/ml, 25 ng/ ml) and E. coli LPS as control (at the same concentrations) as well as ASA (1, 2.5 and 5 mM), LDL (5 and 50 µg/ml) and 7-kCh (2.5 and 20 µg/ml) (Sigma, St Louis, Michigan, US).The primary solution of LDL in 0.85% NaCl/1%EDTA was diluted in RPMI-1640.This solvent was not cytotoxic as shown in a previous study.The stock solution of lyophilized 7-kCh (1mg/ml) was prepared in 96% ethanol and then diluted in a culture medium.The cells were incubated in the milieu of stimulators for 24 h, at 37°C and 5% CO 2 .Then, the wells were emptied and filled with the culture medium without stimulators.The cytotoxic effects were assessed after 24, 48 and 72 h of a continued cell culture and assessed in comparison to control, i.e. untreated cells.
MTT reduction assay.The cytotoxic activity of stimulators was estimated on the basis of the live target cells' ability to reduce MTT, using the TACS TM MTT Cell Proliferation Assay (R&D Systems, USA), as recommended by the manufacturer.The intensity of MTT reduction was estimated spectrophotometrically at 570 nm.The correlation between the number of viable target cells and the absorbance intensity was used for the standard curve construction.The level of cytotoxicity was expressed as downregulation of absorbance values and a percentage of dead target cells.
Cell nuclei staining by DAPI.Cell nuclei damage was considered as a sign of apoptosis, which was determined by staining the cells 24 h after the challenge with 4',6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MI, USA), a fluorescent dye, which has a strong affinity to the AT base pairs in DNA.The cells were fixed with 4% formaldehyde, and stained with a DAPI solution (2.5 µg/ml) for 15 minutes, at room temperature.The samples were viewed under a fluorescent microscope at 358 nm (excitation) and 461 nm (emission).A percentage of cells with damaged nucleus was assessed.
Statistical analysis.The Statistica 10 PL program with non-parametric tests was used: the Mann-Whitney U test (for impaired data) to verify the hypothesis that two samples being compared came from two statistically different populations.

Cytotoxic effects of H. pylori antigens, ASA, LDL and 7-kCh towards Kato III cells assessed by MTT reduction
Our study demonstrated the cytotoxic effect of H. pylori CagA, UreA and LPS on Kato III cells.These antigens diminished the number of cells reducing MTT in the cell cultures 24 h, 48 h and 72 h after antigen challenge (Fig. 1).CagA significantly reduced the percentage of viable cells by 21.6%, 27.6% and 16.37%, respectively, and UreA by 23.3%, 10.4% and 11.2%, respectively.The differences for UreA 48 h and 72 h after challenge were significant.Among H. pylori antigens used in this study, the LPS of H. pylori at a concentration of 25 ng ml caused the greatest loss of MTT reducing cells.The percentages of cells which did not reduce MTT after the challenge with H. pylori LPS at this concentration, were 88.5%, 74.3% and 78.5% in 24 h, 48 h and 72 h cultures, respectively.By comparison, the percentages of MTT non reducing cells in the cultures treated with E. coli LPS at a concentration of 25 ng/ml at 24 h, 48 h and 72 h before the evaluation were 14.9%, 64.2%, 62.5%, respectively.E. coli LPS at a lower concentration (1 ng/ml) showed cytotoxic effect only 24 h after the challenge.In contrast, the cytotoxic effect of a lower dose of H. pylori LPS was shown after 24 h and persisted after 48 and 72 h.The mixture of H. pylori antigens in GE did not decrease the viability of Kato III cells as assessed by MTT reduction.
The cytotoxic effect of ASA used at concentrations of 1, 2.5 and 5 mM towards Kato III cells was dose dependent.The strongest impact was observed in the cell cultures treated with 5 mM ASA 24 h, 48 h and 72 h before the assessment (Fig. 2).In these cell cultures, 88.9%, 75.9% and 85.6% of the primary cell population, respectively, did not reduce MTT.The cytotoxic effect of ASA at a concentration of 1 mM or 2.5 mM was weaker as compared to 5 mM ASA, but the percentage of dead cells was significantly higher than in the control culture in RPMI-1640 culture medium alone.Also, 7-kCh and LDL downregulated the ability of Kato III cells to reduce MTT (Fig. 3).The effect of 7-kCh was time and dose dependent.In the cell cultures 24 h after the challenge with 2.5 µg/ml of 7-KCh, the number of inactive cells was equal to 49.2% and was significantly lower than in the cultures treated with the 7-kCh solvent alone.By comparison, in 48 h and 72 h cell cultures treated with 2.5 µg/ml of 7-kCh, the percentages of dead cells were 16.9% and 15.0%, respectively.
Both, lower (5 µg/ml) and higher (50 µg/ml) doses of LDL affected the metabolic activity of Kato III cells to a similar degree: 58.4% and 56.3%, 24 h after challenge; 18.5% and 19.9%,48 h after challenge; 25.6% and 27.6%, 72 h after challenge, respectively.With regard to the control cultures, the differences were statistically significant.

Genetic material damage in Kato III cells treated with H. pylori antigens, ASA, LDL or 7-kCh visualized by DAPI staining
Figure 4 shows the percentage of Kato III cells with damaged genetic material in cultures treated 24 h before assessment with H. pylori antigens, ASA, LDL and 7-kCh.Figure 5 presents the representative images of the cells exposed for 24 h to H. pylori antigens, LDL, 7-kCh or ASA, stained by DAPI.In the cultures treated with CagA, UreA, and GE, the percentage of cells with damaged nuclei was above 20%, whereas in the control cell cultures in RPMI-1640 culture medium alone it was 8.67%.The diminished number of viable cells after treatment with 1 ng/ml and 25 ng/ml H. pylori LPS was correlated with the increased number of cells with the signs of DNA destruction.The percentage of the cells with damaged DNA was equal to 30.67% and 89.33%, respectively.E. coli LPS also showed genotoxic activity, but it was lower than that of the LPS of H. pylori.Genotoxic properties of ASA detected by DAPI staining were in the range of 46.67% and 59.33% of the cells with disrupted DNA.The strongest genotoxic effect (over 90%) in the cells with damaged DNA was demonstrated in the cultures treated with 20 µg/ml 7-kCh.It was reduced up to 40% in the cultures treated with 2.5 µg/ml 7-kCh.In the cell cultures exposed to 5 µg/ml or 50 µg/ml LDL, the cell nuclei were affected in 25% and 41% of the cells.

DISCUSSION
H. pylori infection and nonsteroidal anti-inflammatory drugs (NSAID)/low-dose aspirin use are associated with a peptic ulcer disease (Sosters et al., 2015).The results of many studies indicate that long-term infections with H. pylori may contribute to the development of CHD (Mendall et al. 1994;Longo-Mbenza et al., 2012;Ayada et al., 2009).CHD pa-   et al., 2006).Given that H. pylori infections remain the most common chronic bacterial infections worldwide, a synergistic or additive effect of H. pylori and ASA in peptic ulcer development has been suggested (Sosters et al., 2014;2015).This correlation has been considered to be of great clinical importance since eradication of the bacterium would reduce the risk of upper gastrointestinal complications in infected ASA-treated patients (Cekin et al., 2012).It can be assumed that changes in the gastric epithelium induced by a H. pylori infection may be intensified by ASA.On the other hand, its toxicity may predispose to H. pylori infections and accumulation of infection related deleterious effects.Persistent epithelial cell defects can promote deeper penetration of H. pylori antigens, where they can interact with the immune cells and even enter the circulation, provoking the development of a systemic inflammatory response.
In view of the chronic nature of H. pylori infection and its putative association with the development or maintenance of the CHD symptom, in this study we focused on the influence of H. pylori antigens and ASA on the gastric epithelial cells using an in vitro cell culture model of Kato III cells.
Our in vitro study demonstrated that H. pylori CagA, UreA and LPS were cytotoxic to Kato III cells.The cytotoxicity of these stimulators was related to a diminished percentage of MTT reducing cells and to an increased number of cells with DNA damage, as visualized by DAPI staining.Various mechanisms can be involved in the cell cytotoxicity.DNA damage often reflects cell apoptosis.Apoptosis induced by H. pylori in vivo may  stimulate an excessive hyperproliferative response.Increased cell apoptosis has been observed in gastric tissues isolated from patients infected with H. pylori as well as in guinea pigs infected experimentally with these bacteria (O'Connor et al., 1997;Miszczyk et al., 2014).Alternatively, apoptosis may be viewed as the response to hyperproliferation in an attempt to reduce tissue growth (Neuman and Crabtree, 2004).Whether apoptosis is the primary or secondary event is not clear.The induction of excessive apoptosis by H. pylori could be related to secondary hyperproliferative response in order to maintain cellular mass.Once hyperproliferation is established, the increased rate of cell cycling might predispose gastric epithelial cells to genotoxic damage.It is possible that gastric epithelial cells exposed to carcinogenic H. pylori bacteria are more sensitive to apoptosis.In our previous study we demonstrated using the in vitro model of epithelial HeLa cells that exposition of these cells to H. pylori antigens was related to cell cycle arrest in the S (GE) or G2/M (CagA and LPS) phase (Miszczyk et al., 2014a).Some experimental data indicate that individual H. pylori components may promote their effects in vivo.Previously Wessler and Backert (2008) showed that soluble factors of H. pylori, such as urease and VacA, can open the tight and adherens junctions allowing direct contact between CagA ligand and beta1 -integrin in the basolateral membrane of epithelial cells (Wessler and Backert, 2008).At the apical side of polarized cells, CagA might translocate in a soluble form via phosphatidylserine and cholesterol.Phosphorylated CagA dysregulates signal transduction pathways, leading to alterations in gene expression, and interferes with the cytoskeletal rearrangement, which is important for the mitogenic response to H. pylori antigens.Another H. pylori compound, i.e. peptidoglycan, binds Nod1 and activates NF-κB signaling (Viala et al., 2004;Backert et al., 2011).Local H. pylori induced inflammation in gastric epithelium might be reflected on the periphery by systemic inflammatory markers, which may enhance the development of inflammatory lesions in vascular endothelium.Also, it cannot be excluded that certain H. pylori components crossing the epithelial barrier in the stomach or intestine could have a direct influence on the vascular endothelial cells, as well as circulating immune cells, maintaining their constant activation.
Among H. pylori antigens, LPS showed the strongest cytotoxic and genotoxic properties towards Kato III cells.In a previous study we have shown that H. pylori LPS affected the cell cycle of HeLa cells and guinea pig fibroblasts (Miszczyk et al., 2014a), and downregulated the cytotoxic activity of NK cells and proliferation of human and guinea pig T lymphocytes (Grebowska et al., 2008;Grebowska et al., 2010;Rudnicka et al., 2015;Miszczyk et al., 2014;Chmiela et al., 2014).It is possible that in vivo domination of antigens negatively modulating the growth of epithelial cells and the activity of immune cells, comprising the first line of an immune response, promotes the development of a chronic infection.In this study, GE showed no cytotoxic and genotoxic effects against Kato III cells.This might be due to the low concentration of individual antigens in GE preparation, which is composed of surface antigens that may probably modulate the effects of each other.Several surface antigens have been reported for H. pylori: 120 kDa, 110 kDa, 94 kDa, 83 kDa, 63 kDa, 61 kDa, 28 kDa, and multiple determinant antigenic cell fragments have been found throughout the H. pylori outer membrane proteins and flagellar structures (Lelwala-Guruge et al., 1992).Major proteins in GE, recognized by sera from H. pylori positive patients, were 120 kDa, 80 kDa, between 66-42 kDa and 29-26 kDa.It is worth mentioning that many H. pylori surface components are lectin-like molecules, which play a role of adhesins and modulate both, proliferative and cytokine activities of the immune cells, without any cytotoxic effects (Chmiela et al., 1996;Hynes et al., 2003;Walz et al., 2009).
The intriguing question is how H. pylori and low dose ASA exert the gastric damage.For instance, different phenotypes of H. pylori may induce various types of gastritis and gastric damage in low dose ASA users.Furthermore, there are various possibilities how low dose ASA damages the gastric mucosa, such as enhanced exposure of gastric epithelial cells to luminal acid, reduction of mucosal blood flow, increase of apoptosis of epithelial cells and recruitment of polymorphonuclear cells, generating the conditions of oxidative stress.Moreover, H. pylori may also further increase local injury by impairing the gastric adaptation to ASA (Sostres et al., 2014).Several clinical studies proved the benefit of H. pylori eradication in ASA users (Lim & Hong, 2014).However, the exact pathophysiological relationship between a H. pylori infection and ASA use has not yet been fully elucidated.Probably, through different mechanisms both, H. pylori components and ASA, may interact in a synergistic or antagonistic manner.
In this study, cytotoxic and genotoxic effect of LDL and 7-kCh, which is a product of cholesterol oxidation, was demonstrated on Kato III cells.During H. pylori infection, the conditions of oxidative stress are exacerbated because the gastric mucosa is extensively infiltrated by polymorphonuclear cells, mainly neutrophils and eosinophils.These cells provide soluble inflammatory mediators that aggravate the influx of other immune cells, resulting in gastric epithelial cell damage.Granulocytes, which are excessively activated and undergo degranulation, provide conditions of oxidative stress due to a release of reactive oxygen species (ROS).They can also secrete excessively proteolytic enzymes.ROS affect the expression of proand anti-apoptotic proteins and represent a major cause of cellular damage and death due to lipid peroxidation.ROS are also responsible for oxidative DNA damage (Yoshikawa & Naito, 2002).Previously, a significant association between H. pylori infections and high LDL-cholesterolemia, low HDL-cholesterolemia and elevated levels of C-reactive protein was shown, indicating a potential impact of a chronic infection on lipid metabolism.By modulation of lipid metabolism, H. pylori can create an atherogenic lipid profile promoting atherosclerosis (Laurilla et al., 1999;Ansari et al., 2010;Buzas, 2014).It was also revealed that H. pylori VacA could specifically utilize the low-density lipoprotein receptor-related protein-1 (LRP1), acting as a receptor which promotes cell autophagy and apoptosis (Cover and Blanke, 2005;Yahiro et al., 2003Yahiro et al., , 2012)).Laurilla et al (2001), using LDL receptor negative (LDLR-/-) mice as model for H. pylori infection, provided for the first time evidence that a standard high-fat, high-cholesterol diet used for atherosclerosis induction is associated with a high incidence of gastritis, which is independent on infection.In these animals, NF-κB, a common signaling factor, was upregulated in both, coronary vascular endothelium and gastric mucosa, in response to high level of cholesterol (Laurilla et al. 2001).These studies indicate that the role of endogenous factors, such as LDL or 7-kCh, in the development of local inflammation in the stomach is possible.On the other hand, our results allow to suggest that epithelial cell damage due to these factors can be involved in the initiation or maintenance of a pre-existing inflammatory response.Whether the cells are damaged directly or indirectly by inflammato-The influence of H. pylori, lipid transport-associated molecules and ASA on gastric epithelial barrier ry mediators secreted by stimulated cells should be addressed in a further study.
In conclusion, the results obtained in this in vitro cellular study, using the Kato III cell line, suggest that in vivo damage to the gastric epithelial cells due to H. pylori infection can be modulated by LDL and 7-kCh, as well as pharmacological agents, such as ASA.The limitation of this study is that the findings do not explain the molecular mechanism of cell damage.However, diminished metabolic activity of the cells and DNA damage indicate that these factors may contribute in vivo to impairment of gastric epithelial barrier function, diminishing of wound healing and upregulation of inflammatory response, with a potentially stronger pathological effects both local and systemic.Further studies are necessary to explore the nature of these processes more deeply.

Figure 1 .
Figure 1.Cytotoxic effects of H. pylori antigens towards Kato III cells assessed by the MTT reduction assay.Human gastric epithelial cells Kato III were stimulated for 24 h with H. pylori antigens: glycine acid extract (GE), subunit A of urease (UreA), cytotoxin associated gene A antigen (CagA), H. pylori lipopolysaccharide (LPS) or control E. coli LPS.Then, stimulators were removed and the cytotoxic effects were assessed after 24 h (a), 48 h (b) and 72 h (c) of continued culture of the cells on the basis of cells' ability to reduce tetrazolium salt (MTT).Statistical significance: *p<0.05;**p<0.001.

Figure 2 .
Figure 2. Cytotoxic effects of acetylsalicylic acid (ASA) towards Kato III cells assessed by MTT reduction assay.Human gastric epithelial cells Kato III were incubated in the milieu of various ASA concentrations for 24 h.Then, stimulators were removed and the cytotoxic effects were assessed after 24 h (a), 48 h (b) and 72 h (c) of continued cell culture on the basis of cells' ability to reduce tetrazolium salt (MTT).Statistical significance: *p<0.05;**p<0.001.

Figure 3 .
Figure 3. Cytotoxic effects of low density lipoprotein (LDL) and 7-ketocholesterol (7-kCh) towards Kato III cells assessed by MTT reduction assay.Human gastric epithelial cells Kato III were stimulated for 24 h with different 7-kCh and LDL concentrations.Then, stimulators were removed and the cytotoxic effects were assessed after 24 h (a), 48 h (b) and 72 h (c) of continued culture of the cells on the basis of cells' ability to reduce tetrazolium salt (MTT).Statistical significance: *p<0.05;**p<0.001.