Direct Action of Helicobacter pylori on the Freshly Isolated Rat Gastric Mucosa Cells (GMCs)

mU/min/10

The aim of this chapter is to give a summary of the observations that are not in agreement with the main line of H. pylori infection and the necessity of its eradication treatment (Mózsik et al., 2014 a, b, c, d).

Testing of Helicobacter pylori (H. pylori) cultures on the isolated gastric mucosal cell
As the letters of Warren and Marshall in Lancet (1983) were published, a very active research chain was started throughout the world. Researchers tried to find the explanation on how H. pylori can lead to inflammation in the stomach and peptic ulcer.
The existence of H. pylori as the cause for direct damaging effect in the stomach was one of the possibilities. To answer the question, we analyzed the direct cellular effect of sonicated H. pylori cultured (at the level of membrane, mitochondrion and DNA) alone and with different combinations on the freshly isolated gastric mucosal cells of rat. The aggravation effect of this sonicated H. pylori was also examined in ethanol-induced cell injury model to evaluate the changes in cell resistance.

H. pyloric culture
Bacteria were obtained from human gastric and duodenal biopsy samples. Urease test and histological positivity were both required to be definite in the tissue samples. Bacterial suspensions (10 6 bacteria/mL in 20 Tris/HCl buffer, pH 7.0) were sonic on ice (30 W) in six consecutive treatments lasting 30 seconds.

Preparation of mixed Gastric Mucosal Cells (GMCs)
Gastric mucosal cells from 1 or 2 unfasted Sprague-Dawley strain rats were isolated by the method of Nagy et al. (1994). Briefly, the segments of glandular stomach without blood vessels surrounding the connective tissue were sequentially incubated in a physiological solution containing 0.5 mg/mL Pronase E (Type XXV Sigma Chemical Co.) and 10 -3 EGTA. After washing for several times, the cells were resuspended and kept in shaking water bath at a temperature of 37°C in a solution (0.157 M, pH 7.4) freshly produced with the following ingredients: 98.0 mM NaCl, 5.8 mM KCl, 2.5 mM NaH 2 PO 4 , 5.1 mM Na pyruvate, 6.9 mM Na fumarate, 2 mM glutamine, 24.5 mM HEPES Na, 1.0 mM Trizma base, 11.1 mM D-glucose, 1.0 mM CaCl 2 , 1.0 mM MgCl 2 and 2 mg/mL (w/v) bovine serum albumin. Mixed population of isolated rat GMCs contained at least three types of cells: parietal (20-25%), chief (40%) and epithelial cells (40-45%). An initial viability of 85-95% of the isolated cells was maintained for 6-7 hours. The examinations were carried out through the same media of solution.

H. pylori culture
Cells were incubated with sonicated H. pylori (10 6 -10 8 bacteria /mL) for 30 minutes in the shaking water bath at 37°C. Equivalent volume of both diluted toxic agent and cell suspension were added. At the end of 30 minutes of incubation, the cells were separated from the supernatant by centrifugation. Cell pellet was carefully resuspended and incubated for 10 minutes in the shaking water bath, before it was centrifuged again (500 g, 10 minutes) to obtain the toxic-agent-free supernatant. Cells were resuspended for further biochemical examinations.

Ethanol
Pretreated and washed cells of sonicated H. pylori were incubated with 15% ethanol (EtOH) for 5 minutes in shaking water bath at 37°C after the cells were treated as described above.

Indomethacin
Pretreated and washed cells were incubated with 10 -8 -10 -4 M indomethacin (IND) (Chinoin, Hungary) for 5 minutes in shaking water bath at 37°C. Then the cells were treated as described above.

Determination of cell viability by Trypan Blue exclusion test
Trypan Blue (TB) is excluded by viable cells, while being taken up by damaged cells, staining the cytoplasm blue (Bauer et al., 1972). A solution of 0.4% TB (Sigma Chemical Co., St Louis, USA) was mixed with the same volume of cell suspension, and 5 minutes later the rate of stained (dead) and unsustained (viable) cells were calculated as percentage of counting 100 cells in a hemocytometer.

Lactate dehydrogenase assay
The enzyme, lactate dehydrogenase (LDH) can be found in the cytoplasm of the cells. If it is present in the supernatant, it indicates membrane damage of cells. Its activity was determined in samples of both toxic-agent-free supernatant and the cell pellet destroyed by freezing. The calorimetric assay was based on the reduction of NAD + to NADH, catalyzed by LDH in the presence of lactate as a substrate (Bergmayer and Bern, 1972). The color produced by the reduction of phenazine methosulfate (Sigma Chemical Co., St. Louis, USA) and tetrazonium salt isinicotinic acid hydrazide (Sigma Chemical Co., St. Louis, USA) was measured at 520 nm by a Hitachi 124 spectrophotometer. The results were expressed as mU/min/10 6 cells.

Succinic dehydrogenase assay
The enzyme succinate dehydrogenase (SDH) can be found in the mitochondria. The mitochondrial integrity was tested in 2×10 6 previously treated and redispersed cells (Mosmann, 1983). The callus formazon product was quantified using a Hitachi 124 spectrophotometer at 500 nm and calculated as nmol/min/2×10 6 cells.
When the IND and H. pylori (10 7 sonicated bacteria/mL) were applied in combination, the number of viable cells was not changed compared with IND treatments alone (Figure 283), and no changes were obtained by biochemical assays (Figures 284, 285).

Combined effect of EtOH and IND
Using these toxic agents, IND and all doses significantly aggravated the 15% EtOH-induced cell injury determined by Trypan blue exclusion (Bódis et al., 1995(Bódis et al., a, b, 1996(Bódis et al., , 1998. In these studies, the direct effects of sonicated H. pylori were examined under different experimental conditions (when it was given alone or in combination with EtOH and IND).  et.al., (1996) This model was suitable to eliminate the additional effect of immune systems, which is usually involved in a bacterial infection. We found that sonicated H. pylori had no direct cellular toxicity.
These types of observations with freshly isolated gastric mucosal cells from rat stomach were widely used to study the direct actions of various aggressive and protective compounds under experimental conditions.