Model organisms and conditions
C57BL/6 mice of 4 weeks of age without certain pathogens are purchased from Orient Bio (Seongnam, Korea) at an amount of 20 males and females each. Normal and healthy mice without any weight loss are used in experiment by clinical observation during 7 days of education. Feeds are the following; solid feeds for laboratory animal are freely offered, and drinking water. The filtration-purified water is also freely offered to mice. The mice were house in groups with ad libitum access to food and water and a 12 h light / 12 h dark cycle. Also, P. acidilactici J9 has been prepared through Industry Promotion Administration.
Configuration of test group and set of dosage setting
Dosage was set by MFDS (Ministry of Food and Drug Safety) standards. Maximum dosage is set to 2000 mg/kg/day for both male and female, with the geometric ratio of 1/2, low dose group, medium dose group, and high dose group are set at 500, 1000, and 2000 mg per body weight (kg) respectively. 500, 1000, 2000 mg of dosage corresponds to 5 × 108 CFU, 1 × 109 CFU, and 2 × 109 CFU, however, we used the mg/ml unit which is more commonly used in the animal experiments. The number of mice in each group are set to 5 males and females each. Dosage is set to not exceed 0.2 ml per 10 g and calculated according to the body weight measured just before administration. Test materials are well-mixed to sterile distilled water before administration, and they are directly injected into the stomach by sonde for oral administration for once a day during 2 weeks. Sterile distilled water for injection is used as reference material.
Normal symptoms and observation of lethality in mice
Observation is conducted for 6 h after oral administration and starting from the next day, to observe change of general condition, as well as expression of addiction. This was held in presence of dead mice and symptoms that can be expressed by the test materials are observed carefully. In the case of abnormality, the type and the extent of symptoms are recorded individually. All mice were checked for death or critical condition.
Measurement of weight, feed and water intake
For every animal, change of weight is measured just before the administration of test materials once a week at a certain time during 2 weeks. Intake of feeds and water is measured and calculated weekly.
Necropsy and naked eye examination
Mice were anesthetized by CO2; the air atmosphere of chamber that contains mice was replaced to CO2 with the volume displacement rate of 20%/min, and all surgical procedures were performed under general anesthesia. Euthanasia of mice was done by collecting 0.5 ml – 0.8 ml of blood from the heart under anesthesia. The protocols were in accordance with official governmental guidelines, and all efforts were made to minimize the number of mice used and their suffering. Also, other organs was obtained by mice. The brain, kidney, liver, lung, reproductive organ, heart, spleen, and thymus are extracted and weighed. External findings such as abnormality of subcutaneous, internal organs and brain were observed with the naked eye.
Blood biochemical
The hematologic analysis of the serum is performed the same day of the necropsy, which is collected from a 3000 rpm, 20 min long, centrifugation of the blood and conducted by auto-analyzer (Hitachi-747, Hitachi Medical Co., Tokyo, Japan). Glucose; GLU, Blood Urea Nitrogen; BUN, Creatinine; CREA, Total cholesterol; T-CHOL, Albumin; ALB, Total Bilirubin; T-BIL, Alkaline Phosphatase; ALP, Aspartate Aminotransferase; AST (GOT), Alanine Aminotransferase; ALT (GPT), Triglyceride; TG, and Total protein; TP are measured.
Hematology
Mice were fasted for 8 h followed by the anesthetization before the necropsy. Part of the blood from the exsanguination is EDTA-treated and stored in tubes and then analyzed by blood auto-analyzer (System SE-9000, TOAMedical Electronics Co., Ltd., Kobe, Japan). Red blood cells, RBC, hematocrit, HCT, hemoglobin, Hb, mean corpuscular volume, MCV, mean corpuscular hemoglobin, MCH, mean corpuscular hemoglobin concentration, MCHC, white blood cells, WBC, Hemoglobin, HGB, Cellular Hb Concentration Mean, CHCM, Red Cell Distribution Width, RDW, Hb Distribution Width, HDW, Cellular Hb content, CH, Cellular Hb Distribution Width, CHDW, Platelet, PLT, Platelet Distribution Width, PDW, Plateletcrit, PCT, Neutrophil, NEUT, Neutrophil, NEUT%, Lymphocyte, LYMPH, Lymphocyte %, LYMPH%, Monocyte, MONO, Monocyte %, MONO%, Eosinophil, EOS, Eosinophil %, EOS%, Basophil, BASO, Basophil %, BASO%, Large Unstained Cells, LUC, Large Unstained Cells, LUC%, Reticulocyte Count, Retic#, Reticulocyte %, Retic%, Mean Corpuscular Volume of Retics, MCVr, Mean Corpuscular Volume of Retics %, MCVr%, Red Cell Distribution Width of Retics, RDWr*, Hb Distribution Width of Retics, HDWr*, Cellular Hb of Retics, CHr, and Cellular Hb Distribution Width of Retics, CHDWr* are measured.
Histopathology
Liver and kidney were extracted and fixed with a 10% neutral buffered formalin solution the day of final necropsy, after the observation of gross lesions on every animal which were administered with test materials. Then paraffin embedding wass conducted and hematoxylin & eosin dye performed with the sections of 3 ~ 4 um sections. Mijung Lee had validated the findings.
H. pylori preparation
H. pylori (ATCC 43504) used in this study were obtained and inoculated onto chocolate media, incubated for 5 ~ 7 days at 37 °C in a 10% CO2 incubator under aerobic conditions and then used for the examination. When the chocolate media is filled over 90%, H. pylori is swabbed with sterilized swabs and suspended in 20 ml of RPMI-1640 media to form the H. pylori suspension.
Cell culture
The human gastric adenocarcinoma cell lines AGS (KCLB 21739; Korea) cells were seeded at a density of 1 × 105 cells in 2 ml of RPMI-1640 (RPMI-1640; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Invitrogen, USA) into 6 well culture plates (SPL) and cultured for 2 ~ 3 days at 37 °C in a 5% CO2 incubator.
Adhesion assay
When the AGS cell reach a density of 80% of the seeding plate, we eliminate the media from the plate and wash with phosphate buffered saline (PBS: Welgene, Daegu, Korea) 3 times. Experimental groups are as follows. For negative control, only AGS is seeded. For positive control, AGS is treated by 1 ml of H. pylori suspension. For the measurement of suppression of attachment, AGS is treated by 1 ml of H. pylori suspension (1 × 108 CFU/ml) and P. acidilactici J9 (1 × 108 CFU/ml) at a multiplicity of infection of 100. The culture plates seeded with AGS treated by H. pylori and P. acidilactici J9 are incubated for 90 min at 37 °C in a 5% CO2 incubator. The culture media is eliminated and the cells are carefully harvested. The cells are suspended in 500ul of PBS then examined with FACS.
Flow cytometry
The culture media is eliminated and the cells were washed and carefully harvested in PBS (phosphate buffered saline, WelGene, Daegu, Korea) using a cell scraper. Cells were counted and 1 × 106 cells were suspended in 1 ml cold PBS. Cells (5 × 106 or 1 × 107) were centrifuged at 1200 rpm for 5 min. The cells are suspended in 500ul of PBS then flow cytometric analysis was performed (FACS Calibur, BD Bioscience, CA, USA). The data were analyzed using Flowing Software (www.flowingsoftware.com).
Statistical analysis
All values shown in the figures are presented as mean ± standard error. A 2-tailed probability value below 0.05 was considered statistically significant. Data were analyzed using SPSS version 17.0 (SPSS Inc., USA).