Model organisms and conditions
C57BL/6 mice of 4 weeks of age without certain pathogens are purchased 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.
Configuration Of Test Group And Set Of Dosage Setting
Dosage was set by MFDs standards. Maximum dosage is set to 2,000 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. 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 hours 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.
Autopsy And Naked Eye Examination
After administration going up to 14 days, the body weight of the surviving mice is measured, before anesthesia with CO2 and autopsy. External findings such as abnormality of subcutaneous, internal organs and brain were observed with the naked eye. The brain, kidney, liver, lung, reproductive organ, heart, spleen, and thymus are extracted and weighed.
The hematologic analysis of the serum is performed the same day of the autopsy, which is collected from a 3,000 rpm, 20 minutes 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.
Mice fasted for 8 hours before the autopsy are slightly anesthetized with CO2. 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.
Liver and kidney were extracted and fixed with a 10% neutral buffered formalin solution the day of final autopsy, 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.
Helicobacter Pylori Preparation
Helicobacter pylori (ATCC 43504) used in this study were obtained and inoculated onto chocolate media, incubated for 5 ~ 7 days at 37oC in a 10% CO2 incubator under aerobic conditions and then used for the examination. When the chocolate media is filled over 90%, Helicobacter pylori is swabbed with sterilized swabs and suspended in 20 ml of RPMI-1640 media to form the Helicobacter pylori suspension.
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℃ in a 5% CO2 incubator.
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 and Pediococcus acidilactici J9 (200ug/ml). The culture plates seeded with AGS treated by H. pylori and Pediococcus acidilactici J9 are incubated for 90 minutes at 37oC in a 5% CO2 incubator. The culture media is eliminated and the cells are carefully harvested. The cells are suspended in 500 ul of PBS then examined with FACS.
All values shown in the figures are presented as mean ± standard error. Western blot results were analyzed by Student's t-test. A 2-tailed probability value below 0.05 was considered statistically significant. Data were analyzed using SPSS version 17.0 (SPSS Inc., USA).