4.1 Preparation of the fucoidan
Using a specific ratio mixed the powder of Sargassum hemiphyllum (g) and ddH2O (mL), then heating to 100℃ for one hour. After cooling, adding the specific enzymes reacted for three hours in the shaking incubator, then heating to terminate the reaction of the enzymes. It filtered the extracts to remove the impurities and Centrifuged (5000 x g, 4 ℃,10 min) to collect the supernatant. Finally, fucoidan was acquired by freeze-drying.
4.2 Analytical method of polysaccharide
Molecular weight was measured by gel permeation chromatography (GPC) using the OHpak SB-804 HQ column (Shodex, Tokyo, Japan)25. Fucoidan was analyzed by the phenol-sulfuric acid method26 to identify the total sugar content. Sulfate content was determined by the turbidimetric method27; moreover, the protein content and total phenolic content were measured by Bradford assays28 and Folin–Ciocalteu method29, respectively. Monosaccharide analysis was determined by the naphthim-idazole (NAIM) saccharide labeling method30 and used a purchased polysaccharide component assay kit (SugarLighter Corporation, Taiwan).
4.3 Cell culture
AGS (BCRC 60102) cells were provided from National Yang-Ming University (Taipei, Taiwan) and cultured in the RPMI-1640 medium supplement with 10 % fetal bovine serum (FBS), which added the extra essential amino acid, and then cultured in the 37 ℃, 5 % CO2 atmosphere. RAW264.7 cells (ATCC TIB-71TM) were bought from ATCC (Manassas, USA) and cultured in the DMEM medium supplemented with 10 % FBS in the 37 ℃, 5% CO2 atmosphere.
4.4 The culture of H. pylori
H. pylori (BCRC15415) was bought from the Food Industry Research and Development Institute Bioresearch Collection and Research Center (Hsinchu, Taiwan). H. pylori was cultured under 37℃, microaerophilic for 3–4 days. Then, used 10 % FBS-BHIB and directly scraped the colony from the plate for collection, and then maintained it in TSA-blood plate.
4.5 MTS assay
Cytotoxicity of fucoidan was measured by MTS assay. Cells were incubated in 96 well plates and treated 100 µL different concentrations of fucoidan in a medium containing 2 % FBS in sequence at 37 ℃ for 24 hours. At due time, 20 µL the MTS reagent was added into 96 well plates, which incubated for 1 hour at 37 ℃. Absorbance was measured at 490 nm.
4.6 NO assay
Cultured RAW264.7 cells with 2 × 105 cells / well into 96 well plate. After the cells adhesion, added fucoidan with different concentrations into the well plate, and separately added 1 µg/mL LPS to induced cells producing nitrate for 24 hours. And then, transferred part of the supernatant to the other 96 well plate. After mixed with Griess reagent A and Griess reagent B reacting in the dark, measured the absorption under 550 nm.
4.7 The adhesion test of H. pylori
First, cultured AGS cells (1 × 105 cells/well) into 96-well plate overnight, removed the medium next day, and then washed the plate with PBS twice. After that, proceeded the experiences with three methods:
(1) Pre-treatment: First of all, added different concentrations of fucoidan to re-acted with AGS cells for 2 hours, after that washed with PBS triple times, then added H. pylori suspension to infected the AGS cells for 2 hours.
(2) Co-treatment: Cultured different concentrations of fucoidan and H. pylori suspension at the same time for 2 hours, then added the mixed medium into the cell, and then cultured for 2 hours.
(3) Post-treatment: Added H. pylori suspension to cause the infection for 2 hours at first. Next, washed PBS for triple times, then separately added the fucoidan in distinct concentrations, and then co-cultured for 2 hours.
The multiplicity of infection of three experiments was 100:1 (M.O.I = 100). All the treatment of H. pylori suspension and fucoidan was essentially washed with PBS triple times, then reacted in the urease broth for 4 hours, and the absorbance was measured under 560 nm. Calculated the ratio of the infection which was the cells infection by H. pylori.
4.8 The experimental design of the infection of H. pylori in vivo
The five-week-old BALB/c mice were purchased from National Laboratory Ani-mal Center (Taipei, Taiwan). All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of National Taiwan Ocean University (NTOU), and the study conformed to the guidelines of the protocol IACUC-101006 approved by the IACUC ethics committee of NTOU. This study was conducted in accordance with the ARRIVE guidelines. 24 mice were divided into four groups, which were respectively represented Control, Infected, Pre-treatment group (Pre-Fu), and Post-treatment group (Post-Fu), six mice for each group. Before proceeded the treatment, mice were acclimated to the new environment and the diet for one week. Before proceeded with the treatment, mice were acclimated to the new environment and the diet for one week. Mice were challenged by H. pylori (1 × 109 CFU/mL) on Day 0 and 2. In Pre-Fu group, before mice were challenged by H. pylori, treated 800 mg/kg/day fucoidan to the mice for one week, then continuously treated fucoidan until mice were sacrificed. On the other hand, after mice were challenged by H. pylori for one week, mice were treated 800 mg/kg/day fucoidan in Post-Fu group. Control and Infected group fed deionized water. At the end of experience, sacrificed the mice and took of the stomach under the aseptic environment.
4.9 Detection of H. pylori and cytokine RNA expression in AGS cells and mouse gastric tissues
According to the different situations of the experiment. After H. pylori infected cells and added fucoidan incubated for 6 hours. Then, washed the cells with PBS, collected the cell pellet to extract RNA. In vivo, trimmed the stomach tissue between 25 to 30 mg to extract the total RNA with RNeasy mini kit (QIAGEN, Germantown, USA). The RNA primers were 16s rRNA, BabA, AlpA, IL-8, IL-4, IL-5, IL-6, IL-1β, TNF-α, and IFN-γ, and the primer sequences were used: H. pylori 16s rRNA forward 5’-CGATGGATGCTAGTTGTTGGAG-3’, H. pylori 16s rRNA reverse: 5’- GTCCCCGTCTATTCCTTTGAGTT-3’, H. pylori BabA forward: 5’- GGTGGGGTTTT-GGAATGTCTTA-3’, H. pylori BabA reverse: 5’- AAAGAACAGGTGATGGAAGTG-GA-3’, H. pylori AlpA forward: 5’- GGTAGGCTCTGGGACTTGTG-3’, H. pylori AlpA reverse: 5’- TGGTGTTCGTGCCGTAGTTA-3’, AGS IL-8 forward: 5’- ACA-CYGCGCCAACACAGAAATTA-3’, AGS IL-8 reverse: 5’- TTTGCTTGAAGTTTCAC-TGGCATC-3’, AGS GAPDH forward: 5’- GCACCGTCAAGGCTGAGAAC-3’, AGS GAPDH reverse: 5’- TGGTGAAGACGCCAGTGGA-3’, mouse IL-4 forward: 5’- CCAAGGTGCTTCGCATATTT-3’, mouse IL-4 reverse: 5’- ATCGAAAAGCCCGAAA-GAGT-3’, mouse IL-5 forward: 5’- GTGGGGGTACTGTGGAAATG-3’, mouse IL-5 re-verse: 5’- ACCAAGGAACTCTTGCAGGT-3’, mouse IL-6 forward: 5’- TCCATCCAG-TTGCCTTCTTG-3’, mouse IL-6 reverse: 5’- TTTCTCATTTCCACGATTTCCC-3’, mouse IL-1β forward: 5’- CGCAGCAGCACATCAACAAGAGC-3’, mouse IL-1β reverse: 5’- TGTCCTCATCCTGGAAGGTCCACG-3’, mouse TNF-α forward: 5’- AGCCCCCAC-TCTGACCCCTTTAC-3’, mouse TNF-α reverse: 5’- TGTCCCAGCATCTTGTGTTTCT-3’, mouse GAPDH forward: 5’- TGCACCACCAACTGCTTAG-3’, mouse GAPDH reverse: 5’- GGATGCAGGGATGATGTTC-3’. RT-PCR was set at 95 ℃ initially for 30s, fol-lowed by 40 cycles of 95 ℃ for 15 s, 55 ℃ for 15 s, 72 ℃ for 45 s. the gene expression level in each sample was normalized using GAPDH.
4.10 Histopathological analysis
After the mice were sacrificed, the stomach tissue was sampled from each mouse. The samples were fixed in 10 % formalin for 24 h at room temperature. The fixed stomach tissue was trimmed into an appropriate size; and paraffin sections were cut at 4 µm. The sections were stained with hematoxylin-eosin (H & E) and Giemsa for observing the presence of inflammatory response in the muscularis mucosae and eosinophil granulocytes in the specimen. Microscopic observations were carried out at 400 x magnifications.
4.11 Statistical analysis
All the data of experiences were expressed as the mean ± SD, and the statistical comparisons were made by Student’s t-test. P-values < 0.05 were considered statistically significant, which would mark with “ * “.