Cell Culture
Three human cell lines, (purchased from the American Type Culture Collection Cell-ATCC-American Type Culture Collection) were used in this investigations: HaCaT - normal, immortal cell line of the transformed phenotype in vitro, the first stable line of adult human keratinocytes presenting normal differentiation. That is typical adherent cell line, growing in monolayer. A549 - human epithelial lung cancer cell line, derived from the 58-year-old Caucasian man. That is typical adherent cell line, growing in monolayer. H69AR - human multidrug resistant small cell lung cancer cell line, derived from the 55-year-old Caucasian man. H69AR cells begin to grow as aggregates which attach as domed patches. Most cells flatten into an epithelial monolayer, however some areas have piling of cells. Even when cells are healthy and well-attached, there are many viable floating cells.
All cell lines were grown in polystyrene flasks with 25 cm2 cell culture surface (Eppendorf, Germany) as a monolayer in Dulbecco modified Eagle medium (DMEM, Sigma-Aldrich, USA) for HaCaT and A549 cell line or RPMI-1640 Medium, (Gibco, USA) for H69AR cell line. Mediums were enhanced 10% (DMEM) or 20% (RPMI-1640) fetal bovine serum (FBS, Sigma-Aldrich, USA) and 50 μg/ml streptomycin (Sigma-Aldrich, USA). Cells were incubated at 37 °C in 5% CO2. Before the every experiments the cells were removed by 0.25% trypsin with 0.02% EDTA (Sigma-Aldrich, USA).
HMW and LMW Beta-Glucans Recovery Procedure
In this examination beta-glucan from oats was derivedin form of white powder. HMW and LMW beta-glucan was obtained at the University of Economics in Wroclaw. HMW oat beta-glucan was obtained due to procedure described elsewhere [17] with beta-glucanase inactivation during lipid removal step, alkaline extraction, protein removal in isoelectric point, solution neutralization to pH = 7,0 and beta-glucan precipitation with ethanol. Low molecular oat 1–3, 1–4–D-beta-glucan was manufactured due to procedure described elsewhere [10] with multistep freeze-milling of raw materials (20% beta-glucan fiber, Microstructure, Poland), fat removal with ethanol extraction, alkaline extraction (pH 8–10) of beta-glucan and oat proteins, protein precipitation and separation in isoelectric point, and finally beta-glucan precipitation with ethanol in equilibrium. Beta-glucan preparations was then dried for 24 h. Purity was determined with according to AOAC 995.16 method with test kit (Megazyme, Ireland) and was 84%. Molecular weight of oat beta-glucan was measured with HPLC-SEC with guard column (OHpak SB-G, Shodex), a GPC column (SB-806 M HQ, Shodex) and was 69,650 g/mol. Firstly the stock solution of beta-glucan was prepared. Two mg of beta-glucan was dissolved in 1 ml of sterile distilled water and one drop of 10% NaOH was added. Then the stock solution was incubated at 37 °C for 24 h. The different concentrations of this compound were used to the studies (5 μg/ml, 10 μg/ml, 20 μg/ml, 25 μg/ml, 50 μg/ml, 75 μg/ml, 100 μg/ml, 150 μg/ml and 200 μg/ml).
Cellular Viability - MTT Assay
The viability of cells was determined by MTT assay (Sigma-Aldrich, USA) after experiments with different concentrations of beta-glucan. The MTT assay was used to estimation of mitochondrial metabolic function through the measurement of mitochondrial dehydrogenase. For the experiment the cells were seeded into 96-well micoculture plates at 1 × 104 cells/well and grown overnight. After incubation with selected concentrations of LMW and HMW beta-glucan the experiments were realized according to the manufacture’s protocol. Those cells were incubated 24 h. The absorbance was determined using a multiwell scanning spectrophotometer at 570 nm (Enspire Perkin Elmer Multiplatereader, USA). Mitochondrial metabolic function was expressed as a percentage of viable treated cells in relation to untreated control cells.
Lipid Peroxidation
The measurement of lipid peroxidation assay is based on the reaction of malondialdehyde with thiobarbituric acid (TBA). The cells were treated with selected concentrations of LMW and HMW beta-glucan. After 24 h incubation the cells were removed by trypsinization and suspended in phosphate buffered saline (PBS, Sigma Aldrich, USA). The final product of lipid peroxidation, malondialdehyde (MDA), reacts with TBA to form a colored complex. The level of MDA was measured by the absorbance at a wavelength of 535 nm. The concentration of MDA was quantified spectrophotometrically based on a set of MDA standards of known concentration [18].
Immunofluorescencent Assessment of Cytoskeleton – CLSM Study
The confocal laser scanning microscopy (CLMS) was used to assess the morphology of treated cells. A549, H69AR and HaCaT cells were prepared for immunofluorescence reaction. The cells were grown on coverslips, than fixed 4% paraformaldehyde (PFA, Sigma-Aldrich, USA) in PBS, permeabilized with 0.5% Triton X-100 (Sigma-Aldrich, USA) in PBS (v/v) for 5 min. And blocked with 1% FBS in PBS (for 30 min. at room temperature). The cells were washed in PBS on the every steps of procedure. The following antibodies were used: primary antibody monoclonal anti-F-actin antibody produced in mouse (overnight incubation at 4 °C; 1:100; Sigma-Aldrich, USA); secondary antibody goat anti-mouse IgG FITC conjugated (for 60 min. at room temperature; 1:50; Sigma-Aldrich, USA). DNA was stained with DAPI (4,6-diamidino-2-phenylindole; 0.2 μg/ml in mounting medium with Mowiol-0.1% and DABCO). For imaging, Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Japan) was used. The images were recorded by employing a Plan-Apochromat 60× oil-immersion objective.
Immunocytochemical MnSOD Evaluation
Immunocytochemistry was performed using the ABC method. The cultures were fixed and dehydrated using 4% PFA during 10 min. The samples were then permeabilized and blocked by incubation with 0.1% Triton X-100 in PBS. The enzymes expression were visualized with polyclonal antibody (1:100, anti-MnSOD; SOD 2; Santa Cruz, USA). For conventional bright-field microscopy (peroxidase-ABC labelling), the samples were incubated with a diaminobenzidine-H2O2 mixture to visualize the peroxidase label and counterstained with haematoxylin for 30 s. The samples were analyzed with the upright microscope (Olympus BX51, Japan). Stained cells were determined by counting 100 cells in randomly selected fields. The result was judged positive if staining was observed in more than 5% of cells. The intensity of immunohistochemical staining was evaluated as: (−) negative, (+) weak, (++) moderate and (+++) strong.
Red Blood Cells Hemolysis
The level of hemolysis was determined by spectrophotometry. Red blood cell hemolysis was induced using sodium chloride solutions of various degrees of hypotonicity. The effect of each type of beta-glucan (LMW and HMW beta-glucan fraction) on erythrocytes hemolysis was examined at two concentrations (300 μg/ml and 400 μg/ml) and incubation periods of 24 h.
Preparation of red Blood Cells Suspensions
Blood samples from healthy volunteers were obtained in heparinized tubes. The red blood cells were separated from leukocytes by centrifugation (3000 rpm for 10 min.) and subsequently, washed three times with 0.9% sodium chloride solution (saline). Then prepared mixture of erythrocytes was filling by saline until the correct hematocrit for an individual subjects (approximately 40% in healthy females and 43% in healthy men) [19].
Preparation of Beta-Glucan Solutions
The solutions of beta-glucan (LMW and HMW beta-glucan fraction) were prepared by dissolving the requisite amount of solute in mixture of erythrocytes to obtain the desired concentrations (300 μg/ml and 400 μg/ml).
Each of the prepared test mixtures of red blood cells with beta-glucan (0.02 mL) was introduced into a tube with respective concentration of hypotonic sodium chloride solution. Then the tubes were incubated for 0.5 h at 23 °C (at room temperature) and then centrifuged (2000 rpm for 5 min.). The absorbance of the supernatant solution was measured spectrophotometrically at 540 nm using a microplate reader (EnSpire, PerkinElmer, USA) [20].
Statistical Analysis
Data are reported as mean ± standard deviation (SD). Analysis utilized the one-way repeated measures analysis of variance (ANOVA). As a post-hoc test when results of the above test were significant Fisher’s Least Significant Difference (LSD), Tukey’s Honestly Significant Difference (HSD) and Scheffe’s test were applied, where Scheffe’s is the most conservative of the three. A level of P < 0.05 was considered to be statistically significant. The results from analysis are presented in supplementary material (SM).