However, in case of noninflamed hepatocytes, considering the results of

glutathione peroxidase activity, the application of the product did not result in severe oxidative distress. In accordance with the

abovementioned findings, FWGE as a redox modulator, applied in the appropriate concentration, can serve as a promising

candidate in the supplementary therapy of patients suffering from various inflammatory diseases, decreasing the free radical

generation, thus avoiding the occurrence of cytotoxic effects.

2.2. Treatments of Cultured Cells. After 24 h, culturing cells

were treated using cell culture media supplemented with 0

(control) or 10 μg/mL Salmonella enterica serovar. Typhimurium derived lipopolysaccharide (LPS) for 2 and 8 h incubation time. Further, in both of the control and LPSchallenged cultures, subgroups were prepared using 0.1%

and 1% FWGE prepared from Immunovet®, silymarin

(50 μg/mL), or ursodeoxycholic acid (UDCA, 200 μg/mL)

containing cell culture medium. In the latter two cases, cultures were treated with proved hepatoprotective and antioxidant substances.

To gain the FWGE working solutions (Immunovet®), 1 g

of FWGE granules was homogenized using a mortar until a

fine powder was received and dissolved in 10 mL sterile phosphate buffered saline (PBS) solution. The gained stock solution (100 mg/mL; 10%) was filtered in different steps, using

gauze sheets (3 layers, 2 times filtering), a cell strainer

(70 μm pore size), and a sterile filter (0.22 μm pore size) in

the end (Merck Millipore, Burlington, MA, USA). Stock solution (10%) was diluted with PBS to 1% and 0.1%

concentrations.

2.3. Measurements of Cellular Metabolic Activity,

Extracellular H2O2 and Malondialdehyde Concentrations,

and Glutathione Peroxidase Activity. Following the treat-ments, metabolic activity of cells cultured on 96-well plates

was checked using CCK-8 assay (Dojindo, Rockville, USA),

monitoring the total amount of NADH+H+ produced in

the cellular catabolic reactions, successfully reflecting also

to the potential cytotoxic effects. According to the manufacturer’s instructions, 10 μL CCK-8 reagent and 100 μL Williams’ Medium E were added to the cultured cells, and after

2 h of incubation at 37°

C, the absorbance was measured at

450 nm with a Multiskan GO 3.2 reader (Thermo Fisher Scientific, Waltham, MA, USA).

Extracellular H2O2 concentration was detected in the culture medium using the fluorimetric Amplex Red method

(Thermo Fisher Scientific, Waltham, MA, USA). After

30 min incubation of 50 μL freshly prepared, Amplex Red

(100 μM) and HRP (0.2 U/mL) containing working solution

with 50 μL culture medium at room temperature (21°

C),

fluorescence (λex = 560 nm; λem = 590 nm) was detected

using a Victor X2 2030 fluorometer (Perkin Elmer, Waltham,

MA, USA).

2.4. Statistics. All the data analysis was performed using the R

3.5.3. software (GNU General Public License, Free Software

Foundation, Boston, MA, USA). On both of 96- and 6-well plates, six wells were included in one treatment group. Normal distribution and homogeneity of variance were checked

by Shapiro-Wilk test and Levene’s test, respectively. Differences between various groups were assessed using one-way

analysis of variance (ANOVA) and Tukey’s post hoc tests

for pairwise comparisons. Results were assessed as the

mean ± standard error of the mean (SEM). Differences were

assumed significant at P < 0:05. Results of the FWGE, silymarin, and UDCA treated groups were compared to the

respective control groups (LPS free or LPS supplemented

control groups). The effects of LPS supplementation were

considered as main effect compared to the control groups

without LPS treatment.