Procurement and care of the rats
Our study was conducted with 32 Wistar albino male rats procured from İnönü University Experimental Animal Breeding and Research Center. Rats were fed ad libutum with standard pellet feed and tap water throughout the experiments.
Group 1: (Control group): Corn oil administration.
Group 2: (Tartrazine group): 100 mg/kg/day tartrazine was administered (Sigma-Aldrich-1934-21-0, St. Louis, USA) (Balta et al. 2019).
Group 3: (Thymoquinone group): 50 mg/kg/day Thymoquinone was administered (Sigma-Aldrich-490-91-5, St. Louis, USA) (Kong et al. 2015).
Group 4: (Tartrazine + Thymoquinone group): 100 mg/kg/day tartrazine + 50 mg/kg/day thymoquinone were administered.
Tartrazine was applied by gavage after it was dissolved in physiological saline and thymoquinone was dissolved in corn oil and 1 ml/kg/day solution was administered to each rat. The solutions were administered at the same time every day for 21 days.
Collection Of The Samples And Preparations
After the experiments, the abdominal region of the rats were opened under anesthesia (xylazine and ketamine) with the protocol prescribed by the ethics committee, and blood samples were obtained from the heart tissue and transferred into adequate tubes. Liver tissues were incised and washed with physiological saline to remove the excess blood. Certain liver tissues were placed in sterile containers and quickly frozen to -80°C for biochemical analyzes. The rest of the liver tissues were placed in containers that included 10% formol solution for histopathological analyses.
Liver tissues were removed from − 80°C freezer and quickly weighed before thawing. Phosphate buffer that equaled to 9 times the tissue weight was added. They were homogenized at 15000 rpm for 1 minute at + 4°C (IKA, Germany). Malondialdehyde (MDA) levels were determined with these homogenates. The tissue homogenates were centrifuged at 4000 rpm for 25 minutes at + 4°C to obtain the supernatants. Glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI), and protein levels were determined on these supernatants. Blood samples were centrifuged at 600g at 4°C for 20 minutes to obtain the serum, and the serum was used to determine tumor necrosis factor alpha (Tnf-α) and interleukin-6 (IL-6).
0.25 ml tissue homogenate was mixed with 1.5 ml 1% H3PO4 and 0.5 ml 0.6% thiobarbituric acid. The product was heated to 30º to completely dissolve the TBA. The mixture was heated at 100ºC for 45 minutes. Then, the formation of the red color was observed, and then the tubes were cooled in tap water and 2ml n-butanol was added to each sample. Each tube was vortexed for 5 minutes. Then, the samples were centrifuged for 25 minutes at 5000 rpm at 25ºC. 25µl supernatant (n-butanol phase) was carefully collected and transferred to individual quartz microplate wells. Readings were conducted at 535 nm. The findings are presented as nanomole/gram wet tissue (Ohkawa et al. 1979).
The supernatant was deproteinized with tricarboxylic acid (TCA). 125 µl TCA was added to 125 µl sample. After the mixture was vortexed, it was centrifuged for 20 minutes at 5000 rpm and + 4ºC to obtain the protein-free supernatant. 29 µl deproteinized sample, 29 µl DTNB and 235 µl Na2HPO4 were added to each microplate well separately, gently vortexed and kept for 5 minutes. Readings were conducted at 410 nm within 5 minutes. The findings are presented as nanomole/gram wet tissue (Ellman 1958).
Sod Enzyme Activity
SOD enzyme activity is determined with the measurement the intensity of this color formed by the reduction of NBT by superoxide radicals (xanthine + xanthine oxidase system) with the spectrophotometer. The reduction forms a blue colored formazan with a maximum absorbance at 560 nm. In the absence of enzyme, this reduction is maximal, and a dark blue color is observed. In the presence of SOD, the enzyme converts the superoxide radical to hydrogen peroxide; and thus, the reduction of NBT is lower and no blue formazan is observed or the intensity of the color is quite light. SOD activity was calculated based on the absorbance of the blue colored formazan at 560nm. The results are presented as U/mg protein (Sun et al. 1988).
Cat Enzyme Activity
Hydrogen peroxide (H2O2) is a substance that provides absorbance in the UV spectrum and its maximum absorbance wavelength is 240 nm. A decrease in absorbance is observed at 240 nm due to the breakdown of the added hydrogen peroxide into water and oxygen by catalase. This decrease in absorbance was recorded for 1 min via kinetic readings and the enzyme activity was measured. The enzyme activity is presented as K/g protein (Aebi 1974).
GSH-Px is an enzyme that catalyzes the conversion of hydrogen peroxide to water by reduced glutathione. After the reaction, reduced glutathione is converted to the oxidized form. For the conversion of another hydrogen peroxide into water, the oxidized glutathione should be converted back to the reduced form. This occurs in the presence of reduced NADP and reduced glutathione in the medium. Then, reduced NADP is converted into oxidized NADP, while oxidized glutathione is converted into reduced glutathione. The maximum absorbance of the reduced NADP is at 340 nm. The absorbance drops at 340nm as glutathione reductase catalysis continues, since the reduced NADP is not converted into the oxidized form. GSH-Px activity is calculated by recording the decrease in absorbance for 3 min. The findings are presented as U/mg protein (Paglia and Valentine 1967).
Tissue protein content is required to calculate enzyme activities. The Lowry method was used to determine the tissue protein content. The absorbance of the resulting color was measured at 660 nm in the spectrophotometer to determine the protein content (Lowry et al. 1951).
Rel Assay brand kit (Rel Assay Diagnostics, Gaziantep Turkey) was used to determine the TAS. The measurement is based on the discoloration of the antioxidant molecules. Sequential steps were conducted based on the kit instructions. TAS was determined with the measurement of the absorbance at 660 nm in the device set for 37ºC as specified in the kit. The findings are presented as mmol Trolox Equiv./l (Erel 2004).
TOS Rel Assay brand kit (Rel Assay Diagnostics, Gaziantep Turkey) was employed to determine the TOS. Ferric ions form a colored chromogenic solution when the oxidants in the sample convert the ferrous ion chelator complexes into ferric ions. TOS is determined with the measurement of the absorbance of the colored complex at 530 nm at 25°C as specified in the kit. The findings are presented as µmol H2O2 equiv/l (Erel 2005).
The OSI was calculated with the formula OSI (arbitrary unit) = TOS (µmol H2O2 eqv/L) / TAS (mmol Trolox eqv/L) X 10. Results are presented as arbitrary units (AU).
Tnf- α And Il-6 Analysis
Bioassay Technology Laboratory ELISA Kit was employed to determine the serum interleukin-6 and TNF-α levels. All reagents were at room temperature before the tests, and the tests were performed at room temperature. Sample and ELISA reagent were transferred to the wells, and these were incubated for 1 hour at 37°C. Then, they were washed five times in the microplate washer with the lavage solution. Substrate solutions A and B were added and incubated at 37°C for 10 minutes. Then, the stop solution was added, and the color change was observed. The readings were conducted within ten minutes. The findings are reported as ng/L.
For histopathological analysis, liver tissue samples were fixed in 10% formaldehyde for 48 hours. Then, liver tissue samples were dehydrated through an incremental ethanol series (50%, 70%, 80%, 96%, Absolute). The liver tissue samples were then transparentized through xylene series and embedded in paraffin blocks after they were passed through melted paraffin series at 62°C for infiltration. 6 µm thick sections were obtained from the paraffin blocks with a microtome and placed on slides (Bancroft and Gamble 2002). They were stained with hematoxylin–eosin (H-E) and anti-caspase-3 [Cleaved-CASP3p17 (D175) Polyclonal Antibody] (Elabscience, Texas, USA) for immunohistochemical (IHC) analysis. Stained sections were examined with Nikon Eclipse Ni-U light microscope, Nikon DS-Fi3 microscope camera and Nikon NIS-Elements Documentation 5.02 image analysis program (Nikon Corporation, Tokyo, Japan).
Histopathological changes in the liver sections stained with hematoxylin–eosin (inflammatory cell infiltration, necrosis, periportal edema, vascular congestion) were scored between 0 and 3 (0; absent, 1; mild-rare, 2; moderate, 3; severe- common), where the maximum histopathological score was 12.
Cleaved caspase-3 expression was considered as an indicator of apoptosis in liver tissue. Sections stained for immunohistochemical analysis were placed on polylysine-coated slides. Sections were initially deparaffinized. Then, they were heat-treated in the retriever (Retriever 2100) (Aptum, Southampton, UK) for 15 min with citrate buffer with a pH of 7.6 (Thermo Scientific, Fremont, CA) for retrieval of the antigens. After the sections were cooled to room temperature for 20 minutes, they were first washed with distilled water and then with phosphate buffered saline (PBS) for 1–2 minutes. Sections were drawn using a hydrophobic pen and lined up on the platform and treated with 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity, then washed with PBS. Sections were incubated with protein-V blocking reagent (Thermo Scientific) for 5 minutes.
Sections were incubated with 1:200 diluted primary rabbit polyclonal cleaved caspase-3 antibody (Cleaved-CASP3p17 (D175) Polyclonal Antibody) (Elabscience, Texas, USA) for 1 hour, then rinsed in PBS, and incubated with biotinylated goat anti-polyvalent secondary antibody for 10 minutes and transferred into PBS. It was then incubated with streptavidin peroxidase (HRP) for 10 minutes and transferred into PBS. The polyvalent HRP kit (Thermo Scientific) was employed in compliance with the manufacturer's instructions. Finally, sections were treated with chromogen (AEC; Thermo Scientific) + substrate buffer (AEC) (Thermo Scientific) for a maximum of 15 minutes. After they were washed with PBS and distilled water, counterstaining was conducted with Mayer's hematoxylin for 1 minute. Sections were rinsed in tap water and then distilled water and covered with a water-based sealer and coverslip (Thermo Scientific, Cheshire, UK).
The caspase-3 immunoreactivity H score (H Score = Pi (i + 1), Pi, is the percentage of stained cells in each density category (0-100%) in sections stained with anti-caspase-3 in the immunohistochemical method, and i denotes weak (i = 1), moderate (i = 2), or strong staining (i = 3) (Budwit-Novoty et al. 1986).
Numerical data were summarized with median, minimum and maximum values. Kruskal-Wallis test was used for independent group comparisons and Friedman test was used for dependent group comparisons. After both universal tests, pairwise comparisons were made with the Conover method. The significance level was accepted as 0.05 in all tests.