2.1 Mouse model
Experiments were performed using homozygous Nrf2 deficient (Nrf2-/-) mice that were originally produced in the laboratory of M. Yamamoto (32) and backcrossed to C57BL/6J at least 9 times. The knockout breeder mice were purchased from Riken BioResource, Tsukuba, Japan. The homozygous mutant (Nrf2-/-) and wildtype (Nrf2+/+) littermates from both genders, produced from heterozygous breeding, were used in the present study at an age of 10-12 weeks. A pilot kinetic study was performed in a limited number of animals, using C57BL/6J mice exclusively as no effect of Nrf2 deficiency on the intestinal uptake kinetics of quercetin was anticipated. Animals were kept in an in-house pathogen-free and environmentally controlled facility (safety level 1) according to guidelines of the Society for Laboratory Animals Science (GVSOLAS). A 12 hours light/dark cycle was maintained and food and water provided ad libitum. The animal study was carried out following approval by the North Rhine Westphalia State Agency for Nature, Environment and Consumer Protection (NRW-LANUV (Az.: 87-51.04.2010.A276)) under the provisions of the German Animal Protection Law (Tierschutzgesetz: 18 Mai 2006 (BGBl.IS.1206.1313).The study was conducted following the principle of the 3Rs (refinement, reduction and replacement), minimizing the pain and using standardized approved procedures for analgesia and euthanasia according to the Law.
2.2 Quercetin diet and evaluation of effectiveness
Animals had ad libitum access to either the control diet (AIN893, ssniff Spezialdiäten, Soest Germany) or the control diet enriched with 800 mg/kg quercetin (Merck, Darmstadt, Germany) starting from 7 days prior to the bleomycin challenge until they were sacrificed. Quercetin was stabilized (I) by adding 0.1 gram citric acid per liter water while preparing both diets and (II) by replacing food pellets by a freshly thawed stock on a daily basis while providing both diets. Based on a daily intake of 5 grams food per day and an average weight of 20 grams, this quercetin-enriched diet will result in an average daily intake of 200 mg quercetin/kilogram BW. Weight gain, general signs of (dis)comfort and illness as well as survival were recorded on a daily basis throughout the entire dietary supplementation to ensure the safety of this intervention. To evaluate its effectiveness, six C57BL/6J mice were fed for one week with quercetin enriched or control diet and sacrificed to determine plasma and pulmonary quercetin concentrations as well the expression of Nrf2 and Nrf2-responsive antioxidant genes.
2.3 Intervention study
After 1 week of quercetin-enriched or control diet, Nrf2-/- and Nrf2+/+mice were exposed to 1 µg/2µl per gram BW bleomycin in 0.9 % NaCl or 0.9 % NaCl by a single pharyngeal aspiration under isoflurane anaesthesia. After 14 days, animals were brought under deep anaesthesia using 50 mg/kg body weight pentobarbital and euthanized via exsanguination after which blood and lungs were collected by respectively cardiac puncture and surgical removal. Left lungs were used for histology and processed as described below. The lobes of the right lungs were equally divided in 2 parts, one for RNA extraction and one for homogenate preparation, and snap-frozen until further analysis. Blood was centrifuged (10’, 1200 g, 4°C) after which plasma aliquots were also snap-frozen until further analysis.
A schematic overview of the experimental setup, including the number of animals per group, is provided in Figure 1. For all groups, littermates were used to ensure optimal comparison of the influence of both factors on the primary and secondary endpoints of this study.
2.4 Histopathological evaluation of lung tissues
Left lungs were fixed in 4 % paraformaldehyde /PBS, pH 7.4, dehydrated in a series of ethanol and subsequently xylol and embedded in paraffin. Sections of the left lungs were cut at a thickness of 4 μm, rehydrated and stained with H&E (hematoxylin and eosin) and Masson’s Trichrome. Subsequently, slides were evaluated histopathologically applying a semiquantitative grading : 1=minimal, 2=slight, 3=marked, 4=severe, 5=massive.
2.5 RNA isolation
To isolate RNA from mouse lungs, the tissue was first homogenized using a tissue homogenizer in 700 µL Qiazol (Invitrogen, Carlsbad, CA). Afterwards, the samples were incubated for 5 min at room temperature, 150 µL chloroform were added and the solutions were mixed. The samples were centrifuged for 15 min at 12.000 rpm and the upper aqueous layer was used for RNA isolation.
RNA was isolated and purified using the RNeasy mini kit (Qiagen, Venlo, the Netherlands) according to the manufacturer’s instructions. A nanodrop spectrophotometer (Thermo Scientific, Waltham, MA) was used to determine the RNA concentration.
2.6 Real-time PCR
cDNA was synthesized from 300 ng isolated RNA using IScript (Biorad, Hercules, CA) according to the manufacturer’s instructions. Next, RT-PCR was performed using SYBR Green PCR Supermix (BioRad) with 2.5 μL of 10 times diluted cDNA and 0.4 μmol/L predesigned primers. PCR amplifications were carried at 95°C for 10 sec for denaturation and 40 cycles of annealing/elongation (60°C, 30 sec) for selected genes (Table 1). The gene expression was normalized to the house keeping gene β-actin and quantified according to the 2−∆∆Ct method to relatively quantify the expression of the genes of our interest. The influence of quercetin on the redox-effects of bleomycin was analyzed by measuring the expression of Nrf2 and the Nrf2-responsive genes HO-1, ƴ-GCS, SOD2, and CAT. The alleged anti-inflammatory effects of the dietary intervention were investigated by including CXCL-1 (KC) and TNF-α, whereas COL1A2 and FN1, were added as pro-fibrotic genes of interest.
2.7 Homogenate preparation
From the collected right lung tissue, homogenates were made by crushing with liquid nitrogen. Before crushing, the weight of the tissue was determined. Afterwards, 3 parts of sodium phosphate buffer (145mM, pH=7.4) was added to the crushed tissue and protein content was measured using Pierce BCA protein assay kit according to manufacturer instructions.
2.8 Determination of total plasma quercetin concentration
Total plasma quercetin concentration was measured as previously described (33) after enzymatic hydrolysis using high performance liquid chromatography (HPLC) with colorimetric array-detection.
2.9 Luminex (Bio-Plex cytokine assay)
Plasma cytokine profiles were determined with Bio-Plex assays (Bio-Rad) using Luminex xMAP-technology. To quantify the concentrations of 11 different cytokines, we used a Bio-Plex murine cytokine multiplex panel including IL-4, TNFα, KC, MIP2, MCP-1, IL-13, IL-1β, IL-17a, IL-10, MIP1-α and MIP-1β. All assays were performed as described by the manufacturer’s instructions. Briefly, 50 µL of the antibody magnetic beads were added to each well and unbound antibody was removed. Next, 25 µL plasma were added to 25 µL universal assay buffer and incubated for 2 h. The plate was washed and 25 µL detection antibody, which binds to its corresponding analyte present in the sample, were added for 30 min. 50 µL Streptavidin-PE were added after washing and incubated for 30 min. After washing, the beads were resuspended in 120 µL reading buffer. Next, the bead and reporter quantity of the antibody sandwich formed around the analyte was determined by a laser detector. Finally, the beads and reporter quantities measured were compared to those of an internal standard corresponding to each specific cytokine. Data analysis was done with a Luminex 100 IS 2.3 system using the Bio-Plex Manager 4.1.1 software.
2.10 Trolox antioxidant capacity
The trolox equivalent antioxidant capacity (TEAC value) is a measurement for the total antioxidant status, assessing the capacity of a compound to scavenge ABTS radicals. This assay has been performed as described earlier (34) with some minor modifications. In short, blood was centrifuged (3000 rpm, 5’ at 4ºC) and the obtained plasma deproteinized by adding 10% TCA (1:1) before centrifugation (13.000 rpm, 5’ at 4ºC). To generate ABTS radicals (ABTS•), a solution of 0.23 mM ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and 2.3 mM ABAP (2,2′-Azobis-(2-amidinopropane)HCl) was incubated at 70°C until the absorbance at 734 nm reached 0.70 ± 0.02. The antioxidant capacity of the samples was measured by mixing 50 ml of the plasma sample with 950 ml ABTS• solution followed by 5 min incubation at 37°C. The decrease in absorbance was measured at 734 nm and the trolox equivalent of the samples was calculated using the absorbance of trolox standards.
2.11 Malondialdehyde-dG DNA adducts (M1-dG)
The presence of M1-dG as was evaluated using 32P-postlabeling as previously described (Peluso et al, 2013). To this end, DNA was first hydrolysed by incubating in 2.5 mM calcium chloride for 4.5 hours at 37°C (pH 6.0) in the presence of 21.45 mU/µl micrococcal nuclease and 6.0 mU/µl spleen phosphodiesterase in 5.0 mM sodium succinate. Upon hydrolysis, samples were incubated with 0.1 U/µl NP1 in 46.6 mM sodium acetate (pH 5.0) and 0.24 mM ZnCl2 for 30 minutes at 37°C. Next step after this NP1 treatment was the generation of 32 P-labelled DNA adducts in bicine buffer (20 mM bicine, 10 mM MgCl2, 10 mM dithiotreithol, 0.5 mM spermidine, pH 9.0) by adding 1.8 µl of 0.16 mM Tris base and incubating at 37°C for 30 minutes with 7–25 µCi of carrier-free [ƴ-32 P]ATP (3000 Ci/mM) and polynucleotide kinase T4 (0.75 U/µl). These generated 32 P-labeled samples were then applied to polyethyleneimine cellulose thin layer chromatography plates (Macherey-Nagel, Germany) and resolved in a low-urea solvent system as previously described (35). Storage phosphor imaging techniques employing intensifying screens from Molecular Dynamics (Sunnyvale, California) were used to detect and quantify M1–dG adducts as well as normal nucleotides, i.e. diluted untreated samples. Levels of M1-dG adducts were expressed as relative adduct labelling, i.e. as pixels in adducted nucleotides/pixels in normal nucleotides, and corrected across experiments based on the recovery of an MDA-treated DNA adduct standard.
All quantitative data are represented as means ± SEM. Statistical differences between groups were evaluated by the nonparametric Mann–Whitney U-test using GraphPad Prism software (version 7.3; GraphPad Software, La Jolla, CA), and considered significant at a P value of less than 0.05.