CHO-K1 (Chinese Hamster Ovary cell), purchased from Lonza (Basel), were cultured in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM-F12; Sigma, Milan, Italy) supplemented with 10% fetal bovine serum (FBS, Sigma, Milan, Italy), 2 mM glutamine and 1% penicillin/streptomycin (Sigma, Milan, Italy) and incubated at 37 °C, 5% CO2, and 95% humidity . When the cells reached 80–90% of confluence were seeded for different experiments; 1 × 104 and 2.5 × 104 cells were plated in a 96-well for MTT test and ELISA activation assay, respectively; 1 × 105 cells plated on 24-well plates to analyzed ROS production; for Western blot analysis and SOD activity the cells were seeded in 6 wells and maintained until 85% of confluence.
Experimental protocol in vitro
The cells before treatments were placed overnight in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, Milan, Italy) without red phenol and FBS in incubator at 37 °C, 5% CO2, and 95% humidity. The cells were treated with a range of Resveratrol (RES) 10-100 μM to determine an optimal concentration; 50 μM was chosen and its efficacy verified in a time-course study (from 2 min to 48 h). The RES concentration was chosen basing on previous studies about the therapeutic range of ovarian evidence [37, 38] and on the experiments of dose-response study. RES was prepared in lipidic solvent that was also tested alone in CHO-K1 cultures. The cooperative activity of RES with vitD (active form of vitamin D, 1,25-dihydroxyvitamin D3), was also tested, evaluating the effects of the co-stimulation with RES 50 μM and vitD 100 nM  in CHO-K1 cells during time.
MTT-based In Vitro Toxicology Assay Kit (Sigma-Aldrich) was performed as described in literature  to determine cell viability after stimulations. Cells were incubated in DMEM without red phenol 0% FBS with 1% MTT dye for 2 h at 37 °C in incubator  and then cell viability was determined measuring the absorbance through a spectrometer (VICTORX4 multilabel plate reader) at 570 nm with correction at 690 nm. The results were obtained comparing the results to control cells (100% viable).
The rate of superoxide anion production was determined as a superoxide dismutase-inhibitable reduction of cytochrome C, following a standard technique [41, 42]. In both treated and untreated cells, 100 μl of cytochrome C were added and in another sample, 100 μL of superoxide dismutase were also added for 30 min in incubator (all substances from Sigma-Aldrich). The absorbance was measured at 550 nm by spectrometer (VICTORX3 Multilabel Plate Reader) and the O2 was expressed as nanomoles per reduced cytochrome C per microgram of protein.
Akt/ERK activation assay
The InstantOne™ ELISA is specifically engineered for accurate measurement of phosphorylated ERK 1/2 and AKT in cell lysates, following the manifacturer’s instructions (Thermo-Scientific). Cells at the end of treatments were lysated with 100 μL Cell Lysis Buffer Mix, shaken for 10 min at RT and 50 μL/well of each sample were tested in InstantOne ELISA microplate strips including the 50 μL/well Positive Control Cell Lysate and 50 μL/well negative control. At each well 50 μL of prepared Antibody Cocktail were added and the strips incubated for 1 h at room temperature on a microplate shaker and washed 3 times with 200 μL/well of Wash Buffer (1X). At the end, 100 μL of the Detection Reagent were added to each well and after 20 min the reaction was stopped adding to each well 100 μL of Stop Solution. The strips were measured by a spectrometer (VICTOR X4 multilabel plate reader) at 450 nm. The results were expressed as means Absorbance (%) compared to control.
SOD activity assay
Cayman’s Superoxide Dismutase Assay Kit utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The SOD assay measures all three types of SOD (Cu/Zn, Mn, and FeSOD). The cells and tissue were lysed after treatments following manufacturer’s instructions (Cayman). In a 96 well, at every sample of 10 μl were added 200 μl of the diluted Radical Detector. At the same time, a standard curve was prepared (0.05–0.005 U/ml). Then, 20 μl of diluted Xanthine Oxidase were added at all wells and the plate mixed for 30 min at RT and then the absorbance measured through a spectrometer (VICTOR X4 multilabel plate reader) at 480 nm. The results were expressed as a means (%) compared to control.
Western blot of cell lysates
CHO-K1 cells were lysed in ice Complete Tablet Buffer (Roche) supplemented with 2 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich), 1:50 mix Phosphatase Inhibitor Cocktail (Sigma-Aldrich) and 1:200 mix Protease Inhibitor Cocktail (Calbiochem). 35 μg of proteins of each sample were resolved on 10% SDS-PAGE gel. Polyvinylidene difluoride membranes (PVDF, GE, Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-VDR receptor (1:400, Santa-Cruz) and anti-ERβ (1:500, Santa-Cruz). Protein expression was normalized to the specific total protein (if possible) and verified through β-actin detection (1:5000; Sigma-Aldrich) and expressed as a mean ± SD (%).
Female Wistar rats weighing 300 to 350 g (n = 94) purchased from Envigo++++ (Bresso, Italy), were housed in a room at a constant temperature of 25 °C on a 12-h/12-h light/dark cycle with food and water available ad libitum. All experiments were conducted in accordance with local ethical standards and prospectively approved by the University OPBA (Organismo Preposto al Benessere degli Animali, Animal Wellness Committee). Experimental protocols were approved by national guidelines (Ministero della Salute authorization number 914/2015-PR) and in accordance with Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86–23, 1985 revision).
In vivo experimental protocol
In order to study the bioavailability 0.5 mg RES were administrated by gavage following a standard technique [43, 44]; the quantity of RES was calculated by the conversion formula (animal-man) approved by FDA . For each animal, anesthesia was performed via isoflurane (1.2–1.5 Mean Alveolar Concentration) in oxygen and gavage was carried out using probe-ended stainless-steel gastric tubes (80 × 1.5 mm, length × outer diameter). After treatment, rats were placed in individual cages and housed separately for the duration of the study and daily monitored. The animals were randomized in different groups: n = 36 treated with RES lipophilic formula; n = 36 with RES plus vitD 0.4 μg lipophilic formula; n = 18 treated with vitD 0.4 μg alone; n = 4 untreated (control) and sacrificed at T0. Time-point for each treatment (2, 5, 15, 30, 60, 180, 360, 720, 1440 min) was conducted in triplicates. The animals were euthanized by CO2 asphyxiation at each time point and the organs (liver, stomach, intestine, heart, kidneys and ovaries) were withdrawn to evaluate biodistribution of the different RES formulations, and to evaluate the ovarian tissue integrity by Western-blot. In addition, blood samples used for RES determination by HPLC analysis, the oxygen radical species (ROS) and vitD quantification (by ELISA kit) were collected at each time-point using CBC tubes to obtain plasma by centrifugation at 3000 rpm for 15 min at room temperature.
Plasma vitamin D quantification
Vitamin D3, the active form of vitamin D, is very short-lived and rapidly metabolized to the deactivated forms 24,25(OH)2D3 and 1,24,25(OH)3D3. For this reason a competitive EIA assay kit has been used that primarily detects the more metabolically stable forms, 25(OH)D3 and 25(OH)D2 (Cayman’s Vitamin D EIA Kit). At the end of each time point, plasma samples were collected using EDTA-Na2 as an anticoagulant, centrifuged for 15 min at 1000×g at 4 °C within 30 min and then the supernatant used immediately. Before adding to wells, the SABC working solution and TMB substrate were equilibrate for at least 30 min at room temperature and the strips of the plate washed twice before adding standard, sample and control. For the quantification it is necessary to plot a standard curve including control (zero well). 0.1 ml of each sample and standard were added into test sample wells, the plate sealed with a cover and incubated at 37 °C for 90 min. After the plate content was removed, 0.1 ml of Biotin- detection antibody work solution was added into the standard and test sample at 37 °C for 60 min. After the plate was washed 3 times with Wash buffer 0.1 ml of SABC working solution into each well was added and the plate incubated at 37 °C for 30 min. After the plate was washed 3 more times with Wash buffer 90 μl of TMB substrate into each well was added and the plate incubated at 37 °C in dark within 15–30 min. After this time 50 μl of Stop solution into each well was added and the absorbance measured at 450 nm in a microplate reader immediately after adding the stop solution.
Total plasma antioxidant capacity
The concentration of radical oxygen species (ROS) in plasma was measured in a 96-well plates using the Antioxidant Assay kit (Cayman) following the manufacturer’s instructions . In brief, 10 μl of Metmyoglobin and 150 μl of Chromogen per well were added in plasma and standard samples (Trolox in Assay buffer from 0 mM to 0.33 mM) and the reactions started adding 40 μl of Hydrogen Peroxide Working Solution to all the wells. The 96-well plate was covered, mixed for 5 min at room temperature and the absorbance was measured using spectrophotometer (VICTORX4 Multilabel Plate Reader) at 750 nm or 405 nm. The results were expressed as means ± SD (%).
Western-blot of ovarian tissues
Ovarian tissues were immediately washed with ice 0.9% saline solution (w/v), weighed and homogenized in a volume of 100 mg tissue/300 μL of lysis buffer (0.1 M Tris, 0.01 M NaCl, 0.025 M EDTA, 1% NP40, 1% Triton X100, Sigma-Aldrich, Milan) supplemented with 2 mM sodium orthovanadate, 0.1 M sodium fluoride (Sigma-Aldrich, Milan), 1:100 mix of protease inhibitors (Sigma-Aldrich, Milan), 1:1000 phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, Milan), using an electric potter at 1600 rpm for 2 min. Samples were mixed for 30 min at 4 °C, centrifuged for 30 min at 13000 rpm at 4 °C and 40 μg of proteins for each samples resolved on SDS-PAGE gel at 15%. Proteins transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy) were incubated overnight at 4 °C with specific primary antibody: anti-VDR receptor (1:400, Santa-Cruz), anti-ERβ (1:500, Santa-Cruz), anti-cyclin-D1 (1:1000, Euroclone, Milan, Italy). Protein expression was normalized and verified through β-actin detection (1:5000; Sigma-Aldrich) and expressed as a mean ± SD (%).
RES quantification in CHO-K1
At the end of stimulations cells were placed in ice and supernatants were collected in 1.5 ml centrifuge tubes to determine the rate of extracellular RES. 1 × 106 CHO-K1 cells at the end of stimulations were washed with cold 0.9% saline solution, lysed in ice 0.9% saline solution, mixed for 10 min at 4 °C and centrifuged for 20 min at 13000 rpm at 4 °C. Supernatants were used for quantification of intracellular RES. Samples were diluted with equal volume of acetonitrile, vortexed, centrifuged at 13000 rpm for 10 min, and analyzed by HPLC-UV (Additional file 1).
RES quantification in rat plasma and tissues
RES quantification in rat plasma and tissue samples (liver, stomach, intestine, heart, kidneys and ovaries) was carried out by HPLC-MS analysis. Tissues were homogenized in a volume of 100 mg tissue/300 μl of ice 0.9% saline solution (w/v) at 1600 rpm for 2 min and the lysates were mixed for 20 min at 4 °C, and then centrifuged at 13000 rpm for 30 min at 4 °C. Plasma and tissue supernatants were processed as follows. An aliquot of 50 μl of plasma or tissue sample was mixed with 50 μl of 1 M of sodium acetate buffer (pH = 5.5) and 2.5 μl of β-glucuronidase/arylsulfatase from Helix pomatia in a 1.5 ml centrifuge tube. Ethyl acetate (600 μl) was added, then sample was extracted by vortexing (40s), and centrifuged at 13000 rpm for 10 min. An aliquot (550 μl) of the organic layer was transferred into 1.5 ml centrifuge tube and evaporated at 45 °C under reduced pressure for 40 min. The residue was dissolved in 100 μl of acetone containing the IS (trans-4-hydroxystilbene- final concentration, 200 μg/l), 25 μl of 0.1 N NaOH, and 100 μl of 1 mg/ml Dns-Cl (dansyl chloride) solution in acetone. Sample was shortly vortexed and heated at 45 °C for 20 min. After centrifugation (13,000 rpm for 5 min) the sample was analysed by HPLC-MS (Additional file 1). Calibration curve for RES quantification was prepared by spiking blank matrixes and processed as described above, except for the addition of β-glucuronidase/arylsulfatase.
In vitro results obtained from at least 5 independent experiments conducted in triplicates were expressed as means ± SD, using One-way ANOVA followed by Bonferroni post hoc test. Values of significance for p < 0,05 were considered statistically significant. Data collected from in vivo results obtained from 4 independent experiments were analyzed by two-way ANOVA and one-way ANOVA followed by Bonferroni post hoc test and the comparisons between the two groups were performed using a two-tailed Student’s t-test. Multiple comparisons between groups were analyzed by two-way ANOVA followed by a two-sided Dunnett post-hoc testing. P-value < 0.05 was considered statistically significant.