Bacterial strains
Rhodococcus rhodnii (DSM 43960) was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH company. The master cell bank of Rhodococcus rhodnii was maintained at − 20 °C in cryovials in Plate count broth medium (1 mL) mixed with glycerol (0.5 mL) as a cryoprotectant agent. The working cell bank was conserved at 4 °C in plate count agar (PCA) slants for 6 months and used for seed cultures.
Salmonella typhimurium mutant TA98 and TA100 strains purchased by Molecular Toxicology Inc. (Boone, NC, USA; moltox.com).
Synthesis of SCA and SCB through biotransformation of cortisone
The biotransformation of cortisone (Merck, Germany) with Rh. rhodnii DSM 43,960 was performed as previously described by Zappaterra et al. [15]. A loopful of Rhodococcus rhodnii from a culture on Plate Count Agar (PCA) (Merck, Germany) containing glucose (1 g/L), yeast extract (2.5 g/L), tryptone (5 g/L) and agar (15 g/L) was inoculated in 50 mL Erlenmeyer flasks filled with 20 mL of sterile Plate Count Broth (PCB) (Merck, Germany) containing glucose (1 g/L), yeast extract (2.5 g/L) and tryptone (5 g/L) and incubated at 30 °C and 110 rpm in an orbital shaker for 48 h. The whole cultures were introduced into 500 mL Erlenmeyer flasks containing 200 mL of sterile PCB, and after 48 h incubation at 30 °C and 110 rpm, cortisone (0.2 g) in DMSO (2 mL) was added and the culture was maintained in the same conditions. After 24 h the cells were removed by centrifugation (5242 RCF, 20 min) and the supernatant was extracted with ethyl acetate (3 × 200 mL). The organic layer was dried over anhydrous Na2SO4, the solvent evaporated and the crude mixture purified on a chromatographic column (silica gel, ethyl acetate as eluent).
Mutagenicity assay (Ames test)
Mutagenicity assay was performed following the plate incorporation method with the histidine-requiring Salmonella typhimurium mutant TA98 and TA100 strains. All strains (100 µl per plate of fresh overnight cultures) were checked with and without the addition of 0.5 mL of a 5% S9 exogenous metabolic activator (S9 mix). The lyophilized post-mitochondrial supernatant S9 mix (Aroclor 1254-induced, Sprague–Dawley male rat liver in 0.154 M KCl solution, Molecular Toxicology, Inc., Boone, NC, USA), commonly used for the activation of pro-mutagens to mutagenic metabolites, was stored at− 80 °C before use. The concentration tested for all the samples were 1, 5, 10, 50, 100 µL/plate of a stock solution 100 mg/mL.
A fully-grown culture of the appropriate tester strain (0.1 mL) was added to 2 mL Molten Top Agar (0.6% agar, 0.6% NaCl, 0.5 mM L-histidine/biotin solution) at 46 °C, together with 0.1 mL of each sample solution at different concentrations, and 0.5 mL S9 mix for assays with metabolic activation or 0.5 mL of phosphate buffer pH 7.4 to test without metabolic activation. The ingredients were thoroughly mixed and poured onto minimal glucose agar plates (1.5% agar in 2% Vogel–Bonner medium E with 5% glucose solution).
DMSO was used as a negative control (100 µl/plate). Positive controls were prepared as follows: 2-aminoanthracene (2 µg/plate) and 2-nitrofluorene (2 µg/plate) for TA98 with and without metabolic activator (S9 mix) respectively; 2-aminoanthracene (2 µg/plate) and sodium azide (2 µg/plate) for TA100, with and without metabolic activator (S9 mix) respectively. The plates were incubated at 37 °C for 72 h and then the his+ revertants were checked and counted using a Colony Counter 560 Suntex (Antibioticos, Italy). A sample was considered mutagenic when the observed number of colonies was at least twofold over the spontaneous level of revertants [21, 22, 34]. All determinations were made in triplicate. Relative standard deviations were computed using the statistical software STATISTICA 6.0 (StatSoft Italia srl).
Determination of antioxidant properties
Photochemiluminescence (PCL-ACL) method (Photochem
®
)
Photochemiluminescence assay, based on the methodology of Popov and Lewin [35], was performed to measure the antioxidant activity of cortisone, SCA and SCB compounds against superoxide anion radicals generated from luminol, a photosensitizer, when exposed to UV light at λmax = 351 nm. The antioxidant activity of compounds was measured using ACL kits (Analytic Jena, Jena, Germany) with a Photochem apparatus (Analytic Jena, Jena, Germany). In the PCL-ACL assay, the photochemical generation of free radicals is combined with the sensitive detection using chemiluminescence. In ACL studies, the kinetic light emission curve was monitored for 3 min and expressed as mM Trolox equivalents. The areas under the curves were calculated using the PCL soft control and analysis software. The presence of Trolox (used as a standard for the calibration curve) or any antioxidants from the samples reduces the magnitude of the PCL signal, and hence, the area calculated from the integral. The observed inhibition of the signal was plotted against the concentration of Trolox added to the assay medium. The concentration of the added sample was such that the generated luminescence during the 3 min sampling interval fell within the limits of the standard curve. For ACL assay, 2.3 mL of reagent 1 (solvent and dilution reagent, methanol), 0.2 mL of reagent 2 (buffer solution), 25 μL of reagent 3 (photosensitizer: luminol 1 mmol L−1), and 10 μL of standard or sample solution were mixed and measured. Luminol is used as a photosensitizer and as a detecting substance for free radicals. Trolox were used for the standard calibration curve from 0.25 to 2 nM.
DPPH Assay
The DPPH assay was evaluated according to Aadil (2013) and Bonetti (2016) [36, 37]. Fifty microliters of samples (cortisone, SCA and SCB) were added to 1.450 μL of 0.06 mM of methanolic DPPH· radical solution. The reaction mixture was left to stand at room temperature in the dark for 15 min. The absorbance for the sample (Asample) was measured at 515 nm against methanol blank. A control was the absorbance of DPPH· solution. The percent inhibition of DPPH· radical was calculated according to the equation:
$${\text{Percent inhibition of DPPH}} \cdot {\text{ radical}} = \left[ {{1} - \left( {{\text{Asample}}/{\text{Acontrol}}} \right)} \right] \times {1}00$$
Methanolic solutions with different Trolox concentrations (0.05 − 1 mM/L) were analyzed for calibration curve. The free radical scavenging capacity was expressed as micromolar of Trolox equivalents (TEs)/g of SCA and SCB compounds using calibration curve.
Anti-inflammatory activity
Cell culture and treatments
HaCaT cells were cultured with Dulbecco’s modified Eagle’s medium High Glucose (Lonza, Milan, Italy) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine as previously described [38]. Cells were maintained in a humidified incubator at 37 °C with 5% CO2 and incubated until the monolayer was 80% confluent. Medium was changed every 2 days.
HaCaT cells were pretreated with or without 10 μM of both SCA and SCB for 24 h. Stock solutions of the two compounds were prepared in DMSO immediately before use and diluted to the final concentration in the culture medium. In some sets of experiments, betamethasone-17-valerate (10 µg/mL) served as a positive control; dimethyl sulfoxide (DMSO) served as a solvent control. After the pretreatments, cells were stimulated with 10 ng/mL TNFα for 1 h to induce the NFκB signaling pathway. Then, media were replaced with fresh culture medium. Cells and supernatants were collected at different time points for each set of experiments as described below.
Immunofluorescence
Briefly, HaCaT cells were seeded on coverslips at a density of 1 × 105 cell/mL and incubated with DMEM high glucose supplemented with FBS 10%. Then, HaCaT cells were treated as indicated above. At the end of the proinflammatory stimulus with TNFα, cells were fixed in 4% paraformaldehyde in PBS for 30 min at 4 °C as previously described [39]. HaCaT cells were permeabilized for 5 min at RT with PBS containing 0.2% Triton X-100. After the blocking with 1% BSA in PBS at RT for 1 h, HaCaT cells were incubated with p65 NF-κB primary antibody (1:100, sc-372; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) in PBS containing 0.5% BSA at 4 °C overnight. After washing, coverslips were incubated with appropriate secondary antibody for 1 h at RT. Nuclei were stained with 1 μg/mL DAPI (Sigma- Aldrich, Italy) for 1 min. Coverslips were mounted onto glass slides using anti-fade mounting medium 1,4 diazabicyclooctane (DABCO) in glycerin. Images were acquired with Leica AF CTR6500HS (Microsystems). Negative controls were performed by omitting primary antibodies. Fluorescence was examined using an Epifluorescence microscope (Nikon Eclipse E800; Nikon Corporation, Surrey, UK) equipped with a plan apochromat 100 × 0.5 − 1.3 oil immersion objective and a mercury lamp source.
Real-time PCR
Quantitative real-time PCR was carried out as described in detail previously [40]. Briefly, HaCaT cells were treated as indicated above and harvested for RNA extraction 3 h after the end of the proinflammatory stimulus with TNFα. Total RNA was extracted using an AURUM total RNA Mini Kit with DNase digestion (Bio-Rad, Italy). One microgram of total RNA for each condition was used to synthesize cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, Italy). Quantitative real-time PCR (qPCR) was performed using SYBR green on the CFX Multicolor real-time PCR detection system (Bio-Rad, Italy). Forward and reverse primers used are: 5′-GGTGCAGTTTTGCCAAGGAG-3′ and 5′-TTCCTTGGGGTCCAGACAGA-3′ respectively. Ribosomal proteins L13a (RPL13a) and L11a (RPL11a) and GAPDH were used as the endogenous controls. Forward and reverse primers are: RPL13a, 5′-CCTAAGATGAGCGCAAGTTGAA-3’ and 5′-CCACAGGACTAGAACACCTGCTAA-3’; RPL11a, 5′-TGCGGGAACTTCGCATCCGC-3′ and 5′-GGGTCTGCCCTGTGAGCTGC-3’; GAPDH, 5′-TGACGCTGGGGCTGGCATTG-3′ and 5′-GGCTGGTGGTCCAGGGGTCT-IL-8 3, F: 5′-GGTGCAGTTTTGCCAAGGAG-3′ and R: 5′-TTCCTTGGGGTCCAGACAGA-3′. The primer pairs were obtained from the Real-Time PCR GenBank Primer and Probe Database Primer Bank, RTPrimerDB. Real-time PCR was initiated with a 3-min hot-start denaturation step at 95 °C and then performed for 40 cycles at 95 °C for 3 s and 60 °C for 5 s. Samples were compared using the relative cycle threshold (CT). After normalization to more stable mRNA RPL13a, RPL11a, and GAPDH, the fold increase or decrease was determined with respect to control, using the formula 2 − ΔΔCT, where ΔCT is (gene of interest CT) (reference gene CT), and ΔΔCT is (ΔCT experimental) (ΔCT control).
IL-1α levels
IL-1α secretion, after the treatment with 10 and 20 µM of each compound and together at the same concentrations, was assayed by a commercially available ELISA kit (Thermo Scientific, Italy). At the end of the treatments, HaCaT cells were incubated for other 24 h. Then, cell-free supernatants were collected and assayed for IL-1α using a commercial ELISA kit, according to the manufacturer's instructions. The optical absorbance was measured with a spectrophotometer microplate reader at 450 nm and correction at 530 nm. Final concentrations were calculated by interpolation with a standard curve of Recombinant Human IL-1α provided by the kit and results expressed as pg/mL.
Cyclooxygenase inhibitory assay
Cyclooxygenase (COX) activity assay was determined by using a fluorescence COX activity assay kit (Cayman, No. 700200) for detecting COX-1 and COX-2 activity in purified enzyme and tissue homogenate preparations. The assay was conducted by monitoring the appearance of fluorescent compound resofurin in accordance to the supplier recommendation. Aspirin (acetylsalicylic acid; 50 µM) served as positive control. The test compounds were dissolved in 1–2% DMSO (v/v) at 4 different concentrations (0.1, 1, 10 and 100 µM) and added to a purified COX preparation. A 1–2% DMSO solution was added to control plates. The plates were shaken for a few seconds and incubated for 5 min at 25 °C. The fluorescence was measured by using an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Average fluorescent values were calculated for all the samples (n = 3/group) in order to determine the percentage of inhibition in respect to control (i.e. untreated) plates.
Glucocorticoid receptor transactivation assays
Glucocorticoid receptor transactivation assays kit was obtained from Indigo Biosciences (Human Glucocorticoid Receptor Reporter Assay Kit, Product #IB0020-32; Indigo Biosciences, State College, PA, USA) and was utilized to assess the activation of human glucocorticoid receptor by SCA and/or SCB. Briefly, stocks of the tested compounds were prepared and diluted in a medium provided by the manufacturer. Frozen reporter cells provided in the assay kit were thawed and compound dilutions were added immediately. Cells were incubated for 24 h and the activation response was measured on a luminometer (Perkin-Elmer, MA, USA). The cells consisted of non-human mammalian cells engineered by Indigo Biosciences to provide constitutive high-level expression of full length, unmodified human glucocorticoid receptor (NR3C1). The nonhuman mammalian reporter cells included a luciferase reporter gene functionally linked to a human glucocorticoid receptor-responsive promoter. The cells are engineered so that only interactions with the human receptor will induce luciferase expression in the treated reporter cells to quantitate nuclear receptor activation. Positive control ligand performance was measured by the manufacturer and provided in the technical manuals thus allowing accurate comparison for assay performance. Additionally, the control ligands of the receptors (dexamethasone; 0.1 µM) were tested on the same test plates (to allow statistical analysis) with the tested compounds and controls.
Effects of SCA and SCB on glutamate-induced neurotoxicity
Primary cultures of rat cortical neurons
Primary cultures of cortical neurons have been prepared from 1 day-old male rats [41]. After re-suspension in the plating medium, the cells were counted and then plated on poly-l-lysine (5 μg/mL)-coated dishes at a density of 2.5 × 106 cells/dish or on poly-l-lysine (5 μg/mL)-coated 24-well plates at a density of 0.5 × 106 cells/dish. The plating medium consisted of Eagle's Basal Medium supplemented with 10% inactivated fetal calf serum, 25 mM KCl, 2 mM glutamine and 100 μg/mL gentamycine. Cultures were grown at 37 °C in a humidified atmosphere of 5% CO2/95% air. Cytosine arabinoside (10 μM) was added within 24 h of plating to prevent glial cell proliferation [41]. The cultures were maintained for 8 days in vitro before experiments.
Glutamate-induced neurotoxicity
Following the removal of the growth medium, the cultures were exposed for 10 min to glutamate 3 μM in a Mg2+-free Krebs–Ringer bicarbonate buffer at 37 °C in a 5% CO2/95% air atmosphere. The cultures were then returned to the incubator in their growth medium. 24 h later, the glutamate-induced neurotoxicity was evaluated by measuring the neuronal viability. The different drugs tested were added 15 min before glutamate exposure and were present until the end of the experiment.
Evaluation of the neuronal viability by MTT reduction assay
The integrity of mitochondrial enzymes in viable neurons was determined with a colorimetric assay using 3-[4,5-dimethylthial-2-yl]-3, 5-diphenyl-tetrazolinium bromide (MTT) salt, as described by Mosdam [42]. In live cells, mitochondrial enzymes have the capacity to transform MTT salt into MTT formazan. Briefly, MTT salt (5 mg/mL in PBS 0.1 N) was added to the neuronal cultures and incubated 4 h at 37 °C. The precipitated dye was dissolved in isopropanol with 1 M HCl and colorimetrically (absorbance at 570 nm) quantified. The results were expressed as values of absorbance at 570 nm.
Statistical analysis
For each of the variables tested, statistical analysis was performed using one-way or two-way ANOVA followed by Bonferroni’s multiple comparison test. A significant effect was indicated by a P-value < 0.05. Data are expressed as mean ± SEM of triplicate determinations obtained from three independent experiments.
