Male adult Sprague-Dawley rats (200–250 g) were housed in Plexiglass cages (40 cm × 25 cm × 15 cm), two rats per cage, in climatized colony rooms (22 ± 1 °C; 60% humidity), on a 12 h/12 h light/dark cycle (light phase: 07:00–19:00 h), with free access to tap water and food, 24 h/day throughout the study, with no fasting periods. Rats were fed a standard laboratory diet (3.5% fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value; 3.20 kcal/g).
Housing conditions and experimentation procedures were strictly in accordance with the European Union ethical regulations on the care of animals for scientific research.
According to the recognized ethical principles of “Replacement, Refinement, and Reduction of Animals in Research”, colon, liver, heart, and prefrontal cortex specimens were obtained as residual material from vehicle-treated rats randomized in our previous experiments approved by the Local Ethical Committee of University “G. d’Annunzio” and the Italian Health Ministry (Italian Health Ministry authorization N. 880, delivered on 24th August 2015). Rats were sacrificed by CO2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per min) and colon, liver, heart, and prefrontal cortex specimens were immediately collected and maintained in a humidified incubator with 5% CO2 at 37 °C for 4 h, in DMEM buffer with added bacterial LPS (10 μg/mL) (incubation period).
During the incubation period, tissues were treated with scalar concentrations of MOMAST(®) HY100 (10, 50, and 100 µg/mL) and MOMAST(®) HP30 (22, 110, and 220 µg/mL). Tissue supernatants were collected, and the PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by radioimmunoassay (RIA), as previously reported [13 (link),53 (link)]. Briefly, specific anti-8-iso-PGF2α and anti-PGE2 were developed in the rabbit; the cross-reactivity against other prostanoids is <0.3%. One hundred microliters of prostaglandin standard or sample were incubated overnight at 4 °C with the 3H-prostaglandin (3000 cpm/tube; NEN) and antibody (final dilution: 1:120,000; kindly provided by Prof. G. Ciabattoni), in a volume of 1.5 mL of 0.025 M phosphate buffer. Free and antibody-bound prostaglandins were separated by the addition of 100 μL 5% bovine serum albumin and 100 μL 3% charcoal suspension, followed by centrifuging for 10 min at 4,000× g at 5 °C and decanting off the supernatants into scintillation fluid (Ultima Gold™, Perkin Elmer, Waltham, MA, USA) for β emission counting. The detection limit of the assay method was 0.6 pg/mL. Additionally, tissue supernatants were assayed for lactate dehydrogenase (LDH) activity [54 (link)]. LDH activity was measured by evaluating the consumption of nicotinamide adenine dinucleotide dehydrogenase (NADH) in 20 mM HEPES-K+ (pH 7.2), 0.05% bovine serum albumin, 20 μM NADH, and 2mM pyruvate using a microplate reader (excitation 340 nm, emission 460 nm) according to manufacturer′s protocol (Sigma-Aldrich, St. Louis, MO). LDH activity was measured by evaluating the consumption of NADH in 20 mM HEPES-K+ (pH 7.2), 0.05% bovine serum albumin, 20 μM NADH and 2 mM pyruvate using a microplate reader (excitation 340 nm, emission 460 nm) according to manufacturer′s protocol. In addition, individual prefrontal cortex, colon, liver, and heart specimens were quickly dissected to evaluate cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) gene expression, as previously reported [55 (link),56 (link)]. Tissue specimens were dissected and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA) at −20 °C until further processed. Total RNA was extracted from the tissues using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s protocol. One microgram of total RNA extracted from each sample in a 20-μL reaction volume was reverse transcribed using a high capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA, USA). Reactions were incubated in a 2720 thermal cycler (Life Technologies, Carlsbad, CA, USA) initially at 25 °C for 10 min, then at 37 °C for 120 min, and finally at 85 °C for 5 s. Gene expression was determined by quantitative real-time PCR using TaqMan probe-based chemistry (Life Technologies, Carlsbad, CA, USA). Reactions were performed in MicroAmp Fast Optic 96-well Reaction Plates (Life Technologies, Carlsbad, CA, USA) on an ABI PRISM 7900 HT fast real-time PCR system (Life Technologies, Carlsbad, CA, USA). PCR primers and TaqMan probes were obtained from Life Technologies (Assays-on-Demand Gene Expression Products, Rn01483828_m1 for COX-2 gene, Rn01525859_g1 for TNFα, Rn00561646_m1 for iNOS. β-actin (Life Technologies, Carlsbad, CA, USA, Part No. 4352340E) was used as the housekeeping gene. The real-time PCR was carried out in triplicate. Data were elaborated with the sequence detection system (SDS) software version 2.3 (Applied Biosystems, Foster City, CA, USA). The comparative 2−ΔΔCt method was used to quantify the relative abundance of mRNA and then determine the relative changes in individual gene expression (relative quantification) [57 (link)].
Housing conditions and experimentation procedures were strictly in accordance with the European Union ethical regulations on the care of animals for scientific research.
According to the recognized ethical principles of “Replacement, Refinement, and Reduction of Animals in Research”, colon, liver, heart, and prefrontal cortex specimens were obtained as residual material from vehicle-treated rats randomized in our previous experiments approved by the Local Ethical Committee of University “G. d’Annunzio” and the Italian Health Ministry (Italian Health Ministry authorization N. 880, delivered on 24th August 2015). Rats were sacrificed by CO2 inhalation (100% CO2 at a flow rate of 20% of the chamber volume per min) and colon, liver, heart, and prefrontal cortex specimens were immediately collected and maintained in a humidified incubator with 5% CO2 at 37 °C for 4 h, in DMEM buffer with added bacterial LPS (10 μg/mL) (incubation period).
During the incubation period, tissues were treated with scalar concentrations of MOMAST(®) HY100 (10, 50, and 100 µg/mL) and MOMAST(®) HP30 (22, 110, and 220 µg/mL). Tissue supernatants were collected, and the PGE2 and 8-iso-PGF2α levels (ng/mg wet tissue) were measured by radioimmunoassay (RIA), as previously reported [13 (link),53 (link)]. Briefly, specific anti-8-iso-PGF2α and anti-PGE2 were developed in the rabbit; the cross-reactivity against other prostanoids is <0.3%. One hundred microliters of prostaglandin standard or sample were incubated overnight at 4 °C with the 3H-prostaglandin (3000 cpm/tube; NEN) and antibody (final dilution: 1:120,000; kindly provided by Prof. G. Ciabattoni), in a volume of 1.5 mL of 0.025 M phosphate buffer. Free and antibody-bound prostaglandins were separated by the addition of 100 μL 5% bovine serum albumin and 100 μL 3% charcoal suspension, followed by centrifuging for 10 min at 4,000× g at 5 °C and decanting off the supernatants into scintillation fluid (Ultima Gold™, Perkin Elmer, Waltham, MA, USA) for β emission counting. The detection limit of the assay method was 0.6 pg/mL. Additionally, tissue supernatants were assayed for lactate dehydrogenase (LDH) activity [54 (link)]. LDH activity was measured by evaluating the consumption of nicotinamide adenine dinucleotide dehydrogenase (NADH) in 20 mM HEPES-K+ (pH 7.2), 0.05% bovine serum albumin, 20 μM NADH, and 2mM pyruvate using a microplate reader (excitation 340 nm, emission 460 nm) according to manufacturer′s protocol (Sigma-Aldrich, St. Louis, MO). LDH activity was measured by evaluating the consumption of NADH in 20 mM HEPES-K+ (pH 7.2), 0.05% bovine serum albumin, 20 μM NADH and 2 mM pyruvate using a microplate reader (excitation 340 nm, emission 460 nm) according to manufacturer′s protocol. In addition, individual prefrontal cortex, colon, liver, and heart specimens were quickly dissected to evaluate cyclooxygenase (COX)-2, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) gene expression, as previously reported [55 (link),56 (link)]. Tissue specimens were dissected and stored in RNAlater solution (Life Technologies, Carlsbad, CA, USA) at −20 °C until further processed. Total RNA was extracted from the tissues using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer’s protocol. One microgram of total RNA extracted from each sample in a 20-μL reaction volume was reverse transcribed using a high capacity cDNA reverse transcription kit (Life Technologies, Carlsbad, CA, USA). Reactions were incubated in a 2720 thermal cycler (Life Technologies, Carlsbad, CA, USA) initially at 25 °C for 10 min, then at 37 °C for 120 min, and finally at 85 °C for 5 s. Gene expression was determined by quantitative real-time PCR using TaqMan probe-based chemistry (Life Technologies, Carlsbad, CA, USA). Reactions were performed in MicroAmp Fast Optic 96-well Reaction Plates (Life Technologies, Carlsbad, CA, USA) on an ABI PRISM 7900 HT fast real-time PCR system (Life Technologies, Carlsbad, CA, USA). PCR primers and TaqMan probes were obtained from Life Technologies (Assays-on-Demand Gene Expression Products, Rn01483828_m1 for COX-2 gene, Rn01525859_g1 for TNFα, Rn00561646_m1 for iNOS. β-actin (Life Technologies, Carlsbad, CA, USA, Part No. 4352340E) was used as the housekeeping gene. The real-time PCR was carried out in triplicate. Data were elaborated with the sequence detection system (SDS) software version 2.3 (Applied Biosystems, Foster City, CA, USA). The comparative 2−ΔΔCt method was used to quantify the relative abundance of mRNA and then determine the relative changes in individual gene expression (relative quantification) [57 (link)].
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