Animals and experimental design
This study was approved by the Animal Care and Use Committee of the Feed Research Institute of the Chinese Academy of Agricultural Sciences. A total of 80 Large White × Landrace F1 crossbred piglets (40 barrows and 40 females), with similar initial body weights (BWs, 7.35 ± 0.14 kg) and the same age (23 days), were randomly assigned to 4 treatments, with 5 pens per treatment and 4 piglets per pen according to BW and sex (half male and half female), for a 72-day trial. The dietary treatments included a corn-soybean meal basal diet supplemented with 0 (control group), 25, 50 or 100 mg/kg daidzein. The daidzein (purity ≥ 98%) used in this experiment was purchased from Guanghan Biochemical Products Co., Ltd. (Guanghan, China). The diets were formulated according to National Research Council (2012) nutrient requirements [15], and the composition and nutrient levels in the basal diets are shown in Table 1. The barn was maintained at a temperature between 25 °C and 28 °C with a 12-h light/dark cycle. Throughout the experiment, the pigs were allowed ad libitum access to water and feed.
Sample collection
On days 14, 28 and 42 of the trial, one piglet from each pen was selected randomly to collect blood samples via jugular veins. Then, blood samples were centrifuged at 3,000×g for 10 min at 4 °C to obtain plasma; subsequently, the plasma was stored at −20 °C until analysis.
Growth performance measurement
Pigs were individually weighed on day 0 of the trial. However, BW by pen was measured on days 14, 28, 42 and 72 of the trial. Feed intake was recorded daily, and the residual feed was measured when pigs were weighed. Growth performance was evaluated by calculating the average daily gain (ADG), average daily feed intake (ADFI) and feed conversion rate (FCR) for each pen.
Assay of plasma antioxidant indices
The activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) in the plasma were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
Cell culture
IPEC-J2 cells were obtained from Dr. Guoyao Wu’s laboratory at Texas A&M University and cultured in Dulbeco’s modified Eagle’s medium/F12 (DMEM/F12, Thermo Fisher Scientific, MA, USA) supplemented with 5% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA), 0.1% ITS (5 μg/L insulin, 5 μg/L transferrin and 5 ng/L selenious acid, Corning Inc., NY, USA), 0.01% epidermal growth factor (5 μg/L, Corning Inc., NY, USA) and 1% pen-strep (Thermo Fisher Scientific, MA, USA) at 37 °C in a humidified atmosphere with 5% CO2. Passage 13-15 cells were used in our experiment.
Establishment of cell oxidative stress model
To select the optimal H2O2 concentration, IPEC-J2 cells were seeded at 1×105 cells/mL (100 μL per well) in 96-well plates (Corning Inc., NY, USA) with 6 replications (wells) per treatment. After 48 h of incubation, oxidation was induced by exposing IPEC-J2 cells to 0, 0.2, 0.4, 0.6, and 0.8 mM H2O2 for another 1 h. Subsequently, the supernatant was removed, the cells were washed twice with PBS (pH 7.4, Thermo Fisher Scientific, MA, USA), and cell viability was determined using a cell counting kit (CCK-8) (MedChemExpression, NJ, USA) according to the manufacturer’s instructions. Briefly, 110 μL of FBS-free DMEM/F-12 (containing 10 μL of CCK-8 reagent) was added to each well, and after 3 h of incubation at 37 °C, the absorbance was measured at 450 nm using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., VT, USA). Cell viability was calculated using the following equation: Cell viability = (As - Ab)/(Ac - Ab) ×100%. As represents the absorbance of the H2O2-treated group, Ac represents the absorbance of the H2O2 untreated group, and Ab represents the absorbance of the blank group which contained culture medium and CCK-8 without cells and H2O2. The cell viability of the H2O2 untreated group was considered 100%.
Selection of daidzein concentration
Daidzein was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) at 10 mg/mL and diluted to the final concentration in medium before use. To select the optimal daidzein concentration, IPEC-J2 cells were seeded at 1×105 cells/mL (100 μL per well) in 96-well plates (Corning Inc., NY, USA) with 6 replications (wells) per treatment. After 24 h of incubation, daidzein at different concentrations (0, 20, 40, 60, 80, 100 μM) was added to the wells and incubated for another 24 h. In addition, the daidzein untreated group contained 0.2% DMSO. Then, 0.6 mM H2O2 was added to daidzein treated or untreated wells and incubated for 1 h. Cell viability was tested with the CCK-8 assay as described above.
Measurement of intracellular reactive oxygen species (ROS)
IPEC-J2 cells were seeded at 1×105 cells/mL (100 μL per well) in 96-well plates (Corning Inc., NY, USA) with 6 replications (wells) per treatment, pretreated with or without 40 μM daidzein for 24 h, and then treated with or without 0.6 mM H2O2 for 1 h. At the end of the experiment, cells were incubated with DCFH-DA probes (Beyotime Biotechnology, Shanghai, China) for 30 min and then washed twice with PBS (pH 7.4). The fluorescence was read at 488 nm for excitation and 525 nm for emission with a fluorescence microplate reader (Infinite M Plex, Tecan, Männedorf).
Measurements of SOD, CAT and GSH-Px activity and MDA content
IPEC-J2 cells were seeded at 1.5×105 cells/mL (2 mL per well) in 6-well plates (Corning Inc., NY, USA) with 6 replications (wells) per treatment, pretreated with or without 40 μM daidzein for 24 h, and then treated with or without 0.6 mM H2O2 for 1 h. The supernatant was removed, and the cells were washed twice with ice-cold PBS (pH 7.4) and lysed using RIPA buffer (Thermo Fisher Scientific, MA, USA) containing 1% protease inhibitors and a phosphatase inhibitor cocktail (Thermo Fisher Scientific, MA, USA) for 30 min at 4 °C. The supernatant was collected after centrifugation at 13,000×g for 30 min at 4 °C and stored at −20 °C. The SOD, CAT and GSH-Px activities and MDA content were determined using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.
RNA isolation, reverse transcription and quantitative real-time PCR (qRT-PCR)
IPEC-J2 cells were seeded at 1.5×105 cells/mL (1 mL per well) in 12-well plates (Corning Inc., NY, USA) with 6 replications (wells) per treatment, pretreated with or without 40 μM daidzein for 24 h, and then treated with or without 0.6 mM H2O2 for 1 h. At the end of the experiment, cells were washed twice with ice-cold PBS (pH 7.4); subsequently, total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. The concentration and quality of total RNA were determined using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., VT, USA). Reverse transcription was performed using the TransScript First-Strand cDNA Synthesis Super Mix Reagent Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. qRT-PCR was performed using SYBR Green as a reagent (Thermo Fisher Scientific, MA, USA) on a QuantStudio™ Real-Time PCR System (Thermo Fisher Scientific, MA, USA). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference. The gene expression of superoxide dismutase 1 (SOD1), CAT, glutathione peroxidase 1 (GPX1), nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), zonula occludens-1 (ZO-1), occludin and claudin 1 was measured. The primer sequences used for qRT-PCR are shown in Table 2. The comparative CT method was used [16], determining fold changes in gene expression, calculated as 2-ΔΔCT. The relative expression of each target gene was normalized to the mRNA level of the GAPDH gene.
Western blotting
IPEC-J2 cells were seeded at 1.5×105 cells/mL (2 mL per well) in 6-well plates (Corning Inc., NY, USA) with 4 replications (wells) per treatment, pretreated with or without 40 μM daidzein for 24 h, and then treated with or without 0.6 mM H2O2 for 1 h. At the end of the experiment, cells were washed twice with ice-cold PBS (pH 7.4) and lysed using RIPA buffer (Thermo Fisher Scientific, MA, USA) containing 1% protease inhibitors and a phosphatase inhibitor cocktail (Thermo Fisher Scientific, MA, USA) for 30 min at 4 °C. The supernatant was collected after centrifugation at 13,000×g for 30 min at 4 °C, and the protein concentration was determined using a BCA protein assay kit (Applygen, Beijing, China). For denaturation, 25 μg of protein and 4× loading buffer (Bio-Rad Laboratories Inc., CA, USA) were boiled at 95 °C for 10 min. The denatured proteins were separated by SDS-PAGE (12%) and subsequently transferred to PVDF membranes (Bio-Rad Laboratories Inc., CA, USA) for 2 h at 200 mA using the Bio-Rad Mini-PROTEAN Tetra electrophoresis system (Bio-Rad Laboratories Inc., CA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 3 h at room temperature and then incubated with primary antibodies (Nrf2, ab92946, Abcam, Cambridge, UK, diluted 1:1000; ZO-1: 61-7300, Thermo Fisher Scientific, MA, USA, diluted 1:1000; Occludin: ab31721, Abcam, Cambridge, UK, diluted 1:1000; GAPDH: #2118, CST, Boston, USA, diluted 1:2000) at 4 °C overnight. After washing 3 times with TBST to remove residual primary antibodies, the membranes were incubated with secondary antibodies for 1 h at room temperature. The membranes were washed 3 times with TBST to remove residual secondary antibodies, and an ECL agent was added for chemiluminescence imaging. The images were detected by a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc., CA, USA). GAPDH was used as an internal reference.
Statistical analysis
Data related to growth performance were analyzed by ANOVA using a completely randomized block design with SPSS 20.0. The remaining data were analyzed using the one-way ANOVA procedure of SPSS 20.0. The pen represents the experimental unit for growth performance, and the individual piglet is the experimental unit for antioxidant parameters. Treatment comparisons were performed using Tukey’s honest significant difference test for multiple testing. Significant differences among the treatments were determined at P < 0.05, whereas a treatment effect trend was noted for 0.05 < P < 0.10.