Scientific Papers

All-trans retinoic acid alleviates transmissible gastroenteritis virus-induced intestinal inflammation and barrier dysfunction in weaned piglets | Journal of Animal Science and Biotechnology

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Animals, diets and experimental design

All animal procedures used in this study were approved by the Animal Care and Use Committee of Sichuan Agricultural University (license number: CD-SYXK-2017-015). In a 19-d study, 32 TGEV-seronegative piglets [Duroc × (Landrace ×Yorkshire)] with initial body weight of 7.63 ± 0.33 kg were weaned at 21 days of age and randomly divided into 4 treatments (n = 8): (1) Control group (piglets fed basal diet and infused with the sterile DMEM medium); (2) TGEV group (piglets fed basal diet and infused with TGEV); (3) TGEV + ATRA5 group (piglets fed basal diet and received oral administration of 5 mg ATRA daily and infused with TGEV); and (4) TGEV + ATRA15 group (piglets fed basal diet and received oral administration of 15 mg ATRA daily and infused with TGEV). Pigs were housed individually in metabolic cages (1.5 m × 0.7 m × 1.0 m) of two environmentally controlled rooms (25–28 °C) with strictly control to avoid cross-infection. The basal diet (Table 1) was formulated to meet or exceed the swine nutrient requirements recommended by the National Research Council (NRC, 2012) [17], except for vitamin A, which was not prepared in the vitamin premix. ATRA (≥ 98% HPLC) was purchased from Sigma-Aldrich (Shanghai, China) and dissolved in corn oil at a dose of 5 or 15 mg/mL. On d 14 of the trial, piglets in the challenged groups were orally administered TGEV at a dose of 2 × 108.85 TCID50 (TGEV was provided by Prof. Zhiwen Xu, College of Veterinary Medicine, Sichuan Agricultural University), while those in control group received the same volume of sterile DMEM medium. The dose of TGEV was chosen according to our preliminary studies, which showed that it significantly induced diarrhea in weaned piglets. All piglets were executed on d 19 to collect samples.

Table 1 Composition and nutrient levels of the basal diet (as-fed basis)

Growth performance and diarrhea rate

Piglets were weighed individually on d 0, 14 and 19 of the experiment, and the daily feed intake was recorded. These values were used to calculate average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G). After the TGEV challenge, piglets were observed daily to record the health status and diarrhea incidence. Fecal consistency was scored as described by Pu et al. [18]: 0 = normal, 1 = pasty, 2 = semiliquid, and 3 = liquid. Score ≥ 2 was considered diarrhea. Diarrhea rate (%) = (days of piglet diarrhea/test days) × 100.

Sample collection

On day 19 of the trial, blood samples were collected in 10-mL vacuum tubes without anticoagulant via anterior vein cava of each piglet, and stood at room temperature for 30 min. Blood was centrifuged at 3,500 r/min for 10 min at 4 °C, and serum samples were collected and stored at −20 °C until further assay. All piglets were euthanized by intramuscular injection of Shumianning (Active ingredients: tiletamine, xylazine and midazolam; 0.08 mL/kg body weight; Zhengzhou Huiji Animal Health Care Products Company, Henan, China). Then, the jejunum was quickly isolated and flushed with ice-cold saline. Mucosal samples from the middle portion of jejunum were scraped and collected by using a sterile glass microscope slide, quickly frozen in liquid nitrogen, and then stored at −80 °C until analysis.

Disaccharidase activity measurement

Approximately 0.5 g of frozen jejunal mucosa was homogenized in ice-cold physiological saline at a ratio of 1:9 (w/v) for 15 min. The homogenate was then centrifuged at 3,500 r/min for 15 min at 4 °C, and the supernatant was collected for disaccharidase activity measurement. The activities of maltase, lactase and sucrase in the jejunal mucosa were measured by commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s instructions. The concentration of total protein in the supernatant was determined by the BCA protein assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China). The activities of disaccharidase were presented as U/mg protein.

Determination of serum diamine oxidase activity

The activity of diamine oxidase (DAO) in serum was detected according to the instructions of a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Jiangsu Meimian Biotechnology Co., Ltd., Jiangsu, China).

Determination of cytokines and secretory immunoglobulin A concentrations in jejunal mucosa

The concentration of cytokines (IL-1β, IL-6, IL-8, IL-10 and TNF-α) and secretory immunoglobulin A (sIgA) in jejunal mucosa of piglets were measured by using ELISA kits (Jiangsu Meimian Biotechnology Co., Ltd., Jiangsu, China) specific for swine according to the manufacturer’s instructions. Before assays, about 0.5 g of frozen jejunal mucosa was homogenized in ice-cold saline and prepared into a 10% homogenate, and then centrifuged (3,500 r/min, 15 min, 4 °C). The collected supernatant was used to determine cytokines and sIgA concentration.

Quantitative real-time PCR analysis

Total RNA from the jejunal mucosa was extracted using TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Dalian, China) in accordance with the manufacturer’s instructions. The concentration and purity of total RNA were analyzed by using a spectrophotometer (Beckman Coulter DU800; Beckman Coulter Inc.). 1 µg of total RNA was reverse transcribed into cDNA using the PrimeScripte RT reagent kit (TaKaRa). Then, the synthesized cDNA was diluted (1:4) and quantitative real-time PCR was performed using SYBR Premix Ex Taq™ reagents (TaKaRa) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The primer sequences were synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China) and listed in Table 2. The relative mRNA expression of target genes was calculated with the 2−ΔΔCt method and using β-actin as the reference gene [19].

Table 2 Primer sequences of the target genes

Western blot analysis

Proteins from jejunal mucosa were extracted with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentrations in the supernatants were measured by a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (25 µg) were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes using a wet Trans-Blot system (Bio-Rad). The membranes were blocked with 5% nonfat dry milk for 1 h at room temperature, and then incubated overnight at 4 °C with corresponding primary antibodies: anti-ZO-1 (Invitrogen, USA), anti-occludin (Abcam, UK), anti-claudin-1(Abcam, UK), anti-IκBα (Cell Signaling Technology, Danvers, MA, USA), anti-NF-κB p65 (Cell Signaling Technology), anti-p-NF-κB p65 (Cell Signaling Technology) and anti-β-actin (Santa Cruz, USA). After washing, the blots were incubated with goat anti-rabbit/mouse IgG-HRP secondary antibody (Santa Cruz, USA) for 1 h at room temperature. Visualization of blots was conducted with the ECL chemiluminescence kit (Beyotime Biotechnology, Shanghai, China) and the ChemiDoc™ XRS Imager System (Bio-Rad). The protein bands were analyzed by Image Lab 5.1 software. The results were represented as the ratio of the optical density of the target protein bands to the respective β-actin band.

Statistical analysis

The Shapiro–Wilk test was performed before statistical analysis to determine normality of variance. Data were analyzed by one-way analysis of variance (ANOVA) of SPSS 22.0 software (SPSS Inc., Chicago, IL, USA), using each piglet as the experimental unit. Differences between means were determined using Tukey’s multiple-comparison test. Moreover, the Chi-square test was used to analyze diarrhea rate. Results were presented as mean ± standard error (SE). P ≤ 0.05 was considered statistically significant.

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