Animals, experimental design, and sample collection
The animal protocol was approved by the Animal Care Committee of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. Twenty-four weaned piglets (Duroc×Landrace×Yorkshire, age of 21d) were randomly assigned to 1 of 4 treatments based on BW and sex, each treatment with 6 piglets and 1 piglet per pen in a temperature-controlled room. The control (Control) and NA-treated (NA) groups were administered 20 mL normal saline or 20 mL NA solution (40 mg NA was dissolved in equal volume of normal saline). The K88 challenged (K88) and NA-treated plus K88 challenged group (K88+NA) groups were administered 20 mL normal saline or 20 ml nicotinic acid solution once daily for 3 consecutive days. On the fourth day, the K88 and K88+NA groups were treated with oral administration of 4*109 cfu /mL ETEC K88. All piglets were provided with access to water ad libitum. The piglets were checked daily for signs of diarrhea. At the end of experiment, the animals were individually weighed, weight loss of piglets was counted. Samples of the duodenum, jejunum, ileum, and colon were collected for analysis. Serum was obtained from the separation gel coagulation promoting tubes after centrifugation at 3000×g for 15 min at 4℃ and stored immediately at -20℃.
Bacterial plate counting analysis
About 2.5 g liver and spleen tissues of pigs in 2.25 ml sterilized buffer liquid, and then homogenized. Aliquots of 10 ml of the dilutions to be analyzed are placed into LB agar medium flat plate, test three parallel plates for each sample. Plates are inverted and incubated for18 to 24 h at 37°C in a constant temperature incubator. Calculate the CFUs of bacterial transfer as the weighted mean from the successive dilutions, which contain between 30 and 300 colonies. The calculate result is the weighted means of the successive dilution multiply by dilution factor.
Intestinal morphology analysis
Formalin-fixed duodenum, jejunum, ileum, and colon samples were embedded in paraffin wax. Segment cross sections were microtomed at approximately 5μm thick and stained with haematoxylin and eosin (H&E). In each section, villus height and associated crypt depth were measured using a DM3000 microscope (Leica Microsystems, Wetzlar, Germany). Images were obtained via using a DM3000 microscope (Leica Microsystems, Wetzlar, Germany). For each section, measurements of 6, well-orientated and intact villi were examined in each piglets’ duodenum, jejunum, and ileum. In the end, the mean villus height was then calculated per piglet with Image-Pro software (Media Cybernetics, Rockville, MD). Histopathologic damage scores were determined according to the statement in Feng's publication [11].
Immunoglobulins, cytokines and biochemistry measurements
The concentrations of secretory IgA (SIgA) in the jejunal and ileal mucosa of piglets were determined using the commercially available enzyme-linked immunosorbent assay (ELISA) kits from TSZ ELISA (Framingham, MA) according to the manufacturer’s instructions. The concentrations of IgM, IgA, IgG, IL-6, IL-8, TNF-α, and IFN-β in serum of piglets were determined using ELISA kits from Nuoyuan Co., Ltd. (Shanghai, China). The concentrations of SIgA, IgM, IgA, IgG, IL-6, IL-8, TNF-α and IFN-β were quantified by using a BioTek Synergy HT microplate reader (BioTek Instruments, Winooski, VT), and absorbance was measured at 450 nm. Serum samples were analyzed by ABX Pentra 400 Clinical Chemistry Analyzer (Horiba ABX, Northampton, UK) for total protein (TP), albumin (ALB), globulin (GLOB), glucose (GLU), lactic dehydrogenase (LDH), blood urea nitrogen (BUN), aspartate aminotransferase (AST) and alkaline phosphatase (ALP).
Analysis of intestinal microbiota via 16S rRNA gene sequencing
The contents in the colon of the piglets was aseptically collected, and the total DNA of the colonic contents was extracted using a DNA Kit (SimGEN, Hangzhou, China) according to the instructions provided by the manufacturer. Subsequently, the purity and yield of the DNA samples were quantified using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA) spectrophotometer. Then, twenty-four samples (n= 6) were sequenced on an Illumina HiSeq PE250 platform provided by Novogene (Beijing, China). Paired-end reads from the original DNA fragments were merged by using FLASH. Clustering was performed using the UPARSE pipeline, and sequences were classified into different operational taxonomic units (OTUs) based on the sequence similarity cut-off value (i.e., 97 %). Lastly, the diversity and composition of the bacterial communities were determined by α and β diversity according to Novogene’s recommendations. At the phylum, class and order levels, LEfSe was used to identify metagenomic biomarkers, while linear discriminant analysis (LDA) was used to estimate the effect of abundance of each species on the difference between groups.
Untargeted metabolomic analysis of colonic contents
Metabolite extractions: equal volume of liquid samples was dried on a freeze-drier, then 0.5 mL cold extraction solvent methanol/acetonitrile/H2O (2:2:1, v/v/v) was added to the sample, and adequately vortexed. After vortexing, the samples were incubated on ice for 20 minutes, and then centrifuged at 14,000 g for 20 minutes at 4°C. The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were re-dissolved in 100 μL acetonitrile/water (1:1, v/v) solvent and transferred to LC vials. LC-MS analysis, data analysis and bioinformatics analysis were performed according to the method in our previous study [12].
Relative quantitative in real-time PCR
Total RNA was extracted from the intestinal tissue samples using Trizol reagent (Invitrogen, Carlsbad, CA). The amount of RNA extracted was determined and its purity was verified using NanoDrop 1000 (Thermo Fisher Scientific). Contaminant DNA was removed by gDNA Eraser (Takara, Dalian, China). The cDNA was generated using 1μg aliquot of total RNA with a PrimeScript RT Reagent Kit (Takara). Synthesized cDNA was stored at −20°C prior to real-time PCR analysis.
Real-time PCR was performed using a CFX Connect Detection system (Bio-Rad, Hercules, CA). The sequences of primers used in this study were listed in Table 1. Primers for specific porcine genes were synthesized by Biotechnology Inc. The cDNA was amplified with SYBR® Premix DimerEraser™ (TakaRa Biotechnology Inc.) containing 4-μL 20-fold diluted cDNA, 0.5 μL primers F (10 μM), 0.5 μL primers R (10 μM), 5 μL iTaq Universal SYBR Green Supermix (Bio-Rad), The PCR amplification was performed using the following conditions: 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, 60°C for 30 s, and 72℃ for 30 s. The melting curves were systematically analyzed to evaluate the specificity after each run. All reactions were conducted in triplicate. To evaluate the relative quantification of mRNA expression, the cycle threshold (CT) values of the target genes were normalized to the CT-values of the β-actin, and the results were presented as fold changes using the 2–ΔΔCt method.
Western blot analysis
Total protein was extracted from intestinal tissue samples using lysis buffer (KeyGEN, Nanjing, China). The protein concentrations of each sample was calculated with the BCA protein assay kit. Protein was used for western blot analysis, after adding 6× concentrated sample buffer (0.5M Tris, 30% glycerol, 10% SDS, 0.6M DTT, 0.012% bromophenol blue) and heating the samples for 5 min at 95°C. Proteins in supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride membranes (PVDF) membranes. Next, the membranes were incubated with the primary antibodies for β-actin (Abcam, MA, USA), SIRT1, HDAC7, acH3, acH3K9, acH3K27 and pH3S10 (Abcam) overnight at 4°C after blocked with Tris-buffered saline containing 0.1% Tween-20 (TBS-T) and 5% low-fat milk blocked for 1 h at RT. After washing for 1 h in TBS-T five times, the membranes were incubated for 1 h at RT with horseradish peroxidase (HRP) secondary antibodies. Protein immunoreactive bands were photographed. Each special banding gray value was digitized using ImageJ software, and the gray value of target protein was divided by internal reference β-actin.
Statistical analysis
Statistical significance analysis of the experimental data was determined by the analysis of variance (ANOVA) with Duncan's multiple range test using SPSS 18.0 software (SPSS Inc., Chicago, IL). Values are given as means ± SEM. The difference was considered to be significant at P < 0.05.