Table_1_Effects of Immobilized Antimicrobial Peptides on Growth Performance, Serum Biochemical Index, Inflammatory Factors, Intestinal Morphology, and Microbial Community in Weaning Pigs.docx

This experiment was conducted to investigate the effects of immobilized antimicrobial peptides on growth performance, serum biochemical index, inflammatory factors, intestinal morphology, and microbial community of weaning piglets. A total of 21 weaning piglets [Duroc × (Landrace × Yorkshire)] with initial body weight (7.64 ± 0.65 kg) were randomly allocated to one of three treatments with seven replicates (one pig per replicate) per treatment according to sex and weight in randomized complete block design. Pigs in the three treatments were fed corn–soybean meal-based diet (CON), corn–soybean meal based diet + flavomycin (25 mg/kg) + quinone (50 mg/kg) (AB), and corn–soybean meal based diet + 1,000 mg/kg immobilized antimicrobial peptides (IAMPs), respectively. The experiment lasted for 28 days, including early stage (0–14 days) and late stage (15–28 days). The results showed the following: (1) compared with the CON group, the average daily gain in the whole experimental time (p < 0.05) was significantly increased, and the diarrhea rate of weaning piglets was decreased (p < 0.01) in the IAMPs group; (2) compared with the CON group, the concentrations of serum IgM and superoxide dismutase (SOD) in the IAMPs group were significantly higher than the CON and AB groups (p < 0.01); (3) compared with CON group, the concentrations of serum interleukin (IL)-10 and transforming growth factor (TGF-β) were significantly increased (p < 0.05), and the concentration of IL-12 was significantly decreased (p < 0.05) in the IAMPs group; (4) compared with CON group, the concentrations of serum endotoxin and D-lactate of piglets were significantly reduced (p < 0.05), and the relative expression of ZO-1 and occludin in the jejunum of piglets were significantly increased (p < 0.05) in the IAMPs group; (5) compared with the CON group, the villus height of the duodenum and jejunum of weaning piglets in IAMPs and AB groups was significantly increased (p < 0.05); and (6) compared with CON group, the relative abundance of Escherichia–Shigella in the colon and cecal digesta was decreased. In summary, the addition of 1,000 mg/kg immobilized antimicrobial peptides in the diet effectively relieved weaning stress by showing improved growth performance, antioxidant and immune capacity, intestinal morphology, and microorganisms.</p

Additional file 5: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Figure S2. Effect of adding different level putrescine in the medium on the growth of IPEC-J2. The IPEC-J2 cells were seeded in 2% FBS containing DMEM/F12 supplemented with 0, 12.5, 25, 50, 100, or 200 μmol/L putrescine, cell numbers were determined at 0 h, 24 h, 48 h, 72 h, 96 h and 120 h with the CCK-8 kit. Cell growth was presented with plotting diagram (A), and was presented with histogram (B) to show the detail of difference among groups. Growth differences were presented with bar chart. Values are means ± SE, n = 4. Means with different letters are different (P < 0.05) in the histogram. IPEC-J2, porcine intestinal epithelial cells. (PDF 175 kb

Additional file 7 of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Figure S4. ERK inhibitor inhibited IPEC-J2 cells growth and ERK1/2 phosphorylation. A. The IPEC-J2 cells were seeded in 2% FBS containing DMEM/F12 supplemented with or without 200 μmol/L putrescine in the presence or absence of ulixertinib, a specific inhibitor for ERK. Cell numbers were determined at 0 h, 24 h, 48 h, 72 h, 96 h and 120 h with the CCK-8 kit. Ulixertinib inhibited the growth of IPEC-J2 cells from 48 h to 120 h with or without putrescine. B. Western blotting of Phospho-ERK1/2 for different treatment. C. Western blotting of total-ERK1/2. Values are means ± SE, n = 4. Means with different letters are different (P < 0.05). CCK-8, cell counting kit-8; Ctrl, control; IPEC-J2, porcine intestinal epithelial cells; P-ERK1/2, phospho-ERK1/2; Put, putrescine; Uli, ulixertinib. (PDF 143 kb

Additional file 1: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Table S1. The body weight of slaughtered piglets on day 14 of the trial. (DOCX 25 kb

Additional file 8: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Figure S5. FAK inhibitor inhibited IPEC-J2 cells migration and FAK phosphorylation. A. The IPEC-J2 cells were seeded in 6 well plates, and pretreated for 48 h with or without 200 μmol/L putrescine, followed by the addition of 2 μg/mL Mitomycin C for 24 h. The cells were then treated with or without 100 μg/mL defactinib for 4 h before scratching. Images were taken immediately after scratching (0 h) and at 8 h post scratching to calculate the area covered by cell migration, the scratched borders were enhanced with black lines. B. Western blotting of phospho-FAK. C. Western blotting of total-FAK. Values are means ± SE, n = 4. Means with different letters are different (P < 0.05). Ctrl, control; Def, defactinib; IPEC-J2, porcine intestinal epithelial cells; P-FAK, phospho-FAK; Put, putrescine. (PDF 93 kb

Additional file 4: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Figure S1. Effects of putrescine supplementation on tight junction gene expression in the jejunal mucosa of piglets. A. The mRNA level of ZO-1. B. The mRNA level of claudin-1. C. The mRNA level of occludin. Values are means ± SE, n = 6. Means with different letters are different (P < 0.05). Ctrl, control; Put, putrescine dihydrochloride; ZO-1: zona occludens 1. (PDF 26 kb

Additional file 3: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Table S3. The list of antibodies used in western blotting. (DOCX 26 kb

Additional file 2: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Table S2. The list of primers used for qPCR. (DOCX 30 kb

Additional file 6: of Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Figure S3. Mitomycin C suppressed completely cell growth at 24 h of treatment. 0.25 × 105 cell/mL IPEC-J2 cells were seeded in 24-well plates and treated with or without 2 μg/mL mitomycin C for 24 h. Cell number was measured with the CCK-8 kit. Values are means ± SE; n = 4. Means with different letters are different (P < 0.05). Ctrl, control; IPEC-J2, porcine intestinal epithelial cells. (PDF 93 kb