IPEC-J2 cells were from Dr. Guoyao Wu from Texas A&M University. The cells were cultured as described previously [17]. Briefly, the cells were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA), 0.01% epidermal growth factor (5 $\mu$g/L, Corning Inc., Corning, NY, USA) and 0.1% ITS solution (containing 5 $\mu$g/L insulin, 5 $\mu$g/L transferrin, and 5 ng/L sodium selenite, Corning Inc., Corning, NY, USA) at 37 °C in a humidified incubator containing 5% CO${}_2$. Culture medium was replaced every 24 h. When the cells covered about 80–90% of the bottom of the plastic culture flask (75 cm${}^2$, Corning Inc., Corning, NY, USA), cells were passaged utilizing 0.25% trypsin-EDTA (Gibco, Grand Island, NY, USA) and subcultured for further experiments. We used passage 12–15 cells in these studies.

IPEC-J2 cells were seeded at 0.5 $\times$ 10${}^5$ cells per well into 96-well plates (Corning Inc., Corning, NY, USA) with six replications (wells) per treatment. After the cells adhered to the plate bottom (4 h), each well was washed three times with PBS, followed by adding 100 $\mu$L culture medium (containing 2% FBS) with different concentrations of putrescine (200 $\mu$mol/L, 400 $\mu$mol/L) or spermidine (1 $\mu$mol/L, 2 $\mu$mol/L, 4 $\mu$mol/L, 8 $\mu$mol/L, 16 $\mu$mol/L, 32 $\mu$mol/L, 64 $\mu$mol/L) per well for each treatment. The concentration of putrescine (putrescine dihydrochloride, Sigma-Aldrich, Co., Saint Louis, MO, USA) was selected based on a previous report in our laboratory [16]. To determine the specific effect of putrescine and spermidine on the proliferation of the cells, 5 mmol/L difluoromethyl ornithine (DFMO, a specific inhibitor of ODC, MedChemExpression, Monmouth Junction, NJ, USA) was added in culture media for 72 h, and 1 mmol/L diethylglyoxal bis (guanylhydrazone) (DEGBG, an inhibitor of SAMDC) was added in culture media for 24 h to inhibit the production of putrescine and spermidine in cells, respectively. After the third or fourth day of cell seeding, the growth of IPEC-J2 cells was measured using the cell counting kit (CCK-8, Monmouth Junction, MedChemExpression, Monmouth Junction, NJ, USA) according to the manufacturer’s instructions. Concisely, the cells were washed twice with PBS, 100 $\mu$L media containing 10% CCK-8 solution was added to each well, followed by incubation for 3 h at 37 °C. Optical density (OD) values of culture media at 450 nm were measured employing a microplate reader (BioTek Instruments, Inc., Montpellier, VT, USA). The number of cells was calculated according to the fitted standard curve from the OD value.

IPEC-J2 cells were seeded at 0.5 $\times$ 10${}^5$ cells/mL (2 mL per well) into 6-well plates (Corning Inc., Corning, NY, USA) with four replications (wells) per treatment. The cells were then cultured for 48 h to fully cover the plates’ bottom. Then added 2 $\mu$g/mL mitomycin C (MedChemExpression, Monmouth Junction, NJ, USA) to the culture media for 24 h to inhibit cell proliferation [18]. A 200-$\mu$L aseptic pipette tip was used to make a straight scratch, the cells were then washed twice with PBS, and the scratch area was measured (recorded as 0 h) by inverted microscopy (Axio Vert.A1, Zeiss, Jena, Thuringia, Germany). The cells were treated with putrescine (200 $\mu$mol/L) or spermidine (8 $\mu$mol/L) for 8 h in the presence or absence of DEGBG. The area of the exact location at 8 h post scratching (recorded as 8 h) was again measured by inverted microscopy (Axio Vert.A1, Zeiss, Jena, Thuringia, Germany). The scratch area at 8 h was subtracted from that at 0 h to calculate the cell migration rate (in percentage).

The cell inflammation model was established in vitro by adding lipopolysaccharides (LPS, Beyotime technology, Shanghai, China). IPEC-J2 cells were seeded at 0.5 $\times$ 10${}^5$ cells/mL (1 mL per well) into 12 well plates. After being cultured for 48 h, the cells were incubated with 8 $\mu$mol/L spermidine for 24 h, and then added 100 $\mu$g/mL LPS for another 4 h. The cells were washed with PBS, and intracellular RNA was extracted for further assays.

IPEC-J2 cells were seeded at 0.5 $\times$ 10${}^5$ cells/mL into 6 well plates. After being cultured for 48 h, the cells were treated with DMEM/F12 containing 0 $\mu$mol/L spermidine, 1 mmol/L DEGBG, 8 $\mu$mol/L spermidine, 8 $\mu$mol/L spermidine + 1 mmol/L DEGBG. Samples preparation and polyamines determination were processed according to the method described by Dai et al. [19]. Briefly, the cells were harvested by centrifugation at 1000 $\times$ g for 10 min. The cells were resuspended in 300 $\mu$L pre-chilled PBS and using the sonication method to obtain cell contents. Cell-free supernatant was obtained by centrifuging the sonication mixtures at 13,000 $\times$ g for 5 min. One hundred and fifty $\mu$L of 1.5 mol/L HClO${}_4$ was added dropwise to 150 $\mu$L supernatant of the cell lysis solution or culture media, and then added 75 $\mu$L of 2 mol/L of K${}_2$CO${}_3$ to react for 5 minutes. The supernatant solution obtained by centrifugation (13,000 $\times$ g for 5 min) was used to determine free polyamines by HPLC (Alliance e2695 HPLC system, Waters Corporation, Milford, CT, USA) using the OPA-NAC method, in which polyamines react with o-phthaldialdehyde (OPA) and N-acetyl-L-cysteine (NAC) to form relatively stable derivative products. The in-line derivatization procedure was programmed in the HPLC system to mix 10 $\mu$L of the samples with 10 $\mu$L of the OPA-NAC solution and stop for 1 min to allow a full derivatization reaction before injection of the mixtures into the system. The fluorescence of the derivative products was determined using a Waters 2475 multi $\lambda$ fluorescence detector with excitation $\lambda$ at 340 nm and emission $\lambda$ at 450 nm. Solvent A was 0.1 mol/L sodium acetate (pH 7.2) and solvent B was HPLC-grade methanol. HPLC gradients for solvent A and B were 70/30, 35/65, 30/70, 0/100, 0/100, 70/30, 70/30 at 0, 12, 16, 18, 23, 25, 30 min, respectively.

mRNA abundance in the cells was determined by qPCR according to the procedure described by Li et al. [20]. In brief, total RNA was isolated from IPEC-J2 cells by using the Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by the determination of the content and quality of total RNA using an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc., Montpellier, VT, USA), and the first-strand cDNA synthesis by TransScript First-Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s protocol. qPCR analysis was performed using the cDNA templates, primer pairs for specific genes of the target, and SYBR Green reagent (Thermo Fisher Scientific, MA, USA) on an ABI 6 flex real-time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for qPCR are shown in Supplementary Table 1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize target genes. The calculation of fold changes of the target genes relative to those of GAPDH was acquired by the comparative Ct value method.

Western blot analysis was performed using a routine procedure described by Liu et al. [16] with slight modification. The membranes were incubated with a primary antibody overnight at 4 °C, followed by washing three times (10 min per time) with Tris-buffered saline and Tween 20 (TBST) buffer, incubated with a secondary antibody for 3 h at room temperature. The membranes were rewashed with TBST buffer before adding the reagents from Western Bright ECL Kit (Bio-Rad Laboratories Inc., Berkeley, CA, USA). Digital images were detected by the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). The antibodies of extracellular signal-regulated kinase1/2 (ERK1/2) (1:1000), adhesion kinase (FAK) (1:1000), NF-$\kappa$B (1:1000), phospho-ERK1/2 (Thr202/Tyr204, 1:1000), phospho-FAK (Tyr 397, 1:1000) and phospho-NF-$\kappa$B (Ser536, 1:1000), as well as secondary antibodies (HRP-linked anti-rabbit or anti-mouse IgG, 1:2000), were obtained from Cell Signaling Technology, USA. GAPDH (1:1000, Cell Signaling Technology, Danvers, MA, USA) and $\alpha$-Tublin (1:1000, Cell Signaling Technology, Danvers, MA, USA) were internal references for equality of sample loading. The list of antibodies used in western blotting is shown in Supplementary Table 2.

Data were expressed as mean $\pm$ SE. Statistics were analyzed by one-way analysis of variance (ANOVA) using SAS v. 9.2 (SAS Institute Inc., Cary, NC, USA). Duncan’s multiple comparison test was used to determine the statistical differences among treatments. Differences were considered statistically significant at p 0.05.

The effect of treatment with different concentrations of spermidine on the proliferation of IPEC-J2 cells is illustrated in Fig. 1. Compared with the control treatment, the addition of 2, 4, 8, 16 $\mu$mol/L spermidine to the culture medium increased cell numbers after incubation for 24 h; cell numbers significantly increased at concentrations of 8 and 16 $\mu$mol/L when incubated with spermidine for 48 h; 1, 4, 8, 16 $\mu$mol/L spermidine supplementation increased cell numbers at 72 h; furthermore, only a low concentration of spermidine (2 $\mu$mol/L) supplementation increased the number of IPEC-J2 cells at 96 h (p 0.05). In addition, the concentrations of spermidine above 16 $\mu$mol/L significantly decreased cell numbers at different processing times compared with the control (p 0.05). A concentration of 8 $\mu$mol/L spermidine was used for subsequent experiments because of its positive effect on stimulating cell proliferation.

Fig. 1.

Effects of different spermidine concentrations on the growth of IPEC-J2 cells. IPEC-J2 cells were seeded into 96-well plates at a number of 0.5$\times$10${}^5$ cells per well and then treated with 0, 1, 2, 4, 8, 16, 32, 64 $\mu$mol/L spermidine. Cell number was detected at 24 h, 48 h, 72 h, and 96 h with CCK-8 reagent. The data were expressed as the mean $\pm$ SE, n = 6. Different letters indicate a significant difference among treatments at each time point (p 0.05).

As shown in Fig. 2A, adding 5 mmol/L DFMO significantly inhibited cell proliferation at 72 h, and adding simultaneously exogenous putrescine at a concentration of 200 or 400 $\mu$mol/L completely recovered cell growth (p 0.05). Similarly, pretreatment with a concentration of 8 or 16 $\mu$mol/L spermidine significantly rescued cell growth (p 0.05) (Fig. 2B).

Fig. 2.

Effect of polyamines (putrescine and spermidine) on IPEC-J2 cells proliferation in the presence of DFMO (or/and) DEGBG. IPEC-J2 cells were cultured with putrescine (200, 400 $\mu$mol/L) or spermidine (8, 16 $\mu$mol/L) for 72 h (with or without DFMO) (A,B) and 24 h (with or without DEGBG) (C,D); for combined addition of DFMO and DEGBG, the cells were pretreated for 48 h with or without DFMO before stimulation with DEGBG for 24 h in the presence or absence of putrescine (200, 400 $\mu$mol/L) or spermidine (8 and 16 $\mu$mol/L) (E,F). Then cell numbers were determined with the CCK-8 kit. The data are expressed as the mean $\pm$ SE, n = 6. Different letters indicate a significant difference (p 0.05).

Adding 1 mmol/L DEGBG to the culture medium significantly reduced the intracellular spermidine content and increased the intracellular putrescine concentrations (p 0.05) (Supplementary Table 3 and Supplementary Fig. 1). The response of 1 mmol/L DEGBG to stimulate IPEC-J2 cells for 24 h was similar to that of DFMO to stimulate IPEC-J2 cells for 72 h; the addition of putrescine failed to restore cell proliferation in enterocytes; under the same conditions, spermidine supplementation recovered cell growth in a dose-dependent manner (from 8 to 16 $\mu$mol/L) (p 0.05) (Fig. 2C,D). Similarly, adding putrescine also failed to rescue cell proliferation in DFMO plus DEGBG-treated cells but adding exogenous 8 or 16 $\mu$mol/L spermidine partially recovered cell growth (p 0.05) (Fig. 2E,F).

As shown in Fig. 3, compared with the control treatment, the addition of 8 $\mu$mol/L spermidine or 200 $\mu$mol/L putrescine significantly elevated the rate of cell migration at 8 h after scratching, DEGBG inhibited cell migration. Still, cell migration was partially restored in the presence of exogenous spermidine (p 0.05). In addition, putrescine didn’t affect this inhibition caused by DEGBG.

Fig. 3.

Effect of putrescine and spermidine on IPEC-J2 cells migration in the presence of DEGBG. IPEC-J2 cells were seeded into 6-well plates at a concentration of 0.5 $\times$ 10${}^5$cells/mL and cultured in a medium until reaching 70–75% confluence. Then, the cells were pretreated with mitomycin C for 24 h before scratching, followed by treatment with putrescine (200 $\mu$mol/L) or spermidine (8 $\mu$mol/L) for another 8 h in the presence or absence of DEGBG. Values are means $\pm$ SE, n = 4. Different letters indicate a significant difference (p 0.05).

Compared with the control treatment, DEGBG supplementation decreased the phosphorylation of ERK1/2, but this passive effect was reversed by the simultaneous addition of spermidine (p 0.05) (Fig. 4D); however, putrescine supplementation had no significant effects on ERK1/2 phosphorylation compared with the DEGBG stimulation (p $>$ 0.05) (Fig. 4B); moreover, the addition of DEGBG, putrescine, and spermidine did not alter the abundances of ERK1/2 in cells compared with the control treatment (p $>$ 0.05) (Fig. 4A,C). In Fig. 5, the combined addition of DEGBG and putrescine or spermidine remarkably increased FAK protein abundance (p 0.05), but DEGBG, putrescine, or spermidine supplementation alone did not affect FAK protein abundance in IPEC-J2 cells, compared with the control treatment (p $>$ 0.05) (Fig. 5A,C). In addition, extracellular putrescine decreased protein levels of phosphorylated P-FAK protein abundance in DEGBG-treated cells. Adding spermidine in the presence of DEGBG numerically increased FAK phosphorylation but was not significant compared with that treated with DEGBG alone (Fig. 5B,D).

Fig. 4.

Effect of putrescine and spermidine on activation of the ERK1/2 protein in IPEC-J2 cells. IPEC-J2 cells were incubated with putrescine or spermidine for 24 h in the presence or absence of DEGBG. Then, the protein abundance of ERK1/2 and P-ERK1/2 proteins were detected by western blotting. (A) ERK1/2 protein abundance and (B) the phosphorylated ERK1/2 protein abundance at 24 h after being pretreated with 200 $\mu$mol/L putrescine in the presence or absence of 1 mmol/L DEGBG. (C) ERK1/2 protein levels and (D) the phosphorylated ERK1/2 protein levels at 24 h after being pretreated with 8 $\mu$mol/L spermidine in the presence or absence of 1 mmol/L DEGBG. The data presented as the mean $\pm$ SE, n = 6. Different letters represent a significant difference (p 0.05).

Fig. 5.

Effect of putrescine and spermidine on activation of FAK protein in IPEC-J2 cells. IPEC-J2 cells were incubated with putrescine (200 $\mu$mol/L) or spermidine (8 $\mu$mol/L) for 24 h in the presence or absence of DEGBG. Then, the protein abundance of FAK and P-FAK was determined by western blotting. (A) FAK protein abundance and (B) the phosphorylated FAK protein abundance at 24 h after incubation with 200 $\mu$mol/L putrescine in the presence or absence of 1 mmol/L DEGBG. (C) FAK protein abundance and (D) the phosphorylated FAK protein abundance at 24 h after incubation with 8 $\mu$mol/L spermidine in the presence or absence of 1 mmol/L DEGBG. The data are presented as the mean $\pm$ SE, n = 6. Different letters represent a significant difference (p 0.05).

Detection of the mRNA abundance of pro-inflammatory cytokines in IPEC-J2 cells was conducted by q-PCR, and the results were shown in (Fig. 6). Compared with the control treatment, a significant augment in the abundance of IL-8, IL-6, TNF-$\alpha$, and IL-1$\beta$ genes was obtained in DEGBG-treated cells (p 0.05), the addition of exogenous spermidine was found to suppress the expression of IL-8, IL-6, and TNF-$\alpha$ induced by DEGBG (p 0.05) (Fig. 6A–C). However, no significant effect on the expression of IL-1$\beta$ was found (p $>$ 0.05) (Fig. 6D). DEGBG, spermidine, or putrescine addition alone did not change the total protein abundance of NF-$\kappa$B compared with the control treatment (p $>$ 0.05) (Fig. 7A–C). However, DEGBG significantly increased NF-$\kappa$B protein phosphorylation, adding spermidine to the culture media completely abrogated phosphorylated NF-$\kappa$B (p 0.05) (Fig. 7D), whereas putrescine failed (p 0.05) (Fig. 7B). In addition, the addition of spermidine attenuated the expression of TNF-$\alpha$ induced by LPS (p 0.05) (Supplementary Fig. 2C).

Fig. 6.

Effect of spermidine on the expression of inflammatory genes in IPEC-J2 cells induced by DEGBG. IPEC-J2 cells were incubated with spermidine (8 $\mu$mol/L) for 24 h in the presence or absence of DEGBG. Then, the expression levels of IL-8 (A), IL-6 (B), TNF-$\alpha$ (C), and IL-1$\beta$ (D) were quantified by qPCR. The data are presented as the mean $\pm$ SE, n = 6. Different letters represent a significant difference (p 0.05).

Fig. 7.

Effect of spermidine on the expression of inflammatory genes in IPEC-J2 cells induced by DEGBG. IPEC-J2 cells were incubated with putrescine or spermidine for 24 h in the presence or absence of DEGBG. Then, the abundance of NF-$\kappa$B and P-NF-$\kappa$B proteins was detected by western blotting. (A) NF-$\kappa$B protein abundance and (B) the phosphorylated NF-$\kappa$B protein abundance at 24 h after incubated with 200 $\mu$mol/L putrescine in the presence or absence of 1 mmol/L DEGBG. (C) NF-$\kappa$B protein abundance and (D) the phosphorylated NF-$\kappa$B protein abundance at 24 h after incubation with 8 $\mu$mol/L spermidine in the presence or absence of 1 mmol/L DEGBG. The data are presented as the mean $\pm$ SE, n = 6. Different letters represent a significant difference (p 0.05).