| This study was conducted to investigate the effect of Lactobacillus Plantarum(LP) on the regulation of barrier function, m RNA expression of transporters and immune response in intestinal porcine epithelial cells(IPEC-J2) challenged with enterotoxigenic Escherichia coli(ETEC) K88. The study consisted of the following three experiments:In experiment 1: 1) Different doses of ETEC K88(MOI = 10, 25, 50, 75 and 100) were selected to challenge with IPEC-J2 cells for different time(1, 2, 3 and 4 h). Cell viability and the m RNA expression of IL-6, IL-8 and TNF-α were examined, to deternine the appropriate challenge dose and time of ETEC K88 for establishment of the ETEC K88-IPEC-J2 interaction model. 2) Different concentrations of LP(MOI = 10, 25, 50, 75 and 100) were selected to pre-treated with IPEC-J2 for different time(3, 6 and 9 h), followed by challenging with ETEC K88. Cell viability and the m RNA expression of IL-6, IL-8 and TNF-α were examined, to deternine the appropriate concentration and time of LP for establishment the LP-IPEC-J2 interaction model. 3) The concentration of IL-8 and TNF-α in the cell culture supernatant were examined, to deternine the LP-IPEC-J2-ETEC K88 interaction model. The results showed that:1) After challenging with ETEC K88(MOI = 50) for 3 h, the inflammatory cytokines of IL-6, IL-8 and TNF-α in IPEC-J2 cells were significantly upregulated(P < 0.05) compared with the control group. Therefore, MOI = 50, and challenge time of 3 h, were selected as the best interaction model of ETEC K88-IPEC-J2. 2) After pre-treated with LP(MOI = 75) for 6 h, the inflammatory cytokines of IL-6, IL-8 and TNF-α in IPEC-J2 cells were siginificantly downregulated(P < 0.05) compared with the ETEC K88 control group. Therefore, LP at MOI = 75, and pre-treated time of 6 h, were selected for the interaction model of LP-IPEC-J2. 3) After challenging with K88(MOI = 50 and challenging time was 3 h), the concentration of IL-8 and TNF-α in the cell culture supernatant were significantly increased(P < 0.05) compared with the control group. While the increase of concentration of IL-8 and TNF-α were inhibited(P < 0.05) by the pre-treatment with LP(MOI = 75 and pre-treatment time was 6 h) compared with the K88 group(MOI = 50 and challenging time was 3 h). In conclusion, cells were pre-treated with LP(MOI = 75, about 1× 108 CFU) for 6 h, and then challenged with ETEC K88(MOI = 50, about 6.5× 107 CFU) for 3 h, was determined as the interaction model of ETEC-IPEC-J2-LP.In experiment 2: cells were seeded onto 6-well Transwell inserts which allowed for a cell density of 1.3 × 106 cells per well at confluence. After the cells were fully differentiated, they were pre-treated with LP and challenged with ETEC K88 under the condition determined in experiment 1. Monolayers of IPEC-J2 cells not treated with either LP or ETEC K88 served as the control group(control), cells pre-treated with LP only(LP), cells infected with ETEC K88 only(K88) and those pre-treated with LP then infected with K88(LP + K88) were the treated groups. Each group consisted of 6 replicates(wells). The results showed that:1) Compared with the control group, the TEER value, the m RNA expression of claduin-1, occludin, ZO-1, and the protein abundance of occludin in IPEC-J2 cells were significantly decreased(P < 0.05) after challenged with ETEC K88. While pre-treatment with LP significantly inhibit these decreases caused by ETEC K88(P < 0.05). These results were further confirmed by confocal immunohistochemistry, indicating that tight junction proteins(claudin-1 and occludin) could be damaged by ETEC K88, while these damages could be partially offset by the pre-treatment of LP. 2) Compared with the control group, the m RNA expression of glucose transporters(SGLT1), amino acid transporters(y+LAT1, CAT1 and ASCT2) were significantly downregulated(P < 0.05), and chlorine ion transporters(CFTR and NKCC1) in IPEC-J2 cells were significantly upregulated(P < 0.05) after challenged with ETEC K88, while pre-treatment with LP significantly inhibited these downregulations caused by ETEC K88(P < 0.05). There was no significant effect in the m RNA expression of SGLT3, GLUT2 and b0,+AT, CAT2 in IPEC-J2 cells among treatments(P > 0.05). 3) Compared with the control group, the m RNA expression of pro-inflammatory cytokines(IL-1β, IL-6, IL-8 and TNF-α), TLR5, TLR7 in IPEC-J2 cells were significantly upregulated(P < 0.05), and the m RNA expression of anti-inflammatory cytokine(TGFβ and PPAR-γ), TLR1, TLR2, TLR6, TLR9 and protein abundance of TLR2, the negative regulators of TLRs(SIGIRR, Bcl3 and MKP-1) in IPEC-J2 cells were significantly downregulated(P < 0.05) after challenged with ETEC K88; While pre-treatment with LP significantly inhibit the upregulation of m RNA expression of pro-inflammatory cytokines(IL-1β, IL-8 and TNF-α), TLR4, TLR5, TLR7 in IPEC-J2 cells(P < 0.05), and inhibit the downregulation of m RNA expression of anti-inflammatory cytokine(TGFβ and PPAR-γ), m RNA expression and protein abundance of TLR2, the negative regulators of TLRs(SIGIRR, Bcl3 and MKP-1) in IPEC-J2 cells(P < 0.05). Moreover, treatment with LP alone upregulated the m RNA expression of PPAR-γ, TLR6 and Bcl3 in IPEC-J2 cells(P < 0.05). There was no significant effect in the m RNA expression of TLR3, Tollip, A20 and IRAK-M in IPEC-J2 cells among treatments(P > 0.05).In experiment 3: cells were seeded onto 6-well Transwell inserts which allowed for a cell density of 1.3 × 106 cells per well at confluence. After the cells were fully differentiated, they were pre-treated with LP(1 × 108 CFU/well) for 6 h at 37°C under 5% CO2, followed by removal of the non-attached bacteria and three washes with PBS for treated cells. The monolayer of cells was then challenged with ETEC K88(MOI = 50,about 6.5× 107 CFU). The cells were incubated for 0, 12, 25 and 50 min, respectively, at 37°C under 5% CO2, followed by removal of the inoculums and cells were washed twice with PBS. Cells were collected to determine the protein abundance of P38, p-P38 and IκBα. Cells infected with ETEC K88 only(K88) and those pre-treated with LP then infected with K88(LP + K88) were the treated groups. Each group consisted of 6 replicates(wells). The results showed that: after exposure to ETEC K88 for 12, 25 and 50 min, cells pre-treated with LP showed increased IκBα protein expression compared with K88 alone(P < 0.05). Conversely, cells pre-treated with LP showed significantly decreased p-P38 at 25 and 50 min compared to ETEC K88 alone(P < 0.05). The results indicated that LP partially inhibited K88-triggered activations of NF-κB and MAPK signaling pathways.In conclusion, the results obtained here indicated that pre-treatment of LP effectively diminished the ETEC K88- induced upregulation of pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines gene expression in IPEC-J2 cells, and thereby protected against epithelial barrier and transport of nutrient damage through sustaining the gene expression and subsequent protein content of tight junctions, and the gene expression of glucose transporters, amino acid transporters and downregulated chlorine ion transporters. It was further demonstrated that the protective effects of LP on IPEC-J2, possibly through modulation of TLRs, NF-κB and MAPK signaling pathways. |
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