PCA and Que decrease ETEC K88 adhesion and endotoxin level in IPEC-1 cells
PCA or Que treatment did not inhibit ETEC K88 growth (Supplemental Figure 1). Pretreating with PCA or Que decreased bacterial adhesion compared with control group at 1 h (P < 0.001), 2 h (P < 0.01) and 3 h (P < 0.01) after infection with ETEC K88 (Figure 1a). ETEC K88 infection increased the endotoxin level (P < 0.001) in cell supernatant at 2 h (Figure 1b). A HPE× ETEC K88 interaction (P < 0.001) was found for endotoxin level in which pretreating with PCA or Que decreased endotoxin secretion (P < 0.05) compared with control cells after infection with ETEC K88, however, there was no difference among non-ETEC K88-infected cells.
PCA and Que increase cell number and decrease lactate dehydrogenases (LDH) activity after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection reduced cell number and increased LDH activity (P < 0.001) in supernatant at 2 h (Figure 2). HPE × ETEC K88 interactions were found for cell number (P < 0.001) and LDH activity (P = 0.001) in which pretreating with PCA or Que increased cell number and decreased LDH activity in ETEC-infected cells, however, there was no difference in cell number and LDH activity of non-ETEC K88-infected cells.
PCA and Que protect epithelial cell barrier integrity after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection reduced TEER value (P < 0.001) of IPEC-1 cell at 2 h and 3 h post stimulation (Figure 3a-c). Pretreating with Que increased TEER at 1 h, 2 h and 3 h and pretreating with PCA did not influence TEER at 1 h and 2 h. There was a trend observed for TEER when pretreating with PCA at 3 h. No HPE × ETEC K88 interaction was found for TEER at 1 h, 2 h and 3 h in which Que pretreating improved TEER (P < 0.001) both in non-ETEC K88-treated groups or ETEC K88-treated groups.
ETEC K88 infection also raised FD4 permeability (P < 0.001) from cell apical membrane to basal membrane at 2 h and 3 h (Figure 3d-f). A HPE × ETEC K88 interaction was found for FD4 permeability at 2 h and 3 h (P < 0.05) in which pretreating with PCA or Que decreased FD4 permeability (P < 0.05) compared with control cells in ETEC K88-treated groups, however, there was no difference for FD4 permeability at 2 h and 3 h among non-ETEC K88-treated groups.
PCA and Que enhance tight junction protein expression and rescue distribution of tight junction proteins after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection reduced protein abundance of occludin (P < 0.001), claudin-1 (P < 0.05) and ZO-1 (P < 0.001) compared with control group (Figure 4a-c). HPE × ETEC K88 interactions (P < 0.05) were found for protein abundance of occludin, claudin-1 and ZO-1 in which pretreating with PCA or Que enhanced protein abundance of these three tight junctions in ETEC-infected groups, whereas the protein abundance of occludin, claudin-1 and ZO-1 did not change among non-ETEC K88-infected groups.
ETEC K88 infection broke the distribution of occludin, claudin-1 and ZO-1 around epithelial cells. Pretreating with PCA and Que rescued the location of these three tight junction proteins at cellular membrane after ETEC K88 stimulation (Figure 4d-g). HPE × ETEC K88 interactions were found for occludin (P < 0.001) and ZO-1 (P < 0.05) inflorescences in which pretreating with PCA or Que improved protein inflorescences of occludin and ZO-1 in ETEC-infected groups, whereas the protein inflorescences of occludin and ZO-1 did not change among non-ETEC K88-infected groups.
PCA and Que prevent cell necrosis after infection with ETEC K88 in IPEC-1 cells
IncuCyte ZOOM™ Live Cell Imaging System was applied to detect cell necrosis in this experiment (Supplemental videos 1-6). ETEC K88 infection increased cell necrosis ratio from 27 to 30 h compared with the control group (Figure 5a). A HPE × ETEC K88 interaction was found at 28 and 30 h in which PCA or Que pretreating prevented the increase of cell necrosis in ETEC K88-infected groups, however, no difference was observed in non-ETEC K88-infected groups (Supplemental Figure 2). Images at 30 h after ETEC K88 infection also demonstrated that PCA or Que pretreating alleviate the increase of cell necrosis ratio after ETEC K88 stimulation (Figure 5b).
PCA and Que suppress pro-inflammatory cytokine secretion after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection increased mRNA level of TNF-α, IL-8 and IL-6 (P < 0.001) (Figure 6a-c). HPE × ETEC K88 interactions were found for TNF-α, IL-8 and IL-6 mRNA expression (P < 0.05) in which pretreating with PCA or Que down-regulated mRNA level of TNF-α, IL-8 and IL-6 (P < 0.05) among ETEC K88-infected cells, however, there was no difference observed among non-ETEC K88-infected groups.
ETEC K88 infection also elevated secretion of TNF-α, IL-8 and IL-6 (P < 0.001) in IPEC-1 cells (Figure 6d-f). PUFA ×ETEC K88 interactions were found for TNF-α, IL-8 and IL-6 protein contents in which PCA or Que suppressed the concentration of TNF-α, IL-8 and IL-6 in ETEC K88-infected groups, however, no difference was observed for concentration of TNF-α, IL-8 and IL-6 in non-ETEC K88-infected cells.
PCA and Que inhibit TLR4 signaling pathway after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection increased mRNA level of toll-like receptor 4 (TLR4), LPS binding protein (LBP), myeloid differentiation factor-2 (MD2), cluster differentiation factor-14 (CD14), IL-1 receptor-associated kinase 1 (IRAK1), and nuclear factor-κB (NF-κB) (P < 0.05) compared with control cells (Figure 7a-f). HPE × ETEC K88 interactions were found for TLR4, LBP, MD2, CD14, IRAK1 and NF-κB mRNA abundance in which PCA or Que pretreating decreased these variables in ETEC K88-infected groups, however, no difference was observed for TLR4, LBP, MD2, CD14, IRAK1 and NF-κB mRNA in non-ETEC K88-infected groups.
PCA and Que suppress necroptosis signaling pathway after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection up-regulated protein level of t-RIP1 (P < 0.001), t-RIP3 (P < 0.001), t-MLKL (P < 0.05), p-RIP1 (P < 0.001), p-RIP3 (P < 0.001), p-MLKL (P < 0.001), p-RIP1/t-RIP1 (P < 0.001), DRP1(P < 0.001), PGAM5 (P < 0.001) and HMGB1 (P < 0.001) compared with control group (Figure 8a-n). HPE × ETEC K88 interactions were found for protein expression of t-RIP1 (P < 0.001), t-RIP3 (P < 0.001), t-MLKL (P < 0.001), p-RIP1 (P < 0.001), p-RIP3 (P < 0.001), p-MLKL (P < 0.001), p-RIP3/t-RIP3 (P < 0.05), DRP1 (P < 0.001), PGAM5 (P < 0.05) and HMGB1 (P < 0.001) in which PCA or Que pretreatment down-regulated protein abundance of RIP1, RIP3, MLKL, p-RIP3/t-RIP3, DRP1, PGAM5 and HMGB1 in ETEC K88-infected groups, however, there was no difference observed for these variables in non-ETEC K88-infected groups.
PCA and Que repress pyroptosis signaling pathway after infection with ETEC K88 in IPEC-1 cells
ETEC K88 infection increased protein abundance of NLRP3 (P < 0.001), ASC (P < 0·001), nod-like receptors family CARD domain-containing protein (NLRC4, P < 0·001), GSDMD (P < 0·001) and caspase-1 (P < 0.05) compared with control group (Figure 9a-f). HPE × ETEC K88 interactions were found for ASC (P < 0.001), NLRP3 (P < 0.001), NLRC4 (P < 0.001), GSDMD (P < 0.001) and caspase-1 (P < 0.05) protein expression in which PCA or Que pretreatment down-regulated protein abundance of NLRP3, ASC, NLRC4, GSDMD and caspase-1 in ETEC K88-infected groups, whereas there was no difference for NLRP3, ASC, NLRC4, and GSDMD protein expression in non- ETEC K88-infected groups.