Figure 1.
Effect of increasing temperature on Caco-2 monolayer barrier function.
Caco-2 monolayers were exposed to increasing temperature for 1 h from 37°C to 43°C. A: Increasing temperature decreased TEER. TEER was recorded before (used as a baseline) and after heat stress. TEER was presented relative (%TEER) to baseline. B: Increasing temperature increased HRP flux. The amount of HRP in the basolateral chambers was expressed as a percentage of added HRP (original%). Values are means ± SD. ** P<0.01, compared with 37°C group. N = 6 per group.
Figure 2.
Temperature-course effect of heat exposure (37°C – 43°C) for 1 h on TJ protein expression in Caco-2 monolayers.
Samples were harvested 24 hours after 1 h of heat exposure and analyzed by Western blotting (A, D). B: Heat exposure caused a significant increase in expression of occludin, but decrease was observed when exposed to 43°C. C: The exposure to heat produced a progressive decrease in ZO-1 protein expression. E: Level of claudin-2 protein in total cell extract was not affected by heat exposure. Results were reported as means ± SD from 3 independent experiments. Values were normalized to β-actin. * P<0.05, ** P<0.01 compared with 37°C group.
Figure 3.
Effect of increasing temperature (37°C – 43°C) for 1 h on the gene expressions of occludin (A) and ZO-1 (B) by Real-time PCR.
Cells were cultured for 24 h after 1 h heat exposure. Values were normalized to 37°C group (37°C set to 1). Results were reported as means ± SD. N = 3 per group. ** P<0.01 compared with 37°C group.
Figure 4.
EPA enhances epithelial barrier integrity and ameliorates heat-induced barrier disruption by measuring TEER.
Caco-2 monolayers were treated with heat at 43°C for 1 h after absence (control) or presence of PUFAs for 96 h. TEER measurements were performed at 0, 24, 48, 72 and 96 h of incubation and after heat stress. TEER was presented as percentage (%TEER) of initial resistance (baseline = 1). Values are means ± SD. N = 6 per group. * P<0.05, ** P<0.01 compared with control at same time point.
Figure 5.
EPA decreases paracellular permeability induced by heat stress by determining HRP flux.
Caco-2 monolayers were treated with heat at 43°C for 1 h after absence (control) or presence of PUFAs for 96 h. HRP transport in the basolateral chambers was calculated as a percentage of added HRP after heat stress,. Values are means ± SD. N = 6 per group. * P<0.05, ** P<0.01 compared with 37°C group. # P<0.05, # #P<0.01 compared with 43°C group.
Figure 6.
Effect of PUFAs on heat-induced change in protein expression of whole cells by Western blot analysis.
Caco-2 monolayers were cultured for 24 h after 1 h of heat exposure without (37°C group and 43°C group) or with PUFAs pre-incubation for 96 h. TJ proteins were shown (A, D): occludin (B), ZO-1 (C) and claudin-2 (E). Results were reported as means ± SD from 3 independent experiments. Values were normalized to β-actin. * P<0.05, ** P<0.01 compared with 37°C group. # P<0.05, ## P<0.01 compared with 43°C group.
Figure 7.
Effect of PUFAs on TJ protein expression in the membrane fraction after heat stress.
Cells were cultured for 24(37°C group and 43°C group) or with PUFAs pre-incubation for 96 h. TJ proteins in the membrane fraction were shown (A): occludin (B), ZO-1 (C) and claudin-2 (D). Results were reported as means ± SD from 3 independent experiments. Values were normalized to β-actin. * P<0.05, ** P<0.01 compared with 37°C group. # P<0.05,## P<0.01 compared with 43°C group.
Figure 8.
Effect of PUFAs pretreatment on TJ protein expression in the cytosol fraction after heat stress.
Cells were cultured for 24(37°C group and 43°C group) or with PUFAs pre-incubation for 96 h. TJ proteins in the cytosol fraction were shown (A): occludin (B), ZO-1 (C) and claudin-2 (D). Results were reported as means ± SD from 3 independent experiments. Values were normalized to β-actin. * P<0.05, ** P<0.01 compared with 37°C group. # P<0.05, ## P<0.01 compared with 43°C group.
Figure 9.
Effect of PUFAs pretreatment on the gene expressions of occludin (A) and ZO-1 (B) after heat stress by Real-time PCR.
After pre-incubation with PUFAs or not (37°C group and 43°C group) for 96 h, Caco-2 monolayers were harvested 24 hours after 1 h of heat exposure. Expression of mRNA was normalized with GAPDH mRNA expression. Values were normalized to 37°C group (37°C set to 1). Results were reported as means ± SD from 3 independent experiments. N = 3 per group.* P<0.05, ** P<0.01 compared with 43°C group.
Figure 10.
Effect of PUFAs on junctional localization of TJ proteins by immunofluorescence.
Cells were pre-incubated with PUFAs or without (37°C group and 43°C group) for 96 h with heat exposure for 1 h, and cultured for 24 hours. Results were reported from 3 independent experiments. Magnification was 400×.
Figure 11.
Effect of PUFAs on morphological ultrastructure of tight junction induced by heat stress.
Caco-2 cell monolayers were pre-incubated without (A: 37°C group and B: 43°C group) or with EPA (C), DHA (D) or AA (E) with heat exposure for 1 h. Images were acquired by transmission electron microscopy after culturing for 24 h. Data are representative of 3 independent experiments. Arrows indicate tight junctions. Scale bars = 500 nM.
Table 1.
Fatty acid composition of membrane microdomains from control cells and PUFAs – treated cells.