Differential Effects of Oligosaccharides, Antioxidants, Amino Acids and PUFAs on Heat/Hypoxia-Induced Epithelial Injury in a Caco-2/HT-29 Co-Culture Model

Abstract

(1) Exposure of intestinal epithelial cells to heat and hypoxia causes a (heat) stress response, resulting in the breakdown of epithelial integrity. There are indications that several categories of nutritional components have beneficial effects on maintaining the intestinal epithelial integrity under stress conditions. This study evaluated the effect of nine nutritional components, including non-digestible oligosaccharides (galacto-oligosaccharides (GOS), fructo-oligosaccharides (FOS), chitosan oligosaccharides (COS)), antioxidants (α-lipoic acid (ALA), resveratrol (RES)), amino acids (l-glutamine (Glu), l-arginine (Arg)) and polyunsaturated fatty acids (PUFAs) (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)), on heat/hypoxia-induced epithelial injury. (2) Two human colonic cell lines, Caco-2 and HT-29, were co-cultured and pre-treated with the nutritional components for 48 h. After pre-treatment, the cells were exposed to heat/hypoxia (42 °C, 5% O₂) for 2 h. Epithelial integrity was evaluated by measuring trans-epithelial electrical resistance (TEER), paracellular Lucifer Yellow (LY) permeability, and tight junction (TJ) protein expression. Heat stress and oxidative stress levels were evaluated by determining heat-shock protein-70 (HSP-70) expression and the concentration of the lipid peroxidation product malondialdehyde (MDA). (3) GOS, FOS, COS, ALA, RES, Arg, and EPA presented protective effects on epithelial damage in heat/hypoxia-exposed Caco-2/HT-29 cells by preventing the decrease in TEER, the increase in LY permeability, and/or decrease in TJ proteins zonula occludens-1 (ZO-1) and claudin-3 expression. COS, RES, and EPA demonstrated anti-oxidative stress effects by suppressing the heat/hypoxia-induced MDA production, while Arg further elevated the heat/hypoxia-induced increase in HSP-70 expression. (4) This study indicates that various nutritional components have the potential to counteract heat/hypoxia-induced intestinal injury and might be interesting candidates for future in vivo studies and clinical trials in gastrointestinal disorders related to heat stress and hypoxia.

Keywords: epithelial integrity; heat stress; hypoxia; intestinal epithelial cells; nutritional components; tight junction.

Figure 1

LDH release of Caco-2/HT-29 cell monolayers pre-incubated with GOS, FOS, COS (A), RES, ALA (B), arginine, and EPA (C) for 48 h then exposed to 2 h of hypoxia and heat treatment. LDH release into supernatants was assayed using a LDH assay. All values were presented as means ± SEM (N = 3, n = 2 (GOS, FOS, COS group) and n = 3 (all other groups)). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. LDH, lactate dehydrogenase; GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The detailed raw OD data are listed in Table S1.

Figure 2

Relative TEER values of Caco-2/HT-29 cell monolayers pre-incubated with GOS (A), FOS (B), COS (C), RES (D), ALA (E), Arg (F), and EPA (G) for 48 h then exposed to hypoxia and heat treatment (2 h). After heat/hypoxia exposure, TEER values were determined by using an epithelial volt-ohm meter. All values were presented as means ± SEM (N = 3, n = 3). Same control and model values were depicted in separate figures for different nutritional components. Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus control; # p < 0.05, ## p < 0.01, and #### p < 0.0001 versus model. TEER, trans-epithelial electrical resistance; GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The detailed raw TEER readings are listed in Table S2.

Figure 3

Lucifer Yellow flux of Caco-2/HT-29 cell monolayers pre-incubated with GOS, FOS, COS, (A), RES, ALA (B), Arg, and EPA (C) for 48 h then exposed to hypoxia and heat treatment (2 h). After hypoxia and heat treatment, Lucifer Yellow was added to the apical compartment, and the fluorescent intensity of the medium in the basolateral compartment was measured 4 h after application. All values were presented as means ± SEM (N = 3, n = 2 (GOS, FOS, COS group), and n = 3 (all other groups)). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. ** p < 0.01 and **** p < 0.0001 versus control; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 versus model. LY, Lucifer Yellow; GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The detailed fluorescent readings are listed in Table S3.

Figure 4

Relative ZO-1 protein expression level in Caco-2/HT-29 cell monolayers pre-incubated with GOS, FOS, COS (A), RES, ALA (B), Arg, and EPA (C) for 48 h then exposed to hypoxia and heat treatment (2 h). ZO-1 protein expression was determined by WB and normalized to β-actin. All values were presented as means ± SEM (N = 3, n = 2 (GOS, FOS, COS group), and n = 3 (all other groups)). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. ** p < 0.01 and *** p < 0.001 versus control; # p < 0.05 and ## p < 0.01 versus model. GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The original uncropped bands are included in the Supplementary Materials.

Figure 5

Relative CLDN3 protein expression level in Caco-2/HT-29 cell monolayer pre-incubated with GOS, FOS, COS (A), RES, ALA (B), Arg, and EPA (C) for 48 h then exposed to hypoxia and heat treatment (2 h). CLDN3 protein expression was determined by WB and normalized to β-actin. All values were presented as means ± SEM (N = 3, n = 2 (GOS, FOS, COS group), and n = 3 (all other groups)). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. **** p < 0.0001 versus control; # p < 0.05, ## p < 0.01 and ### p < 0.001 versus model. GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The original uncropped bands are included in the Supplementary Materials.

Figure 6

MDA production of Caco-2/HT-29 cell monolayers pre-incubated with GOS, FOS, COS (A), RES, ALA (B), Arg, and EPA (C) for 48 h then exposed to hypoxia and heat treatment (2 h). After hypoxia and heat treatment, MDA in the supernatant was determined by a thiobarbituric acid assay. All values were presented as means ± SEM (N = 3, n = 2 (GOS, FOS, COS group), and n = 3 (all other groups)). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. * p < 0.05, *** p < 0.001, and **** p < 0.0001 versus control; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus model. MDA, malondialdehyde; GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The detailed raw OD data are listed in Table S4.

Figure 7

Relative HSP-70 protein expression levels in Caco-2/HT-29 cell monolayer pre-incubated with GOS, FOS, COS (A), RES, ALA (B), Arg, and EPA (C) for 48 h then exposed to hypoxia and heat treatment (2 h). HSP-70 protein expression was determined by WB and normalized to β-actin. All values were presented as means ± SEM (N = 3, n = 2 (GOS, FOS, COS group), and n = 3 (all other groups)). Statistical differences were analyzed by two-way ANOVA followed by the Bonferroni’s multiple comparison test. ** p < 0.01 and **** p < 0.0001 versus control; # p < 0.05 and ## p < 0.01 versus model. GOS, galacto-oligosaccharides; FOS, fructo-oligosaccharides; COS, chitosan oligosaccharides; ALA, α-lipoic acid; RES, resveratrol; Arg, l-arginine; EPA, eicosapentaenoic acid. The original uncropped bands are included in the Supplementary Materials.