L-tryptophan Enhances Intestinal Integrity in Diquat-Challenged Piglets Associated with Improvement of Redox Status and Mitochondrial Function

Abstract

Tryptophan (Trp) supplementation has been shown to improve growth performance and enhance intestinal integrity in piglets. However, the effects of dietary Trp supplementation on the intestinal barrier function in piglets exposed to oxidative stress remain unknown. This study was conducted to evaluate whether dietary Trp supplementation can attenuate intestinal injury, oxidative stress, and mitochondrial dysfunction of piglets caused by diquat injection. Thirty-two piglets at 25 days of age were randomly allocated to four groups: (1) the non-challenged control; (2) diquat-challenged control; (3) 0.15% Trp-supplemented diet + diquat; (4) 0.30% Trp supplemented diet + diquat. On day seven, the piglets were injected intraperitoneally with sterilized saline or diquat (10 mg/kg body weight). The experiment lasted 21 days. Dietary supplementation with 0.15% Trp improved growth performance of diquat-challenged piglets from day 7 to 21. Diquat induced an increased intestinal permeability, impaired antioxidant capacity, and mitochondrial dysfunction. Although dietary supplementation with 0.15% Trp ameliorated these negative effects induced by diquat challenge that showed decreasing permeability of 4 kDa fluorescein isothiocyanate dextran, increasing antioxidant indexes, and enhancing mitochondrial biogenesis. Results indicated that dietary supplementation with 0.15% Trp enhanced intestinal integrity, restored the redox status, and improved the mitochondrial function of piglets challenged with diquat.

Keywords: intestinal barrier function; mitochondria; oxidative stress; piglets; tryptophan.

Figure 1

Effects of dietary tryptophan supplementation on intestinal permeability of piglets challenged with diquat. (A) D-lactate concentration; (B) diamine oxidase activity; (C) TER, transepithelial electrical resistance; (D) the flux of FD4, 4 kDa fluorescein isothiocyanate dextran, at the jejunum. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 2

Effects of dietary tryptophan supplementation on mRNA expression of tight junction proteins in piglets challenged with diquat. (A) ZO1, zona occludens1; (B) ZO2, zona occludens 2; (C) OCLN, occludin; (D) CLDN1, claudin1; (E) CLDN2, claudin 2. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 3

Effects of dietary tryptophan supplementation on protein expression of tight junction proteins in piglets challenged with diquat. (A) Representative blots of ZO1, occludin, claudin1, and GAPDH in the jejunal mucosa of piglets; (B) protein expression of ZO1, zona occludens1; (C) protein expression of occludin; (D) protein expression of claudin1. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 4

Effects of dietary tryptophan supplementation on jejunal redox status of piglets challenged with diquat. (A) Superoxide dismutase activity; (B) glutathione peroxidase activity; (C) catalase activity; (D) malondialdehyde concentration. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 5

Effects of dietary tryptophan supplementation on mRNA expression of redox sensitive genes in piglets challenged with diquat. (A) SOD1, copper/zinc superoxide dismutase; (B) HMOX1, heme oxygenase 1; (C) GPX1, glutathione peroxidase; (D) TXNRD1, thioredoxin reductase; (E) NRF2, nuclear respiratory factor 2; (F) KEAP1, kelch like ECH associated protein 1. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 6

Effects of dietary tryptophan supplementation on protein expression of antioxidant enzymes in piglets challenged with diquat. (A) Representative blots of HO1, GPx, and GAPDH in the jejunal mucosa of piglets; (B) HO1, heme oxygenase 1; (C) GPx, glutathione peroxidase. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 7

Effects of dietary tryptophan supplementation on intestinal mitochondrial reactive oxygen species (ROS) production (A) and intestinal mitochondrial membrane potential change (B) of piglets challenged with diquat. The ROS production and mitochondrial membrane potential were expressed as fold changes, calculated relative to the control group. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.

Figure 8

Effects of dietary tryptophan supplementation on mRNA expression of genes involved in mitochondrial biogenesis and mtDNA content in the jejunum of piglets challenged with diquat. (A) NRF1, nuclear respiratory factor 1; (B) TFAM, transcription factor A, mitochondrial; (C) PPARGC1A, peroxisome proliferative activated receptor gamma coactivator 1 alpha; (D) SIRT1, sirtuin 1; (E) SSBP1, single stranded DNA binding protein 1; (F) POLRMT, DNA-directed RNA polymerase, mitochondrial; (G) mtDNA, mitochondrial DNA. Data are means ± standard error; n = 8 for each group. Mean values sharing different superscripts within the same row differ significantly (p < 0.05). Control, piglets fed the basal diet without diquat injection; diquat, piglets fed the basal diet and challenged with diquat; 0.15% Trp + diquat, piglets fed the 0.15% supplemented diet and treated with diquat.