Weaning disrupts intestinal antioxidant status, impairs intestinal barrier and mitochondrial function, and triggers mitophagy in piglets

Abstract

In the present study, we investigated the influence of weaning on antioxidant status, intestinal integrity, mitochondrial function, and the mitophagy level in piglets (weaned at 21 d) during the 1 wk after weaning. The redox status was measured by antioxidant enzymes activities, related genes expression, and malondialdehyde (MDA) content in jejunum. The intestinal barrier function was assessed by the Ussing chamber and expression of tight junction proteins in the jejunum. The function of intestine mitochondria was measured by mitochondrial DNA (mtDNA) content and activities of mitochondria oxidative phosphorylation complexes. The levels of light chain 3-1 (LC3-I), light chain 3-II (LC3-II), PTEN-induced putative kinase 1 (PINK1), and Parkin were determined to investigate whether mitophagy is involved in the weaning process. The results showed that, as compared with the preweaning phase (d 0), weaning suppressed (P < 0.05) the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) on d 3 and d 7 postweaning, decreased (P < 0.05) the expression of copper and zinc superoxide dismutase (Cu/Zn-SOD), manganese-containing superoxide dismutase (Mn-SOD) on d 3 postweaning, declined (P < 0.05) the level of glutathione peroxidase 1 (GPX-1) and glutathione peroxidase 4 (GPX-4) on d 3 and d 7 postweaning, and increased (P < 0.05) MDA content in jejunum on d 3 and d 7 postweaning. The jejunal transepithelial electrical resistance and levels of occludin, claudin-1, and zonula occludens-1 on d 3 and d 7 postweaning were reduced (P < 0.05), and paracellular flux of fluorescein isothiocyanatedextran (4 kDa) on d 3 and d 7 postweaning was increased (P < 0.05). Weaning induced mitochondrial dysfunction, as demonstrated by decreased (P < 0.05) content of mtDNA on d 3 and d 7 postweaning and declined (P < 0.05) activities of mitochondria complexes (I, II, III, IV) in jejunum on d 1, d 3, and d 7 postweaning. Weaning led to an increased (P < 0.05) expression level of mitophagy-related proteins, PINK1 and Parkin, in the intestinal mitochondria, as well as an enhancement (P < 0.05) of the ratio of LC3-II to LC3-I content in the jejunal mucosa on d 1, d 3, and d 7 postweaning. These results suggest that weaning disrupted intestinal oxidative balance, and this imbalance may impair intestinal barrier and mitochondrial function and trigger mitophagy in piglets.

Figure 1.

(a) Effects of weaning on expression of tight junction proteins in jejunal mucosa. (b) Representative blots of claudin-1, occludin, zonula occludens-1 (ZO-1) and β-actin in the jejunal mucosa of piglets. Values are means and SD represented by vertical bars. ^a,bMeans with different letters differ significantly (P < 0.05). The protein expression of all samples was expressed as fold changes, calculated relative to the preweaning (d 0 postweaning) pigs.

Figure 2.

(a) Effects of weaning on mitochondrial DNA (mtDNA) content in the jejunum of piglets. (b) Effects of weaning on the activities of mitochondrial complexes in jejunal mitochondria of piglets. Values are means and SD represented by vertical bars. ^a,b,cMeans with different letters differ significantly (P < 0.05). The mtDNA content and mitochondrial complex activities of all samples were expressed as fold changes, calculated relative to the preweaning (d 0 postweaning) pigs.

Figure 3.

Effects of weaning on expression of mitophagy-related protein in the intestinal mitochondrial of piglets. (a) Representative blots of PTEN-induced putative kinase 1 (PINK1) and Parkin in the intestinal mitochondria of piglets. (b) Relative PINK1 and Parkin expression of piglets. (c) Representative blots of light chain 3-I (LC3-I) and LC3-II in the jejunum mucosa weaning of piglets. (d) Relative LC3-I and LC3-II expression of piglets. Summary of Western blot for n = 6 pigs. Values are means and SD represented by vertical bars. ^a,bMeans with different letters differ significantly (P < 0.05). The protein expression of all samples was expressed as fold changes, calculated relative to the preweaning (d 0 postweaning) pigs.