GFP Reporter Cell Line - IPEC-J2

Deoxynivalenol (DON) is considered one of the most serious mycotoxins affecting the gut health of animals and humans. Phenylethyl isothiocyanate (PEITC) in feed is an anti-nutritional factor that impairs the digestion and absorption of nutrients in the animal gut. Here, researchers investigated the effects of PEITC on DON-induced apoptosis, intestinal tight junction disorder, and its potential molecular mechanisms in the porcine jejunal epithelial cell line (IPEC-J2). The results showed that PEITC treatment significantly alleviated DON-induced cytotoxicity, reduced the apoptosis rate and pro-apoptotic mRNA/protein levels, and increased the mRNA/protein expression levels of tight junction protein-1 (ZO-1), closure protein, and tight junction protein-1. Simultaneously, PEITC treatment improved DON-induced increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) mRNA levels and intracellular reactive oxygen species (ROS) levels, as well as decreases in glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), catalase (CAT), and heme oxygenase 1 (HO-1) mRNA levels. Furthermore, PEITC treatment significantly downregulated the mRNA/protein levels of autophagy-associated protein 5 (ATG5), beclin-1, and microtubule-associated protein 1 light chain 3B (LC3-II), reduced the number of green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) spots and the protein expression of phosphatidylinositol 3-kinase (PI3K), and upregulated the protein expression of phosphorylated protein kinase B (p-Akt) and phosphorylated mammalian target of rapamycin (p-mTOR), thus counteracting DON damage. In summary, PEITC can protect porcine intestinal epithelial cells from DON-induced damage by inhibiting ROS-mediated autophagy.

Here, researchers detailed the effects of PEITC on DON-induced autophagy. Compared to the control group, the 1 μmol/L DON treatment group showed higher expression levels of ATG5, beclin-1, and LC3B mRNA (Figure 1A-C). To further determine whether DON treatment triggered autophagy, researchers transfected IPEC-J2 cells with green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3), a specific marker of autophagic vesicles and autophagy activity. As shown in Figure 1G, the number of GFP-LC3 spots in the 1 μmol/L DON treatment group was higher than that in the control group. DON treatment significantly increased the expression of PI3K, ATG5, and LC3-II proteins and decreased the expression of p-Akt and p-mTOR proteins (Figure 1D-F). However, PEITC supplementation significantly reduced the DON-induced increase in ATG5, beclin-1, and LC3B mRNA levels in a dose-dependent manner. Meanwhile, compared with the 1 μmol/L DON treatment group, the 5 μmol/L PEITC treatment significantly inhibited the protein expression of PI3K, ATG5, and LC3-II, as well as the number of GFP-LC3 spots, and increased the protein expression of p-Akt and p-mTOR. Therefore, these results suggest that activation of the PI3K/Akt/mTOR signaling pathway may be essential for PEITC to alleviate DON-induced autophagy.

Figure 1. Effect of PEITC on DON-induced autophagy in IPEC-J2 cells. (Liu S, et al., 2022)