Roles of JNK, ERK, and PI3K/AKT signaling pathways in zinc-mediated alleviation of thermal stress-induced damage to the integrity and barrier function of primary cultured chick jejunal epithelial cells

Abstract This study investigated the precise alleviating mechanisms of Zn on chick embryonic jejunal epithelial cells under thermal stress (TS) and elucidated signaling pathways directly involved in this process. Experiment 1 identified the effective concentrations of inhibitors or agonists for extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT). The data were analyzed using one-way ANOVA. In experiment 2, a randomized factorial design consisting of 3 controls + 3 cell types × 3 Zn treatments was used to confirm the effect of Zn and cell type on each signaling pathway. Under heat stress (HT, 44°C), 3 cell types in which the signaling pathways were normal, inhibited, or overexpressed were subjected to 3 treatments consisting of supplementation with inorganic Zn sulfate (iZn), organic Zn proteinate (oZn), or no Zn supplementation (containing 3.98 μmol Zn/L). Under normal temperature (NT, 40 °C), the 3 cell types without Zn addition as controls. The significant differences between NT and HT across each cell type were conducted by T-test, data under HT were evaluated using two-way ANOVA. The results showed that TS decreased transepithelial resistance (TEER), claudin-1 and zona occludens-1 (ZO-1) mRNA levels, phosphorylated (p)-ERK/ERK, p-PI3K/PI3K, and p-AKT/AKT levels, and increased phenol red permeability compared with NT in 3 cell types (P < 0.05). Under HT, iZn and oZn decreased phenol red permeability and diamine oxidase (DAO), while increasing cell proliferation, claudin-1, occludin, and junctional adhesion molecule-A (JAM-A) mRNA and p-ERK/ERK level in normal cells, and the above mRNA levels were higher in the oZn group than in the iZn group (P < 0.05); however, different Zn treatments had no effect on DAO and cell proliferation in ERK inhibited and overexpression cells (P > 0.05). The interaction between cell type and Zn treatment did not affect the integrity and barrier function indices and p-JNK/JNK level in the cells for the JNK signaling pathway (P > 0.05). Supplemental iZn and oZn increased TEER, cell proliferation, claudin-1 and p-PI3K/PI3K level in normal cells (P < 0.05), but different Zn treatments had no effect on phenol red permeability, DAO and cell proliferation in PI3K/AKT inhibited and over expression cells, and ZO-1 mRNA and protein levels and p-PI3K/PI3K level in over expression cells (P > 0.05). Therefore, the above results show that Zn could alleviate TS-induced damage to the integrity and barrier function of chick embryonic jejunal epithelial cells via the ERK and PI3K/AKT signaling pathways.