RESUMO
The present study aimed to investigate the effect of glutamine (Gln) addition on the damage of porcine intestinal epithelial cells (IPEC-J2) induced by heat stress (HS). IPEC-J2 cultured in logarithmic growth period in vitro were firstly exposed to 42 °C for 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h for cell viability and cultured with 1, 2, 4, 6, 8, or 10 mmol Gln per L of culture media for heat shock protein 70 (HSP70) expression to determine the optimal disposal strategy (HS, 42 °C for 12 h and HSP70 expression, 6 mmol/L Gln treatment for 24 h). Then IPEC-J2 cells were divided into three groups: control group (Con, cultured at 37 °C), HS group (HS, cultured at 42 °C for 12 h), and glutamine group (Gln+HS, cultured at 42 °C for 12 h combined with 6 mmol/L Gln treatment for 24 h). The results showed that HS treatment for 12 h significantly decreased the cell viability of IPEC-J2 (P < 0.05) and 6 mmol/L Gln treatment for 12 h increased HSP70 expression (P < 0.05). HS treatment increased the permeability of IPEC-J2, evidenced by the increased fluorescent yellow flux rates (P < 0.05) and the decreased transepithelial electrical resistance (P < 0.05). Moreover, the downregulated protein expression of occludin, claudin-1, and zonula occludens-1 was observed in HS group (P < 0.05), but Gln addition alleviated the negative effects on permeability and the integrity of intestinal mucosal barrier induced by HS (P < 0.05). In addition, HS resulted in the elevations in HSP70 expression, cell apoptosis, cytoplasmic cytochrome c potential expression, and the protein expressions of apoptosis-related factors (apoptotic protease-activating factor-1, cysteinyl aspartate-specific proteinase-3, and cysteinyl aspartate-specific proteinase-9) (P < 0.05); however, the reductions in mitochondrial membrane potential expression and B-cell lymphoma-2 expression were induced by HS (P < 0.05). But Gln treatment attenuated HS-induced adverse effects mentioned above (P < 0.05). Taken together, Gln treatment exhibited protective effects in protecting IPEC-J2 from cell apoptosis and the damaged integrity of epithelial mucosal barrier induced by HS, which may be associated with the mitochondrial apoptosis pathway mediated by HSP70.
It has been demonstrated that heat stress (HS) induced damages of the intestinal epithelial cell membrane and tight junction, which ultimately compromises intestinal integrity and increases intestinal permeability and leads to the reduced growth performance and the increased morbidity and mortality. However, glutamine (Gln) contributes to rescuing the phenotype of intestinal barrier dysfunction through decreasing intestinal permeability, regulating the gut tight junction proteins under HS conditions, enhancing the viability, and attenuating cell apoptosis in porcine enterocytes suffered from stress treatment. In addition, it was reported that Gln administration increased the protein expression of intestinal heat shock protein 70 (HSP70), which may play a regulatory role in cellular apoptosis within IPEC-J2 cells. Therefore, we hypothesized that Gln might contribute to alleviating HS-induced damage of porcine intestinal epithelium via inhibiting the mitochondrial apoptosis pathway mediated by HSP70. The results showed that Gln addition alleviated the negative effects on permeability and the integrity of intestinal mucosal barrier induced by HS. In addition, Gln treatment reversed the elevations in HSP70 expression, cell apoptosis, cytoplasmic cytochrome c potential expression, and the protein expressions of apoptosis-related factors (apoptotic protease-activating factor-1, cysteinyl aspartate-specific proteinase-3, and cysteinyl aspartate-specific proteinase-9) induced by HS, and resulted in an increase in mitochondrial membrane potential expression and B-cell lymphoma-2 expression. Taken together, Gln treatment exhibited protective effects in protecting IPEC-J2 from cell apoptosis and the damaged integrity of epithelial mucosal barrier induced by HS, which could be associated with the mitochondrial apoptosis pathway mediated by HSP70.
