ROS/JNK-mediated lysosomal injury in rat intestinal epithelial-6 cells during heat stress

Injury to the intestinal epithelial cells and loss of the intestinal barrier are critical to heatstroke. To reveal the mechanism through which heatstroke leads to intestinal epithelial injury, the relationship between reactive oxygen species (ROS), c-Jun NH2-terminal kinase (JNK), and lysosomes were studied in intestinal epithelial cells subjected to heat stress. Cells of heat stress groups were incubated at 43 degrees C for 1 h, then incubated at 37 degrees C as indicated. Control group cells were incubated at 37 degrees C. Cell-counting kit-8 assay was used to assess cell viability. Cells were labeled with 2 '-7 ' dichlorofluorescin diacetate and acridine orange (AO) staining, respectively, the total ROS and AO were detected by confocal laser scanning microscopy and flow cytometry. Apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/prodium iodide staining, the expressions of mitogen-activated protein kinases were detected by western blotting. Heat stress induced apoptosis and inhibited cell viability, the production of ROS, and lysosomal injury in IEC-6 cells. After pretreatment with the lysosomal cathepsin inhibitor E64, the JNK inhibitor SP600125, or the ROS scavenger NAC, the effect of heat stress on apoptosis or lysosomal injury was significantly attenuated. In conclusion, heat stress induced apoptosis, lysosomal injury, and the accumulation of ROS in IEC-6 cells; mechanistically, this occurred through the ROS-induced activation of JNK signaling, which mediated the lysosomal injury and ultimately apoptosis.