Signal transduction pathways involved in oxidative stress-induced intestinal epithelial cell injury

Abstract

Free oxygen radicals are thought to be involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. We have recently demonstrated that cyclooxygenase (COX)-2 induction occurs in NEC; however, the potential upstream cellular mechanisms involved are still largely unknown. Therefore, the purpose of this study was to determine the intracellular signal transduction pathways involved during oxidative stress-induced intestinal epithelial cell injury. Rat intestinal epithelial cells (RIE-1) maintained in DMEM with 5% fetal bovine serum (FBS) were used for our experiments. Cells were serum-starved overnight and treated with either H 2O 2 or vehicle in the presence of various kinase inhibitors: PD98059 for p44/42 mitogen-activated protein kinase (MAPK), SB203580 for p38 MAPK, SP600125 for c-Jun N-terminal kinase (JNK), and LY290042 for phosphatidylinositol 3-kinase (PI3K/Akt). Treated cells were harvested over a time course and protein was extracted for Western blotting. Cell viability was determined using CCK-8 assay. H 2O 2 treatment resulted in increases in phosphorylation of p44/42 MAPK, p38 MAPK, JNK and Akt in RIE-1 cells in a time- and dose-dependent manner. The peak increases were noted with 200 μM dosage at 5–15 min after treatment. Total MAPK and Akt levels were not affected by H 2O 2 treatment. Additionally, RIE-1 cell viability was significantly decreased when either MAPK or PI3K pathway was selectively inhibited with PD98059 and LY294002 compounds, respectively. Interestingly, RIE-1 cell viability was markedly enhanced when JNK pathway was inhibited by SP600125. Inhibition of p38 MAPK with SB203580 did not influence RIE-1 cell viability after H 2O 2 treatment. Phosphorylation of multiple signaling transduction pathways occurs during oxidative stress-induced intestinal epithelial cell injury. In contrast to JNK pathway, both p44/42 MAPK and PI3K/Akt may be involved as protective cellular signaling pathways in intestinal injury that occurs during NEC. A better understanding of this process may allow us to elucidate molecular mechanisms contributing to NEC.