Silencing of soluble epoxide hydrolase 2 gene reduces H2O2-induced oxidative damage in rat intestinal epithelial IEC-6 cells via activating PI3K/Akt/GSK3β signaling pathway

Abstract

Oxidative stress plays a vital role in the occurrence and development of intestinal injury. Soluble epoxide hydrolase 2 gene (EPHX2) is a class of hydrolytic enzymes. We aim to explore the effects and molecular mechanism of siEPHX2 on H 2 O 2 -induced oxidative damage in rat intestinal epithelial IEC-6 cells. IEC-6 cells were transfected with EPHX2-siRNA and control si RNA plasmids by lipofectamine™ 2000 transfection reagent. The transfected samples were treated with H 2 O 2 (50, 100, 200, 300, 400, and 500 µmol/L) for 12, 24, and 48 h, respectively. Cell viability was determined by cell counting kit-8 (CCK-8). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were assessed by respective detection kits. Mitochondrial membrane potential (MMP), cell apoptosis and reactive oxygen species (ROS) and the levels of factors were determined by flow cytometer, quantitative real-time PCR (qRT-PCR) and western blot assays, respectively. We found that the IC50 of H 2 O 2 was 200 µmol/L at 24 h, and the transfection of siEHPX2 in H 2 O 2 -induced IEC-6 cells significantly promoted the cell viability, SOD activity and MMP rate, and reduced the rates of ROS and apoptosis as well as LDH and MDA contents. siEHPX2 up-regulated the B-cell lymphoma-2 (Bcl-2) level and down-regulated the levels of fibroblast-associated (Fas), Fas ligand (Fasl), Bcl-2 associated X protein (Bax), and Caspase-3. Moreover, the phosphorylation levels of phosphoinositide 3 kinase (PI3K), protein kinase B (Akt), and glycogen synthase kinase3β (GSK3β) were up-regulated. We proved that siEPHX2 had a protective effect on H 2 O 2 -induced oxidative damage in IEC-6 cells through activating PI3K/Akt/GSK3β signaling pathway.