Preventing hypoxia/reoxygenation damage to hepatocytes by p66shc ablation:: Up-regulation of anti-oxidant and anti-apoptotic proteins

Background/Aims:Ischemia/reperfusion damage to the liver remains a serious concern in many clinical situations. Major mechanisms for this certainly include oxidative stress. Methods:The effects of ablating the p66 isoform of ShcA (p66(shc)) on hypoxia/reoxygenation (H/R)-induced oxidative stress and cell injury in hepatocytes were investigated. Results: Immediately after reoxygenation, AML12 cells were clearly under oxidative stress; many cells underwent apoptosis. However, knockdown of p66(shc) by specific RNAi markedly decreased cellular oxidative stress and H/R-induced apoptosis, as well as conferring resistance to H2O2 insult. These data suggest that prevention of apoptosis conferred by ablation of p66(shc) results from changed ROS-scavenging, but not inhibition of ROS generation. These data were also confirmed in fibroblasts from p66(shc) knockout mice. Anti-oxidant molecules, such as MnSOD and Ref-1 and the anti-apoptotic molecule Bcl-xL were up-regulated, and pro-apoptotic FLICE was down-regulated, by ablation of p66. Interestingly, catalase expression was not affected in p66(shc)-knockdown-AML12 cells although it is a major target in other cell types. Conclusions: Our findings suggest that in hepatocytes, ablation of p66 A, is cytoprotective against H/R-induced oxidative stress, with MnSOD and Ref-1 playing critical roles, and with up-regulation of Bcl-xL and down-regulation of FLICE contributing jointly to preventing cells from undergoing oxidant-induced apoptosis. (C) 2007 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.