In Vivo Effects of Intracellular Reactive Oxygen Species and Anti-protease Action on NF-kB Activation in Rats

Abstract

Reactive oxygen species (ROS) have recently been suggested (in vitro) to play a role as second messengers in the signal transduction pathway leading to nuclear factor-kappa B (NF-kB) activation, a critical step for effective inflammatory responses, both beneficial and harmful. To better understand the involvement of ROS in the NF-kB activation cascade and explore possible ways to modulate its undesired activation in vivo, a series of experiments were performed involving chemically-induced intracellular ROS and their effect on NF-kB activation. Redox-cycling agent diquat (DQ) (0.1 mmoI/kg i.p.) was administered in male Fischer rats by itself, as well as in rats pretreated with glutathione-depleting agent phorone (PH) (200 mg/kg i.p.). Rats were also treated with endotoxin (ET) (0.01 mg/kg i.v.) and DQ together. Hepatic glutathione concentrations and plasma alanine aminotransferase (ALT) activities were measured to determine the extent of the induced intracellular oxidative stress (caused by ROS). Since proteases are thought to be involved in the NF-kB activation cascade, we explored the role of anti-proteases such as N-tosyl-L-Iysylchloromethylketone (TLCK) in the attenuation of NF-kB activation in rat hepatocytes. NF-kB activation was determined by running nuclear extracts through electrophoretic mobility shift assays (EMSA). Increases in the percentage of hepatic glutathione disulfide (GSSG) concentrations and plasma alanine aminotransferase (ALT) activities indicated the presence of intracellular oxidative stress in both DQ only and PH + DQ treated animals. PH pretreatment resulted in greater intracellular oxidative stress as indicated by higher ALT activities. NF-kB was activated in both groups. ET pretreatment alone had little effect on intracellular oxidative stress but highly activated NF-kB, whereas the addition of DQ greatly enhanced intracellular oxidative stress without any additive effect on NF-kB activation demonstrating the lack of modulatory, additive effect of ROS on NF-kB activation. The anti-proteases, TLCK decreased endotoxin-induced NF-kB activation. This in vivo study appears to support the hypothesis of ROS as (direct) second messengers in NF-kB activation and further suggests a lack of modulatory (additive) effect of ROS on NF-kB activation. In addition, the study also demonstrates possible regulation of NF-kB activation by anti-protease (TLCK) action.

With honors.