In vivo siRNA delivery of Keap1 modulates death and survival signaling pathways and attenuates Concanavalin A-induced acute liver injury in mice

Abstract

Abstract Oxidative stress contributes to the progression of acute liver failure (ALF). Transcription factor nuclear factor-erythroid 2-related factor (Nrf2) serves as an endogenous regulator by which cells combat oxidative stress. We have investigated liver damage and the balance between death and survival signaling pathways in Concanavalin A (ConA)-mediated ALF using in vivo siRNA delivery targeting Keap1 in hepatocytes. For that goal, mice were injected with Keap1 or Luciferase siRNA-containing liposomes via tail vein. After 48 hours, ALF was induced by ConA. Liver histology, pro-inflammatory mediators, anti-oxidant responses, cellular death and stress/survival signaling were assessed. Keap1 mRNA and protein levels significantly decreased in livers of Keap1 siRNA-injected mice. In these animals, histological liver damage was less evident than in control mice when challenged with ConA. Likewise, markers of cellular death (FasL and caspases 8, 3 and 1) decreased at 4 and 8 hours post-injection. Nuclear Nrf2 and its target hemoxygenase 1 (HO1) were elevated in Keap1 siRNA-injected mice compared to control animals resulting in reduced oxidative stress in the liver. Similarly, mRNA levels of pro-inflammatory cytokines were reduced in livers from Keap1 siRNA-injected mice. At the molecular level, activation of c-jun (NH2) terminal kinase (JNK) was ameliorated whereas insulin-like growth factor I receptor (IGFIR) survival pathway was maintained upon ConA injection in Keap1 siRNA-treated mice. In conclusion, our results have revealed a potential therapeutic use of in vivo siRNA technology targeted to Keap1 to combat oxidative stress by modulating Nrf2-mediated anti-oxidant responses and IGFIR survival signaling during the progression of ALF.