González-Rodríguez Águeda, Reibert Bjorn, Amann Thomas, Constien Rainier, Rondinone Cristina M, Valverde Ángela M
Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain Instituto de Investigaciones Biomédicas "Alberto Sols" (Consejo Superior de Investigaciones Científicas/Universidad Autónoma de Madrid), 28029 Madrid, Spain
Roche Kulmbach GmbH, Kulmbach, D 95326, Germany.
Dis Model Mech. 2014 Sep;7(9):1093-100. doi: 10.1242/dmm.015537. Epub 2014 Jul 4.
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 the tail vein. After 48 hours, ALF was induced by ConA. Liver histology, pro-inflammatory mediators, antioxidant responses, cellular death, and stress and 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 with 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 the 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 antioxidant responses and IGFIR survival signaling during the progression of ALF.
氧化应激促进急性肝衰竭（ALF）的进展。转录因子核因子-红系2相关因子（Nrf2）作为细胞对抗氧化应激的内源性调节因子。我们使用针对肝细胞中Keap1的体内小干扰RNA（siRNA）递送，研究了刀豆蛋白A（ConA）介导的ALF中的肝损伤以及死亡和生存信号通路之间的平衡。为此，通过尾静脉给小鼠注射含Keap1或荧光素酶siRNA的脂质体。48小时后，用ConA诱导ALF。评估肝脏组织学、促炎介质、抗氧化反应、细胞死亡以及应激和生存信号。注射Keap1-siRNA的小鼠肝脏中Keap1 mRNA和蛋白水平显著降低。在这些动物中，用ConA攻击时，肝脏组织学损伤比对照小鼠轻。同样，细胞死亡标志物（FasL和半胱天冬酶8、3和1）在注射后4小时和8小时降低。与对照动物相比，注射Keap1-siRNA的小鼠中核Nrf2及其靶标血红素加氧酶1（HO1）升高，导致肝脏氧化应激降低。同样，注射Keap1-siRNA的小鼠肝脏中促炎细胞因子的mRNA水平降低。在分子水平上，c-jun（NH2）末端激酶（JNK）的激活得到改善，而在注射Keap1-siRNA的小鼠中注射ConA后胰岛素样生长因子I受体（IGFIR）生存通路得以维持。总之，我们的结果揭示了靶向Keap1的体内siRNA技术在ALF进展过程中通过调节Nrf2介导的抗氧化反应和IGFIR生存信号来对抗氧化应激的潜在治疗用途。
