Nrf2‐mediated liver protection by sauchinone, an antioxidant lignan, from acetaminophen toxicity through the PKCδ‐GSK3β pathway

Abstract

BACKGROUND AND PURPOSESauchinone, an antioxidant lignan, protects hepatocytes from iron-induced toxicity. This study investigated the protective effects of sauchinone against acetaminophen (APAP)-induced toxicity in the liver and the role of nuclear factor erythroid-2-related factor-2 (Nrf2) in this effect. EXPERIMENTAL APPROACHBlood biochemistry and histopathology were assessed in mice treated with APAP or APAP + sauchinone. The levels of mRNA and protein were measured using real-time PCR assays and immunoblottings. KEY RESULTSSauchinone ameliorated liver injury caused by a high dose of APAP. This effect was prevented by a deficiency of Nrf2. Sauchinone treatment induced modifier subunit of glutamate-cysteine ligase, NAD(P)H:quinone oxidoreductase-1 (NQO1) and heat shock protein 32 in the liver, which was abolished by Nrf2 deficiency. In a hepatocyte model, sauchinone activated Nrf2, as evidenced by the increased nuclear accumulation of Nrf2, the induction of NQO1-antioxidant response element reporter gene, and glutamate-cysteine ligase and NQO1 protein induction, which contributed to the restoration of hepatic glutathione content. Consistently, treatment of sauchinone enhanced Nrf2 phosphorylation with a reciprocal decrease in its interaction with Kelch-like ECH-associated protein-1. Intriguingly, sauchinone activated protein kinase C-d (PKCd), which led to Nrf2 phosphorylation. In addition, it increased the inhibitory phosphorylation of glycogen synthase kinase-3b (GSK3b), derepressing Nrf2 activity, which was supported by the reversal of sauchinone's activation of Nrf2 by an activated mutant of GSK3b. Moreover, phosphorylation of GSK3b by sauchinone depended on PKCd activation. CONCLUSION AND IMPLICATIONSOur results demonstrate that sauchinone protects the liver from APAP-induced toxicity by activating Nrf2, and this effect is mediated by PKCd activation, which induces inhibitory phosphorylation of GSK3b. AbbreviationsALT, alanine aminotransferase; AMPK, AMP-activated protein kinase; APAP, acetaminophen; ARE, antioxidant response element; AST, aspartate aminotransferase; GCL, glutamate-cysteine ligase; GCLM, modifier subunit of glutamate-cysteine ligase; GSH, glutathione; GSK3b, glycogen synthase kinase 3b; HSP32, heat shock protein 32; Keap1, Kelch-like ECH-associated protein-1; KO, knockout; LDH, lactate dehydrogenase; NAPQI, N-acetyl-p-benzoquinoneimine; NQO1, NAD(P)H:quinone oxidoreductase-1; Nrf2, nuclear factor erythroid-2-related factor-2; PKCd, protein kinase C d; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; WT, wild-type

Pterostilbene Reduces Acetaminophen-Induced Liver Injury by Activating the Nrf2 Antioxidative Defense System via the AMPK/Akt/GSK3β Pathway

“…Emerging evidence indicates that AMPK is a negative modulator of oxidative stress responses that plays a protective role in an APAP-induced mouse model of DILI [19]. Since Nrf2 target genes can detoxify highly active intermediates, chemical activators of Nrf2 might be used to protect hepatocytes from the toxic effects of APAP or other drugs [20].…”

Fyn Inhibition by Cycloalkane-Fused 1,2-Dithiole-3-thiones Enhances Antioxidant Capacity and Protects Mitochondria from Oxidative Injury

“…Cells were plated at a density of 10 6 cells per well in six-well dishes and were transfected the following day, according to previously published methods (Kay et al, 2011). Briefly, the cells were incubated for 3 h with 1 g of NQO1-ARE reporter plasmid and 3 l of FuGENE HD reagent (Roche Diagnostics, Indianapolis, IN) in 1 ml of antibiotic-free minimal essential medium.…”

“…Nuclear extracts was prepared according to a previously published method, as described previously in detail (Kay et al, 2011). The nuclear fractions were stored at Ϫ70°C until use.…”

“…Reduced GSH in the cells was quantified by using a commercial GSH determination kit (GSH-400; OXIS Research, Inc.), as described previously (Kay et al, 2011).…”

“…Treatment with each of the agents enabled cells to restore GSH contents. Many AMPK activators may also enhance Nrf2 activity (Kay et al, 2010(Kay et al, , 2011. Similarly, AMPK activation increases Nrf2 expression levels (Liu et al, 2011).…”