Nrf2 counteracts cholestatic liver injury via stimulation of hepatic defense systems

doi:10.1016/j.bbrc.2009.08.156

Biochemical and Biophysical Research Communications

Volume 389, Issue 3, 20 November 2009, Pages 431-436

https://doi.org/10.1016/j.bbrc.2009.08.156 Get rights and content

Abstract

The transcription factor Nrf2 is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes and Mrp efflux transporters. We aimed to investigate whether Nrf2 activation counteracts liver injury associated with cholestasis. The role of Nrf2 activation in counteracting cholestatic liver injury was studied using a bile duct-ligation (BDL) model of Keap1 gene-knockdown (Keap1-kd) mice that represent the sustained activation of Nrf2 in the liver. Upon Nrf2 activation, Keap1-kd mice showed large increases in Mrp efflux transporters, detoxifying enzymes and antioxidative stress genes in the livers. After BDL, the number of hepatic parenchymal necrosis and the reactive oxygen species content were significantly smaller in the livers of the Keap1-kd mice than in those of the WT mice. Moreover, the increase in serum bilirubin levels was attenuated in the Keap1-kd mice. In conclusion, the results suggest a hepatoprotective role of sustained Nrf2 activation against liver injury associated with cholestasis.

Introduction

Cholestasis leads to hepatic and systemic accumulation of potentially toxic biliary compounds; e.g., bile acids and bilirubin; which in turn results in liver injury and jaundice [1]. Spontaneous defense mechanisms of hepatocytes against cholestasis include down-regulation of influx pumps and adaptive induction of export pumps; e.g., Mrp3 and Mrp4 [2], [3]. This adaptive response may prevent hepatocellular accumulation of bile acids and bilirubin and thereby limit cholestatic liver injury. Moreover, detoxification of biliary compounds accumulated in the liver through phase I hydroxylation and phase-II conjugation may counteract cholestatic liver injury by rendering hydrophobic substrates less toxic and better soluble for biliary and urinary excretion routes [4].

Under cholestatic conditions, oxidative stress plays an important role in the cell death of hepatocytes and links to the development of liver injury. Hydrophobic bile acids stimulate the generation of reactive oxygen species (ROS) in hepatocytes and in liver mitochondria [5]. Evidence of oxidative stress-induced liver injury has been demonstrated in rats receiving intravenously infused hydrophobic bile acids [6] and in the bile duct-ligated (BDL) rat model of obstructive cholestasis [7], [8].

One transcription factor that serves as a cellular sensor for oxidative stress is termed nuclear factor-E2-related factor-2 (Nrf2). Nrf2 is sequestered in the cytosol by Kelch-like Ech-associated protein (Keap1). However, during oxidative challenge, modification of Keap1 sulfhydryl groups results in release and nuclear translocation on Nrf2 [9]. Nrf2 is essential for antioxidant responsive element/electrophile-responsive element (ARE/EpRE)-mediated gene induction of detoxifying enzymes [10] and antioxidative stress genes, and can transcriptionally activate several enzymes involved in cellular protection [9], [10], [11], [12], [13], [14], [15]. Activation of this gene battery serves to decrease the oxidative burden of the cell by increasing GSH concentrations, reducing reactive intermediates, and increasing general detoxification of chemicals via phase-II conjugation reactions [11]. Moreover, the recent studies of our group [16], [17] have shown that the Nrf2 activators induce Mrp efflux transporters in rodent livers.

In this study, we aimed at determining whether Nrf2 activation counteracts liver injury in obstructive cholestasis caused by BDL in Keap1-kd mice. The results indicate that BDL-induced liver injury is significantly attenuated in the Keap1-kd mice compared with that in wild-type (WT) mice, through a stimulation of detoxification and antioxidative stress systems, along with Mrp efflux transport.

Section snippets

Materials and methods

Animals. Pair-matched littermate wild-type and Keap1 knockdown (Keap1-kd) mice on an C57/B6 background, in which cytoplasmic protein Keap1, a repressive factor of Nrf2, is markedly reduced and thereby results in constitutive activation of Nrf2, were engineered and bred at the Center for Tsukuba Advanced Research Alliance and Institute of Basic Medical Science, the University of Tsukuba [16]. All experiments were conducted according to the institution’s guidelines for the care and use of

Effects of constitutive activation of Nrf2 on hepatoprotection of the mouse liver in obstructive cholestasis

Effects of constitutive activation of Nrf2 on hepatoprotection of the mouse liver were tested in Keap1-kd mice with BDL (Keap1-kd BDL mice), a particularly severe model of obstructive cholestasis.

Histological analysis of liver tissue sections to determine the levels of hepatic parenchymal necrosis were conducted in WT mice with BDL (WT BDL mice) and Keap1-kd BDL mice. Keap1-kd BDL mice had significantly decreased numbers of necrotic foci on the sections (Fig. 1A).

Nuclear levels of Nrf2 were

Discussion

The results of this study and those of other recent studies from our laboratories [17], [18] have demonstrated that expression levels of Mrp efflux transporters, phase I detoxifying enzymes, phase II conjugating enzymes and antioxidative stress genes are markedly up-regulated by Nrf2 activation. Especially, since Mrp efflux transporters play a key role in cellular protection by removing xenobics, metabolites, and endogenous substrates that can accumulate in tissues and lead to toxicity,

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Citation Excerpt :
It then binds to antioxidant response elements (AREs) in the promoter of target genes, thereby promoting the transcription of a wide array of cytoprotective genes that contribute to the disposition, detoxification, and clearance of a large number of xenobiotics (Shen and Kong, 2009). The defensive response mediated by the Nrf2 pathway has been one of the key cellular protection mechanisms against harm caused by exposure to xenobiotics in the liver (Ma, 2013; Okada et al., 2009). HQ is a popular herb frequently coadministered with many other herbal or chemical drugs for efficacy improvement in clinics.
Astragali radix (Huang Qi, HQ), a well-known Chinese herbal medicine, is widely coadministered with many other drugs for treating diseases. The potential herb–drug interactions (HDIs) possibly occur during the combination therapy. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are the crucial targets that mediate the production of HDIs. We previously observed that HQ and its three main bioactive compounds, including Astragaloside IV (AS-IV), calycosin (CS) and formononetin (FMNT), could significantly induce the expression of P-gp and BCRP in HepG2 cells in vitro. However, their modulations on the function of P-gp and BCRP remain unknown; their impact on these two proteins expression in vivo is not clear; the exact regulatory mechanism has also not yet been explored.
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Nrf2 counteracts cholestatic liver injury via stimulation of hepatic defense systems

Abstract

Introduction

Section snippets

Materials and methods

Effects of constitutive activation of Nrf2 on hepatoprotection of the mouse liver in obstructive cholestasis

Discussion

Gastroenterology

Gastroenterology

Free Radic. Biol. Med.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Chromatogr. B Biomed. Sci. Appl.

Anal. Biochem.

Hepatology

J. Hepatol.

Gastroenterology

Molecular pathogenesis of cholestasis

N. Engl. J. Med.

Mrp4−/− mice have an impaired cytoprotective response in obstructive cholestasis

Hepatology

Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice

Gastroenterology

Ursodeoxycholic acid protects against secondary biliary cirrhosis in rats by preventing mitochondrial oxidative stress

Hepatology