Luteolin prevents liver from tunicamycin-induced endoplasmic reticulum stress via nuclear factor erythroid 2-related factor 2-dependent sestrin 2 induction

https://doi.org/10.1016/j.taap.2020.115036Get rights and content

Highlights

  • Luteolin decreases ER stress-induced CHOP expression in hepatocyte-derived cells.

  • Luteolin inhibits the activation of three canonical UPR signaling pathways.

  • Nrf2-mediated sestrin 2 expression contributes to alleviate ER stress by luteolin.

  • Luteolin ameliorates tunicamycin-induced acute liver injury in mice.

Abstract

Endoplasmic reticulum (ER) stress designates a cellular response to the accumulation of misfolded proteins, which is related to disease progression in the liver. Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a phytochemical found frequently in medicinal herbs. Although luteolin has been reported to possess the therapeutic potential to prevent diverse stage of liver diseases, its role in hepatic ER stress has not been established. Thus, the present study aimed to determine the role of luteolin in tunicamycin (Tm)-induced ER stress, and to identify the relevant mechanisms involved in its hepatoprotective effects. In hepatocyte-derived cells and primary hepatocytes, luteolin significantly decreased Tm- or thapsigargin-mediated C/EBP homologous protein (CHOP) expression. In addition, luteolin reduced the activation of three canonical signaling pathways related to the unfolded protein response, and decreased mRNA levels of glucose-regulated protein 78, ER DNA J domain-containing protein 4, and asparagine synthetase. Luteolin also significantly upregulated sestrin 2 (SESN2), and luteolin-mediated CHOP inhibition was blocked in SESN2 (+/−) cells. Moreover, luteolin resulted in phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), as well as increased nuclear Nrf2 expression. Deletion of the antioxidant response element in the human SESN2 promoter inhibited increased luciferase activation by luteolin, suggesting that Nrf2 is a critical transcription factor for luteolin-dependent SESN2 expression. In a Tm-mediated liver injury model, luteolin decreased serum alanine aminotransferase and aspartate aminotransferase activities, prevented degenerative changes and apoptosis of hepatocytes, and inhibited CHOP and glucose-regulated protein 78 expression in hepatic tissues. Therefore, luteolin may be an effective phytochemical to manage ER stress-related liver injury.

Introduction

Imbalance between protein synthesis and turnover leads to accumulation of misfolded proteins in the endoplasmic reticulum (ER) and induces the unfolded protein response (UPR). Misfolded proteins sequester molecular chaperones (e.g., glucose-regulated protein 78 [GRP78]) in the ER, and then transmit the cellular signal via activating transcription factor (ATF) 6, inositol requiring enzyme 1 (IRE1)/spliced X-box binding protein 1 (sXBP1), and PKR-like ER kinase/eukaryotic initiation factor 2α (eIF2α)/ATF4 (Dara et al., 2011). These UPR pathways halt general translation, expand the ER, and increase protein expression associated with ER chaperones and ER-associated degradation, to resolve protein overloading in the ER. Because sustained accumulation of misfolded proteins (ER stress) leads to apoptosis via Ca2+ leakage from the ER and induction of proapoptotic proteins including C/EBP homologous protein (CHOP) (Sano and Reed, 2013), ER stress is closely related with progression of many diseases. Importantly, hepatocytes contain highly developed ER, and are sensitive to ER stress. Accumulated evidence has indicated that ER stress is associated with viral hepatitis, nonalcoholic fatty liver disease, cholestasis, alcohol-induced liver injury, and hepatocellular carcinoma (Dara et al., 2011; Malhi and Kaufman, 2011). Thus, the control of ER stress is a promising therapeutic option for the management of liver diseases.

Of the diverse signaling molecules that regulate ER stress, we previously reported that ATF6-dependent induction of sestrin 2 (SESN2) prevents tunicamycin (Tm)-mediated liver injury by inhibiting apoptosis (Jegal et al., 2017). SESN2 is a highly conserved multifunctional protein that contains three functional domains for suppressing reactive oxygen species, regulating the mechanistic target of rapamycin complex, and sensing levels of leucine (Ho et al., 2016). Several transcription factors including nuclear factor erythroid 2-related factor 2 (Nrf2) and ATF4 are known to regulate the expression of SESN2, and the SESN2 induction protects cells from harmful stimuli (Park et al., 2014; Shin et al., 2012). Although we and others have suggested that SESN2 attenuates diverse stages of liver diseases (Yang et al., 2019a; Jegal et al., 2017; Yang et al., 2015; Park et al., 2014), studies are still required to discover candidates that exhibit hepatoprotective effects through the induction of SESN2.

Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a polyphenolic flavonoid found in medicinal herbs, vegetables and fruits, and possesses diverse pharmacological activities including antioxidant activity (Wu et al., 2015). Importantly, luteolin is a promising phytochemical to treat hepatitis, steatosis, fibrosis, and hepatocellular carcinoma in experimental animals (Yang et al., 2019b; Yin et al., 2017; Li et al., 2015; Tai et al., 2015). Although relief of ER stress probably contributes to the beneficial effects of luteolin in the liver, there is controversy about the role of luteolin during ER stress. Luteolin decreases ER stress-mediated CHOP induction and protects cells from acetaminophen and 4-hydroxynonenal (4-HNE) (Tai et al., 2015; Wu et al., 2015), while it also causes apoptosis by activating ER stress in some cancer cells (Wang et al., 2017; Choi et al., 2011).

In the present study, we therefore determined the in vitro and in vivo hepatoprotective effects of luteolin using Tm, a well-defined ER stressor via inhibiting post-translational modification in the ER, and investigated the underlying molecular mechanisms.

Section snippets

Reagents

Anti-ATF6α (Cat. No. 73–500) and anti-Nrf2 (Cat. No. sc-13032) antibodies were obtained from BioAcademia (Osaka, Japan) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Antibodies against GRP78 (Cat. No. ab21685), phosphorylated IRE1 (Ser724) (Cat. No. ab124945), 4-HNE (Cat. No. ab46545), and phosphorylated Nrf2 (Ser40) (Cat. No. ab76026) were purchased from Abcam (Cambridge, UK). Anti-CHOP (Cat. No. 2895), anti-IRE1α (Cat. No. 3294), anti-phosphorylated eIF2α (Ser51) (Cat. No.

Luteolin inhibits ER stress-mediated CHOP induction in hepatocytes

CHOP is a representative protein induced by ER stress (Jegal et al., 2017; Sano and Reed, 2013). To identify valuable natural products regulating ER stress in the liver, we first screened a natural product library by assessing protein levels of CHOP in HepG2 cells treated with Tm and Tg (another ER stressor that inhibits Ca2+ pumps in the ER). Of the 87 natural products tested, luteolin (30 μM) inhibited CHOP expression induced by Tm and Tg (Supplementary Table 2 and Supplementary Fig. 1). To

Discussion

Phytochemicals, including flavonoids, organic acids, carotenoids, alkaloids, lignans, xanthines, and polyphenols, have emerged as alternative pharmaceutical sources, showing fewer toxic effects and greater efficacy (Madrigal-Santillán et al., 2014). There have been many studies supporting the hepatoprotective effects of phytochemicals with antioxidant, anti-inflammation, anti-fibrosis, or anticancer properties. However, in spite of the pathophysiological importance of ER stress in liver

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning of Republic Korea (Grant No. 2015R1D1A1A01060284, 2018R1A5A2025272, and 2018R1A2B6007514). K. H. Jegal would like to thank the Ph.D. program at Daegu Haany University (Gyeongsan, Republic of Korea) for completing the thesis through part of this work.

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