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