Wang et al. Cell Death and Disease (2020) 11:781 https://doi.org/10.1038/s41419-020-02961-5 Cell Death & Disease ARTICLE Open Access (+)-Clausenamide protects against drug-induced liver injury by inhibiting hepatocyte ferroptosis Min Wang1,2,Chun-YuLiu1,3,4,TianWang5,Hong-MinYu1,3,4, Shu-Hua Ouyang1,3,4,Yan-PingWu1,3,4,Hai-BiaoGong1,3,4, Xiao-Hui Ma1,3,4, Gen-Long Jiao6, Lei-Lei Fu7, Qiong-Shi Wu2, Hiroshi Kurihara1,3,4,Yi-FangLi1,3,4,TaoShen5 and Rong-Rong He 1,3,4 Abstract Drug-induced liver injury is the major cause of acute liver failure. However, the underlying mechanisms seem to be multifaceted and remain poorly understood, resulting in few effective therapies. Here, we report a novel mechanism that contributes to acetaminophen-induced hepatotoxicity through the induction of ferroptosis, a distinctive form of programmed cell death. We subsequently identified therapies protective against acetaminophen-induced liver damage and found that (+)-clausenamide ((+)-CLA), an active alkaloid isolated from the leaves of Clausena lansium (Lour.) Skeels, inhibited acetaminophen-induced hepatocyte ferroptosis both in vivo and in vitro. Consistently, (+)-CLA significantly alleviated acetaminophen-induced or erastin-induced hepatic pathological damages, hepatic dysfunctions and excessive production of lipid peroxidation both in cultured hepatic cell lines and mouse liver. Furthermore, treatment with (+)-CLA reduced the mRNA level of prostaglandin endoperoxide synthase 2 while it increased the protein level of glutathione peroxidase 4 in hepatocytes and mouse liver, confirming that the inhibition of ferroptosis contributes to the protective effect of (+)-CLA on drug-induced liver damage. We further revealed that (+)-CLA specifically reacted with the Cys-151 residue of Keap1, which blocked Nrf2 ubiquitylation and resulted in an – 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; increased Nrf2 stability, thereby leading to the activation of the Keap1 Nrf2 pathway to prevent drug-induced hepatocyte ferroptosis. Our studies illustrate the innovative mechanisms of acetaminophen-induced liver damage and present a novel intervention strategy to treat drug overdose by using (+)-CLA. Introduction cell lines and animal models2,3. It is now well-accepted Drug-induced liver injury (DILI) is a major cause of that APAP can induce hepatoxicity through two common acute liver and kidney failures. The yearly incidence rate forms of cell death, namely apoptosis and necrosis4. of DILI is between 13.9 and 19.1 per 100,000 individuals, Notably and intriguingly, a recent study indicated that the and approximately 20% of children who suffer from DILI third form of cell death, termed ferroptosis, might also will develop liver failure1. Acetaminophen (APAP) over- contribute to APAP-induced DILI in vitro5,6. However, dose is the predominant cause of DILI, and as such its in-depth studies are required to define the key role of underlying mechanism has been heavily investigated using ferroptosis in DILI, which will open a new door for developing effective intervention strategies. Ferroptosis, a form of programmed oxidative cell death, Correspondence: Yi-Fang Li ([email protected])or differs from other cell death forms at the morphological, Tao Shen ([email protected]) or Rong-Rong He ([email protected]) biochemical, and genetic levels. The occurrence and 1Guangdong Engineering Research Center of Chinese Medicine & Disease Susceptibility, Jinan University, 510632 Guangzhou, China execution of ferroptosis have been reported to be regu- 2Department of Pharmacy, Hainan General Hospital (Hainan Affiliated Hospital lated by amino acids, lipids, and iron metabolism7. For of Hainan Medical University), 570311 Haikou, Hainan, China example, a classic ferroptosis inducer, erastin, inhibits Full list of author information is available at the end of the article − These authors contributed equally: Min Wang, Chun-Yu Liu system Xc , resulting in suppression of cellular cysteine Edited by M. Agostini © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Wang et al. Cell Death and Disease (2020) 11:781 Page 2 of 15 uptake and depletion of glutathione (GSH). The latter Materials and methods participates in the main defense mechanisms of anti- Chemicals and reagents oxidant against reactive oxygen species (ROS). Glu- Erastin (S7242), fer-1 (S7243), necrostatin-1(nec-1, tathione peroxidase 4 (GPX4) uses GSH to repair lipids S8037), and ZVAD-fmk (S7023) were purchased from and converts toxic lipid hydroperoxides into non-toxic Selleck Ltd. (Shanghai, China). APAP (1003031), GSH lipid alcohols8. Depletion of GSH inactivates GPX4, (PHR1359), cycloheximide (CHX, 239763), dimethyl sulf- leading to overwhelming lipid peroxidation, ultimately the oxide (DMSO, D2650), deferoxamine (DFO, D9533) and induction of ferroptosis. Ferroptotic cell death can be MG-132 (474790) were purchased from Sigma-Aldrich blocked by iron chelators, lipophilic antioxidants, and (Shanghai, China). (+)-CLA was supplied by the China lipid peroxidation inhibitors like ferrostatin-1 (fer-1)7,9. Academy of Chinese Medical Science. Aminotransferase The Keap1–Nrf2 pathway, an essential regulator of (ALT), aspartate aminotransferase (AST), malondialdehyde oxidative stress, has been recently discovered to parti- (MDA), and lactate dehydrogenase (LDH) detection kits cipate in protecting cells against ferroptosis10.Under were purchased from Nanjing Jiancheng Bioengineering unstressed conditions, nuclear factor erythroid 2-related Institute (Nanjing, China). Nicotinamide adenine dinu- factor 2 (Nrf2) is constantly ubiquitinated and degraded cleotide phosphoric acid (NADPH) assay kit was purchased in proteasomes in a Keap1-dependant manner. In from Comin Biotechnology (Suzhou, China). Hematoxylin response to stress, the cysteine residues in Keap1 and eosin (H&E) staining kit, 4′,6′ diamidino-2- (Kelch-like ECH-associated protein 1) react with oxi- phenylindole (DAPI), Annexin V-FITC/PI apoptosis kit, dants or electrophiles, leading to its modification and and lysis buffer were purchased from Beyotime Technology separation from Nrf211. Subsequently, the released Nrf2 (Shanghai, China). In situ cell death detection peroxidase is translocated to the nucleus, forming heterodimers dismutase (POD) kit was purchased from Roche (Ger- with small Maf proteins to bind to antioxidant response many). Pierce BCA protein assay kit and NE-PER™ nuclear elements (ARE) in the promoter region of cytoprotective and cytoplasmic extraction kit were purchased from genes and to enhance their transcription12. Importantly, Thermo Scientific (Shanghai, China). Trizol was purchased a growing body of evidence has indicated that Nrf2 plays from Tiagen Biotechnology (Beijing, China). SYBR Green crucial roles in both ferroptosis-related pathways, kit was bought from Transgen Biotechnology (Beijing, including lipid metabolism, iron homeostasis, and the China). Primers for detecting Ptgs 2, GSTA1, GSTM2, – pathogenesis of DILI13 15.Hence,theKeap1–Nrf2 heme oxygenase-1 (HO-1), glutamate-cysteine ligase pathway has been considered as a potential strategy for (GCLM), NAD(P)H: quinone oxidoreductase 1 (NQO1), the treatment of DILI, and several natural products can Nrf2, glucose transporter 1 (GLUT1), thioredoxin reductase inhibit DILI by modulating the Keap1–Nrf2 pathway in 1(TXNRD1),β-actin, and 18s mRNA were synthesized by – the liver16 18. Generay Biotechnology (Shanghai, China). Anti-Nrf2 anti- Clausenamides (CLA), natural racemic pyrrolidone bodies were purchased from Abcam (ab62352) and Pro- compounds, are isolated from the leaves of Clausena teintech Group (16396-1-AP). Antibodies for solute carrier lansium (Lour.) Skeels, a popular fruit tree in southern family 7 member 11 (SLC7A11, ab37185), GPX4 China. The isolated compounds of Clausena lansium (ab125066), 4-hydroxynonenal (4-HNE, ab46545), and share a wide range of pharmacological activities, and TATA binding protein (TBP) (ab125009) were purchased CLA have been reported to protect against chemical- from Abcam (MA, USA). Anti-Keap1 antibody was pur- induced liver injury independently of its capability of chased from Cell Signal Technology (MA, USA). Anti- – scavenging hydroxyl radicals19 21. The enantiomer glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (+)-CLA (Fig. 1a) has the best effect on promoting the antibody (FD0063) and FDbio-Pico ECL kit were from Fude synthesis of GSH and enhancing the activity of glu- Biological Technology (Hangzhou, China). Alexa Flour 594 tathione S transferase (GST)22. We thus proposed that and antibodies for Keap1(10503-2-AP), GCLM (14241-1- (+)-CLA might hold the potential to regulate