Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice

Journal Pre-proof Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice

Jing Qi, Zixiong Zhou, Chae Woong Lim, Jong-Won Kim, Bumseok Kim PII:

S0041-008X(19)30375-8

DOI:

https://doi.org/10.1016/j.taap.2019.114767

Reference:

YTAAP 114767

To appear in:

Toxicology and Applied Pharmacology

Received date:

1 June 2019

Revised date:

21 September 2019

Accepted date:

24 September 2019

Please cite this article as: J. Qi, Z. Zhou, C.W. Lim, et al., Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice, Toxicology and Applied Pharmacology (2019), https://doi.org/10.1016/j.taap.2019.114767

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© 2019 Published by Elsevier.

Journal Pre-proof

Amlexanox ameliorates acetaminophen-induced acute liver injury by reducing oxidative stress in mice Jing Qi†, Zixiong Zhou†, Chae Woong Lim, Jong-Won Kim*, Bumseok Kim* Biosafety Research Institute and Laboratory of Pathology (BK21 Plus Program), College of Veterinary Medicine, Chonbuk National University, Iksan, 54596, Republic of Korea

†These authors contributed equally to this work.

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*Corresponding Author: Bumseok Kim, D.V.M., Ph.D.

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Laboratory of Pathology, College of Veterinary Medicine, Chonbuk National University, Iksan, Jeonbuk 54596,

Tel: 82-63-850-0953. Fax: 82-63-850-0980.

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Jong-Won Kim, Ph.D.

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E-mail: [email protected]

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South Korea

South Korea

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E-mail: [email protected]

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Laboratory of Pathology, College of Veterinary Medicine, Chonbuk National University, Iksan, Jeonbuk 54596,

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Journal Pre-proof Abstract Amlexanox, a clinically approved small-molecule therapeutic presently used to treat allergic rhinitis, ulcer, and asthma, is an inhibitor of the noncanonical IkB kinase-ε (IKKε) and TANK-binding kinase 1 (TBK1). This study was to investigate the protective mechanism of amlexanox in acetaminophen (APAP)-induced acute liver injury (ALI). Mice were intraperitoneally injected with APAP (300 mg/kg, 12 hours) to induce ALI and were orally administrated with amlexanox (25, 50 and 100 mg/kg) one hour after APAP treatment. Inhibition of IKKε and TBK1 by treatment of amlexanox attenuated APAP-induced ALI as confirmed by decreased serum levels of aspartate aminotransferase and alanine aminotransferase. Furthermore, amlexanox significantly decreased

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hepatocellular apoptosis in injured livers of mice as evidenced by histopathologic observation. Consistently,

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reduced oxidative stress by amlexanox was observed by increased hepatic glutathione concomitant with decreased levels of malondialdehyde. Amlexanox also enhanced expression levels of nuclear factor erythroid 2-

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related factor 2 (Nrf2) target genes including heme oxygenase 1, NAD(P)H:quinone oxidoreductase 1, and

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glutamate-cysteine ligase in injured livers of mice. Mechanistic insights into the mode of action of amlexanox against APAP-induced hepatotoxicity were involved in increasing phosphorylation of AMP-activated protein

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kinase (AMPK) and nuclear translocation of Nrf2, both in vivo and in vitro. Furthermore, the protective effects of amlexanox on APAP-induced hepatotoxicity were abolished by compound C, an AMPK inhibitor. Taken

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together, our findings suggest that amlexanox exerts antioxidative activities against APAP-mediated

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hepatotoxicity via AMPK/Nrf2 pathway.

Key words: Amlexanox; acetaminophen; oxidative stress; AMPK; Nrf2

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Journal Pre-proof Introduction Acetaminophen (N-acetyl-p-aminophenol, APAP) is a safe and effective analgesic and antipyretic drug when taken at therapeutic doses (Yin et al. 2010); however, APAP overdose is the leading cause of severe and acute liver injury (ALI) with a high mortality rate (Tujios and Fontana 2011). In recent mechanistic studies, N-acetylp-benzoquinone imine (NAPQI), a highly reactive and toxic intermediate of APAP by cytochrome P450 (CYP) family members, plays a critical role in APAP-induced hepatotoxicity by depleting glutathione (GSH), producing reactive oxygen species (ROS), and subsequently covalent binding cysteine groups of proteins, resulting in formation of APAP-protein adducts (Ghaffari et al. 2011; Streeter et al. 1984). These adducts induce

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protein impairment resulting in mitochondrial dysfunction, loss of adenosine triphosphate, and consequent

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cellular necrosis (Martin-Murphy et al. 2013). Because imbalance between oxidants and antioxidants plays a critical role in the ALI progression induced by APAP overdose, restoring the imbalance with detoxifying and

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antioxidant molecules is closely associated with ALI severity (Kay et al. 2011). Based on recent clinical trials,

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N-acetylcysteine (NAC) is one of the most effective antidotes to treat APAP-poisoning. However, NAC therapy is effective only for the patients who are given timely treatment, and it can trigger side effects such as severe

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vomiting (Ferner et al. 2011). Hence, new insights in developing novel therapeutic strategies may pave the way for better treatment of ALI induced by APAP overdose.

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Canonical IκB kinase (IKK) complex is comprised of IKKα, β, and γ. This complex could be activated in response to various stimulants including cytokines and endogenous and exogenous ligands via toll-like receptors

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(TLRs). Subsequently, IκBα is phosphorylated and ubiquitinated, which results in its proteasome-mediated degradation. These responses lead to the release of retained cytoplasmic nuclear factor kappa B (NF‐κB) by IκBα and its translocation from the cytoplasm to the nucleus. Consequently, NF‐κB modulates various pathophysiological responses including inflammatory immune responses, wound healing and tissue regeneration, and cell fate decision (Ghosh and Hayden 2008; Perkins 2007). IKKε and TANK-binding kinase 1 (TBK1), known as non-canonical IKKs (which are sequential homologs of IKKα and IKKβ), have recently been focused on as critical regulators of interferon response factor (IRF) 3/7 and as signal transducers and activators of transcription 3 and 6 as well as NF‐κB (Durand et al. 2018; Shen and Hahn 2011). Furthermore, TBK1 is constitutively expressed in many cells or tissues, including most immune cells, brain, lung, liver, gastrointestinal tract, and reproductive organs (Muvaffak et al. 2014). Although specific tissues (pancreas, thymus, and spleen) and immune cells (T cells and peripheral blood leukocyte) constitutively express basal levels of IKKε, other 3

Journal Pre-proof tissues or cell types express it in response to cytokines and TLR stimulation (Verhelst et al. 2013). Indeed, it has been well documented that TBK1 and IKKε are involved in the pathogenesis of numerous diseases including cancers and infectious diseases as well as their associated innate immune responses (Clement et al. 2008; Hiscott 2007). In recent studies, TBK1 and IKKε are well recognized to link chronic inflammation to insulin resistance and metabolic dysfunction. Amlexanox, an inhibitor of these non-canonical IKKs, is clinically used to treat aphthous ulcers and asthma. It has been reported that amlexanox attenuates the severity of obesity and type 2 diabetesassociated insulin resistance accompanied by increasing thermogenesis and energy expenditure (Oral et al.

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2017; Reilly et al. 2013). In line with these findings, inhibiting TBK1 and IKKε accelerates pancreatic β-cell

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proliferation in mice with diabetes (Xu et al. 2018). Also, amlexanox inhibits the receptor activator of NF-κB ligand-induced osteoclastogenesis and attenuates ovariectomy-induced bone loss (Zhang et al. 2015). These

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findings suggest that TBK1 and IKKε play pivotal roles in the pathogenesis of not only cancer and infectious

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diseases but also non-infectious metabolic diseases and tissue injury. Hence, the aim of the current study is to investigate the effects and the possible underlying mechanisms of amlexanox in APAP-induced ALI in mice due

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Animals

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Materials and Methods

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to a yet uncharacterized role of TBK1 and IKKε in ALI.

Seven-week-old C57BL/6 male mice were purchased from Samtako (South Korea). Mice were maintained under standard conditions (24 ± 2°C, 12 h day-night cycle, 50 ± 5% humidity) in a pathogen-free environment. Mice were allowed free access to food pellets and water throughout the study period. Experimental procedures and animal management procedures were undertaken in accordance with the requirements of the Animal Care and Ethics Committees of Chonbuk National University.

APAP-induced liver injury To induce ALI, mice were intraperitoneally (i.p.) injected with 300 mg/kg body weight of APAP (SigmaAldrich, St. Louis, MO, USA) or equal volume of phosphate-buffered saline (PBS) as control (Cont) following 16 hours starvation. For in vivo inhibition of TBK1 and IKKε signaling, mice were administered 25, 50, or 100 4

Journal Pre-proof mg/kg amlexanox (Santa Cruz Biotechnology Inc., Dallas, TX, USA) or equal volume of vehicle 1 hour after APAP administration. After 12 hours, blood was withdrawn from the heart for analysis; liver samples were also collected for histological and hepatic biochemical examinations. To demonstrate the effects of compound C, mice were i.p. injected with APAP (300 mg/kg) and then received amlexanox (50 mg/kg) 1 hour after APAP administration with or without treatment of compound C (25 mg/kg, i.p). After 12 hours, blood was withdrawn from the heart for analysis. Liver samples were also collected for histological and hepatic biochemical examinations.

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Biochemical measurements

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Liver injury was evaluated by measuring serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) determined with AM101-K spectrophotometric assay kits (ASAN Pharmaceutical,

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Hwasung, Korea). The absorbance of samples was quantified at wavelength of 490 nm using an EMax

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spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

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Histopathologic examination

For histological review of hematoxylin and eosin (H&E)-stained liver sections by light microscopy (BX53F,

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Olympus Corp., Tokyo, Japan), livers were fixed in neutral buffered formalin solution, routinely processed, and then embedded in paraffin. Tissue sections (4 µm in thickness) were prepared using a microtome (HM-340E,

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Thermo Fisher Scientific Inc., Waltham, MA, USA) and placed on glass slides. H&E staining was performed according to standard techniques.

Determination of liver apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) Assay To detect apoptotic cells in the liver, TUNEL assay was performed on paraffin-embedded sections using an ApopTaq peroxidase in situ apoptosis detection kit (EMD Millipore, Temecula, CA, USA) according to the manufacturer’s instruction. Positive reactions were visualized with DAB substrate. Next, nuclear counterstaining was performed using methyl green dye. TUNEL-labeled cells were quantified by the percentage of positive area in a high-power field; a total of 10 high-power fields of liver tissues were analyzed for each animal. Data are expressed as percentages of TUNEL-positive areas. Total liver section images were analyzed 5

Journal Pre-proof using a light microscope and digital image software (analySIS TS, Olympus Corp.).

Immunohistochemistry (IHC) For IHC staining, livers were fixed in 10% phosphate-buffered formalin, routinely processed, and then embedded in paraffin. Liver sections were cut and placed onto glass slides. Before the staining protocol, slides were deparaffinized, rehydrated, and submerged in antigen retrieval solution (Dako, Jena, Germany) for 30 minutes at 100 °C. Non-specific binding was blocked with 3% peroxidase solution followed by blocking with Super Block (ScyTek Laboratories, Inc., Logan, UT, USA). These sections were then incubated with rabbit anti-

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Ly6G antibody (Abcam, Cambridge, UK) at 4°C overnight. Negative control slides were incubated with non-

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immune immunoglobulin under the same condition. These sections were further incubated with biotinconjugated rat or rabbit Ig antibody as secondary antibodies. Immune complexes were detected using DAB

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Substrate Kit (Vector Laboratories) according to the manufacturer’s instructions. Finally, tissues were

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counterstained and mounted. Images were analyzed under a light microscope (BX53F, Olympus Corp.) and

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digital imaging software (analySIS TS, Olympus Corp.).

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Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis Total RNAs were isolated from tissues using an Easy-Spin Total RNA extraction kit (GeneAll, Seoul, Korea).

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Following incubation with DNase I-containing RNase inhibitor (Toyobo, Osaka, Japan), samples were transcribed using ReverTra Ace® qPCR RT Master Mix (Toyobo) according to the manufacturer’s instructions. qRT-PCR was performed in the CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR® Green (TOYOBO). After the reaction was complete, specificity was verified by melting curve analysis. Relative quantification was performed by normalizing to the value of glyceraldehyde-3phosphate dehydrogenase. PCR primers used in this study are summarized in Table 1.

Western blotting analysis Liver tissues or cells were directly lysed for 30 min on ice with an extraction buffer (T-PER or RIPA, Thermo Fisher Scientific Inc.). Nuclear protein was extracted using a Nuclear/Cytosol Fractionation Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instruction. Protein concentration was determined by 6

Journal Pre-proof Pierce BCA Protein Assay kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gels were transferred to a polyvinylidene difluoride (PVDF) membrane and then blocked with SuperBlock™ (TBS) Blocking Buffer (Thermo Fisher Scientific Inc.) for 1 h at room temperature. Primary antibodies were diluted 1:1000 in the blocking solution and incubated overnight at 4°C. Antibodies specific for phosphor IKKε (pIKKε, Ser172), pTBK1 (Ser172), IKKε (D20G4), TBK1 (D1B4), AMP-activated protein kinase α (AMPKα), pAMPKα (Threonine 172), pNF-κB p65 (Ser536), NF-κB p65, nuclear factor erythroid 2-related factor 2 (Nrf2), β-actin and Lamin A/C were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against B-cell lymphoma 2 (Bcl2) and Bcl2-

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associated X protein (Bax) were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA) (all 1:200

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dilutions). Antibody specific for heme oxygenase-1 (HO-1, Abcam) was used. Anti-rabbit (Enzo Life Sciences, Farmingdale, NY, USA) or anti-mouse (Abcam) horseradish peroxidase-conjugated secondary antibodies were

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used to detect antigen-antibody complexes on PVDF membranes. Immunoblot images were visualized with

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ImageQuantTM LAS 500 (GE Healthcare Life Science, Pittsburgh, PA, USA). Protein band expression was

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quantified with ImageQuantTM TL software (GE Healthcare Life Science).

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Glutathione (GSH), Glutathione disulfide (GSSG) and malondialdehyde levels in liver tissues The levels of hepatic GSH and GSSG were determined using a commercially available GSSG/GSH

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quantification kit (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Hepatic content of malondialdehyde (MDA), a marker of oxidative stress, was measured using OxiSelect™ TBARS Assay Kit (MDA Quantitation, Cell biolabs Inc., SD, CA, USA) according to the manufacturer’s instructions.

Isolation of primary hepatocytes and non-parenchymal cells (NPCs) Primary hepatocytes were isolated as previously described (Kim et al. 2018). Briefly, the in situ digestion of mouse livers was performed using collagenase 1 (Worthington Biochemical Corporation, Lakewood, NJ, USA) perfusion (1 mL/min). After enzymatic digestion of livers, the cell suspension was suspended in DMEM (Welgene Inc., Seoul, Korea), filtered, and centrifuged at 50g for 3 minutes. The pellet containing the hepatocytes was immediately used for RNA extraction or protein extraction or resuspended and washed several times with DMEM. The viability was assessed using trypan blue (Sigma-Aldrich). Finally, cells were seeded in 7

Journal Pre-proof collagen-coated wells (rat-tail collagen type I, Corning Life Sciences, Corning, NY, USA) and cultured in DMEM supplemented with 10% fetal bovine serum (Welgene Inc.), 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO2 in a humidified incubator. Following centrifugation of digested liver cell suspension at 50 g for 3 minutes, supernatant containing hepatic NPCs was collected, washed with PBS, and resuspended in 40% Percoll in RPMI-1640 media. The cell suspension was gently overlaid onto 70% Percoll and centrifuged at 750 g for 20 min with off-brake setting.

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NPCs were collected from the interface, washed twice with PBS, and immediately used for RNA extraction.

Cell viability assay

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Isolated primary hepatocytes were seeded into a 96-well plate (100 μl per well, 1×104 cells/well) and incubated

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at 37°C in a 5% CO2 incubator for 24 h to allow them to adhere and grow. They were then treated with various concentration of amlexanox or compound C (Cayman Chemical, Ann Arbor, MI, USA) with or without 10 mM

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APAP. After 24 h of treatment, cell viability was evaluated using MTT assay (Promega, Madison, WI, USA).

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The absorbance of sample was quantified at wavelength of 570 nm using an EMax spectrophotometer (Molecular Devices). Hepatocyte death was evaluated using a Cytotoxicity Detection Kit (Sigma-Aldrich) based

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on lactate dehydrogenase (LDH) released from the cytosol into the culture medium following the manufacturer’s instruction. Absorbance of sample was measured at wavelength of 490 nm using an EMax

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spectrophotometer (Molecular Devices).

Cell culture and treatment

Primary hepatocytes (5.0 × 105 cells/well) were plated into 12-well plates and then cultured overnight at 37°C in a 5% CO2 incubator with serum-free DMEM media. They were then treated with amlexanox (50 μM) or compound C (2.5 μM) with or without 10 mM APAP treatment. After 24 h of treatment, cells or cell culture supernatants were collected and immediately frozen at -70°C for future analysis.

Statistical analysis 8

Journal Pre-proof All data are expressed as means ± standard errors of the means. The statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Differences between groups were compared using two-tailed Student’s t test in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). A p value of less than 0.05 is considered statistically significant.

Results Phosphorylation of IKKε and TBK1 increases significantly in the mouse livers after APAP treatment

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To determine IKKε and TBK1 expression in livers of mice with ALI, liver tissues were collected from mice

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treated with APAP, and the results showed significantly greater mRNA expression of IKKε, although not TBK1 (Fig. 1A). To more clearly demonstrate these results, protein levels of pIKKε and pTBK1 were evaluated in the

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liver tissues or isolated hepatocytes upon APAP treatment. As shown in Figs. 1B, there were significantly higher

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protein levels of pIKKε and pTBK1 in isolated hepatocytes or liver tissues of mice from APAP group compared to Cont group. Based on these results, we assumed that IKKε and TBK1 might be critical mediators in the

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progression of APAP-induced ALI and hepatic inflammation. Following treatment with amlexanox (50 mg/kg), a dual IKKε and TBK1 small molecule inhibitor, phosphorylation of IKKε and TBK1 was markedly reduced in

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injured livers induced by APAP (Fig. 1C). In addition, the differences in histopathologic lesions between vehicle- and amlexanox-treated injured liver were more obvious at 12 hours than at 24 hours after APAP

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treatment (Supplemental Fig. 1). Therefore, we performed subsequent in vivo experiments and analysis at a single time point (12 hour).

Inhibition of IKKε and TBK1 by amlexanox attenuates the severity of APAP-induced ALI Decreased these non-canonical IKKs are closely associated with reduced liver injury as confirmed by the levels of serum biochemicals (Fig. 2A). Consistent with this finding, we found similar patterns in histopathologic observation using H&E staining (Fig. 2B). Furthermore, we observed markedly less APAP-induced hepatocellular death in mice treated with amlexanox than in mice without amlexanox treatment as determined by TUNEL staining and analysis of its positive area (Fig. 2B and C). Next, we measured pro- or anti-apoptotic molecules to support decreased cell death by amlexanox (50 mg/kg); as shown in Fig. 2D, protein levels of proapoptotic Bax decreased with concurrent elevation of anti-apoptotic Bcl2, resulting in an overall reduction of 9

Journal Pre-proof disease severity. Because the NF-κB signaling pathway is a typical inflammatory pathway and plays a vital role in animal models of APAP-induced ALI (Xiahou et al. 2017), we next verified hepatic inflammation and NF-κB activation. As shown in Fig. 3A, treatment with amlexanox significantly down-regulated the mRNA levels of tumor necrosis factor α, interleukin-1β, and interleukin-6 in injured livers. Also, amlexanox significantly decreased the mRNA levels of C-C motif chemokine ligand 2 (CCL2), C-X-C motif ligand (CXCL) 1 and CXCL2 (Fig. 3B). Consistently, we found decreased hepatic neutrophil infiltration in injured livers of mice treated with amlexanox (50 mg/kg) as revealed by IHC staining of Ly6G, one of the neutrophil markers (Fig. 3C). In support of these

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results, injured mouse livers treated with amlexanox (50 mg/kg) showed significantly lower phosphorylation

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(activation) of NF-κB p65 than did injured livers without amlexanox treatment (Fig. 3D). Taken together,

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inhibition of IKKε and TBK1 by amlexanox suppresses NF-κB activation and its related inflammation, which

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reduced APAP-induced ALI.

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Amlexanox attenuates oxidative stress via AMPK/Nrf2 pathway To further clarify the mode of action of amlexanox on APAP-induced ALI, we evaluated whether amlexanox

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affected hepatic oxidative stress in injured livers and found that it significantly increased hepatic GSH in a dosedependent manner in APAP-treated mice, which reflected the antioxidant capacity of amlexanox (Fig. 4A). This

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result was further supported by reduced MDA levels, showing that treatment with amlexanox reduced lipid peroxidation in injured livers (Fig. 4B). Based on a previous study showing that 100 mg/kg amlexanox had no side effects in experimental mice (Reilly et al. 2013), we used multiple doses of amlexanox (25, 50, and 100 mg/kg) in our preliminary in vivo experiments. We found significantly less hepatic injury in APAP-treated mice administered with amlexanox at a dose of 50 mg/kg compared with that in APAP-treated mice without amlexanox administration. Also, there was no statistical difference in liver injury among the APAP-treated groups at different doses of amlexanox (Fig. 2A). Thus, we performed further investigation at a single dose (50 mg/kg) of amlexanox. Because AMPK accelerates nuclear accumulation of Nrf2 and subsequent activation of antioxidant response element target genes (Joo et al. 2016), we next assessed hepatic protein levels of AMPKα1 and its activation (phosphorylation). As shown in Fig. 4C, protein levels of pAMPKα1 and nuclear Nrf2 were increased by 10

Journal Pre-proof treatment of amlexanox in livers of mice from APAP and Cont group. Furthermore, inhibiting IKKε and TBK1 dramatically up-regulated protein levels of HO-1, known as an Nrf2-target enzyme, in injured livers (Fig. 4C). We next measured the expression levels of Nrf2-target genes including HO-1, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase modifier subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC), and we found markedly increased NQO1 expression by inhibiting IKKε and TBK1 in injured livers (Fig. 4D). Overall, amlexanox exerts antioxidative activity in mice with ALI.

Amlexanox alleviates oxidative stress and its related hepatotoxicity induced by APAP

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To further confirm the protective effects of amlexanox on APAP-induced ALI, primary hepatocytes were treated

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with APAP to induce in vitro hepatotoxicity. As shown in Fig. 5A, APAP increased hepatotoxicity as revealed by

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reduced cell viability, whereas such effects were reversed by treatment with 25 and 50 μM amlexanox without cellular toxicity (Supplemental Fig. 2A). In addition, amlexanox-treated mice showed decreased levels of

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hepatic MDA with concurrent elevation of hepatic GSH, resulting in an overall improving of oxidative stressrelated hepatotoxicity by APAP (Fig. 5B and C). Also, amlexanox elevated the levels of pAMPKα1 and nuclear

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Nrf2 in APAP-treated hepatocytes (Fig. 5D), which is consistent with its ability to activate AMPK in normal hepatocytes (supplemental Fig. 2B). Amlexanox also promoted the expression of Nrf2-target genes including

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HO-1, NQO1, and GCLC in APAP-treated hepatocytes (Fig. 5E). Based on our in vivo and in vitro results, IKKε

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and TBK1 are closely associated with hepatic oxidative stress via affecting AMPK/Nrf2 signaling pathway.

AMPK is a key mediator by which amlexanox increases Nrf2 nuclear translocation in APAP-induced hepatotoxicity To further confirm whether AMPK activity is affected by IKKε and TBK1 in APAP-induced hepatotoxicity, we used compound C to inhibit the activity; we observed that inhibiting AMPKα1 abolished the therapeutic effects of amlexanox as confirmed by significantly higher serum biochemical levels (Fig. 6A). We observed similar results for hepatic MDA (Fig. 6B), showing that AMPKα1 plays a critical role in the protective effects of amlexanox in APAP-induced ALI. Also, amlexanox-treated mice with ALI exhibited significantly higher pAMPKα1, nuclear Nrf2, and HO-1 expression in hepatic proteins, and these effects were completely abrogated by co-treatment with compound C in injured livers (Fig. 6C). We observed consistent findings for expression levels of Nrf2-target genes (Fig. 6D) as well. To further support in vivo findings, we conducted additional 11

Journal Pre-proof experiments using an in vitro ALI model. First, we determined adequate in vitro concentration of compound C without cellular toxicity (Supplemental Fig. 2C); as shown in Fig. 7A, amlexanox markedly decreased cell death and cytotoxicity in APAP-treated primary hepatocytes, while compound C abolished the protective effects of amlexanox upon APAP treatment. Western blot analysis revealed that amlexanox-induced phosphorylation of AMPKα1 and the nuclear translocation of Nrf2 were suppressed by treatment with compound C (Figure 7B). As expected, amlexanox-increased expression levels of NQO1, GCLM, and GCLC decreased significantly with compound C treatment (Fig. 7C). Collectively, the mode of action of amlexanox in APAP-induced ALI is

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mediated by the AMPK/Nrf2 dependent pathway.

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Discussion

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APAP overdose is one of the major causes of drug-induced ALI, and cellular GSH conjugating with toxic APAP metabolite (NAPQI) plays a key role in detoxification following overdose. When APAP overdose overwhelms

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hepatocyte defense mechanisms, excessive and uncontrolled ROS-mediated oxidative stress and subsequent inflammatory responses are known to be major drivers of the progression of APAP-induced hepatotoxicity

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(Yoon et al. 2016). Because damaged cell-derived endogenous ligands trigger sterile inflammation, leading to

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greater liver injury with concomitantly lower liver regeneration in late-stage ALI (Yang and Tonnesseen 2019), key targets regulating oxidative stress and inflammation may pave the way for developing effective treatments

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for APAP-induced ALI.

In the present study, APAP-induced hepatic oxidative stress and subsequent lipid peroxidation were confirmed by increased hepatic levels of GSSG, GSSG/GSH ratio and MDA (supplemental Fig. 3B and C), which is consistent with findings from a previous study (Wendel and Feuerstein 1981). Increased hepatic levels of MDA were significantly reversed by amlexanox treatment, which might contribute to hepatic antioxidant capacity; therefore, it is intriguing to explore the downstream targets of IKKε and TBK1. Among many signaling molecules related to oxidative stress, AMPK is known to be closely associated with a variety of metabolic stresses, including hypoxia, ischemia, and oxidative and hyperosmotic stresses (Long and Zierath 2006). Consistently, AMPK activation attenuates oxidative stress-related pathology by improving redox balance, autophagic flux, and nicotinamide adenine dinucleotide homeostasis (Han et al. 2016). Of note, recent studies have shown that pharmacological activation of AMPK ameliorates APAP-induced hepatotoxicity by improving 12

Journal Pre-proof loss of intracellular adenosine triphosphate and mitochondrial dysfunction (Hwang et al. 2015; Kang et al. 2016). Furthermore, accumulating evidence has shown that AMPK activation is closely associated with the increasing expression of Nrf2 leading to regulating oxidative stress (Shirwany and Zou 2014; Zimmermann et al. 2015). In support of these findings, Nrf2-lacking mice are more susceptible to APAP overdose by affecting antioxidant responsive element-mediated gene expression (Enomoto et al. 2001). In this study, amlexanox activated AMPK and Nrf2, and promoted the expression of Nrf2-target genes in livers of mice treated with APAP. In addition, pretreatment of compound C has been reported to inhibit AMPK activity in mice (Hu et al. 2017; McCullough et al. 2005). Recent studies showed that compound C down-regulated pAMPK and thus

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promoted APAP-induced hepatotoxicity in hepatocytes (Saberi et al. 2014; Wang et al. 2016). Thus, we used

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compound C, and found that treatment of compound C exacerbated APAP-induced hepatotoxic damage in mice via inactivating AMPK (supplemental Fig. 4). Furthermore, as expected, compound C-induced AMPK inhibition

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abolished the protective effects of amlexanox against APAP-induced ALI, and reduced Nrf2 nuclear

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translocation and Nrf2-target genes expression. Based on these results together with a recent finding that TBK1 regulates phosphorylation of AMPK (Zhao et al. 2018), amlexanox-mediated hepatoprotective effects on APAP-

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induced ALI might be at least in part mediated by modulating the AMPK/Nrf2 signaling pathway (Fig. 8). It has been well documented that IKKε and TBK1 phosphorylate IRF3, which induces translocation of IRF3

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into the nucleus to trigger type I interferon (IFN) production (Meylan et al. 2006). Studies have shown that

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hepatic immune cells produce type I IFN by sensing necrotic cell-derived DNA, which amplified APAP-induced liver damage by increasing release of nitric oxide derived from inducible nitric oxide synthase (Araujo et al. 2018; Bachmann et al. 2017). Therefore, we sought to evaluate whether amlexanox treatment would affect type 1 IFN production in injured livers. We observed significantly decreased IFNβ production in hepatocytes isolated from injured livers treated with amlexanox, with no significant difference in NPCs, which is inconsistent with findings above (Supplemental Fig. 5). Because CYP isotype enzymes including CYP1A2, 2E1, and 3A11 produce NAPQI, a highly toxic electrophilic intermediate (Woolbright and Jaeschke 2015), we further measured expression levels of these enzymes. The results showed that CYP3A11 expression decreased significantly with amlexanox treatment (Supplemental Fig. 6). These collective observations support the conclusion that inhibiting IKKε and TBK1 attenuates the severity of drug-induced ALI. Recently, Lafont et al. demonstrated that IKKε- and TBK1-mediated phosphorylation of receptor interacting 13

Journal Pre-proof protein kinase 1 (RIPK1) prevents TNF-induced cell death or lethal shock in vivo by preventing TNF-induced RIPK1 activation (Lafont et al. 2018). Therefore, those authors suggested that patients with deregulated TBK1 and IKKε activity are prone to developing TNF-induced cell death and associated diseases. In contrast, we herein showed a pivotal and differential role of IKKε and TBK1 in promoting APAP-induced ALI progression: In the present study, we demonstrated that amlexanox inhibition of IKKε and TBK1 reversed APAP-induced hepatic oxidative stress by the AMPK/Nrf2 signaling pathway. Therefore, we assumed that oxidative stress at an early stage of APAP-induced ALI in mice is more vital than inflammatory responses induced by damaged hepatocytes. This assumption is in consistent with considerable evidence that sterile inflammatory response

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following APAP does not contribute to the liver damage phase in mice (Cover et al., 2006; Williams et al.,

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2014), though it may be necessary for later liver recovery and regeneration (Holt et al., 2008; Jaeschke et al., 2012). This notion is further supported by recent clinical approaches to treating APAP-induced ALI, indicating

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that NAC is the first suggested and the only standard antidote for APAP overdose through recovering GSH

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depletion, resulting in increased detoxification of toxic intermediates of APAP (Yan et al. 2018). Furthermore, the severity of APAP-induced ALI was similar between hepatocyte-specific RIPK1-depleting mice and WT

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littermates, indicating that RIPK1 does not mediate hepatotoxicity of APAP in mice (Schneider et al. 2016). One recent study has provided new insights into the metabolic factors in the pathogenesis of APAP-induced ALI

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(Teratani et al. 2017); authors found that high cholesterol diet-induced accumulation of free cholesterol in

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endolysosomes of liver sinusoidal endothelial cells increased hepatotoxic damage by increasing the TLR9 signaling pathway. In addition to cholesterol loading-induced TLR9 activation, released mitochondrial DNA sensing-mediated TLR9 activation also plays a key role in the pathogenesis of hepatotoxicity induced by APAP overdose by increasing type 1 IFN-mediated oxidative stress and inflammasome-mediated inflammatory responses (Araujo et al. 2018; Bachmann et al. 2017; He et al. 2017; Imaeda et al. 2009). Moreover, the accumulation of free cholesterol in intracellular endolysosomes is one of the critical metabolic risk factors in patients with obesity and metabolic dysfunction (Teratani et al. 2017). Based on these findings, it seems that abnormal metabolic responses, unwanted cell death, and related immune responses are closely associated with each other. This notion is further supported by our present study, in which inhibiting IKKε and TBK1 with amlexanox attenuated acute liver damage induced by drug overdose as well as already known metabolic diseases. In conclusion, our findings showed that treatment with amlexanox, which has recently received great attention for treating metabolic diseases such as type 2 diabetes, ameliorated APAP-induced ALI by reducing 14

Journal Pre-proof TBK1 and IKKε-mediated oxidative stress and inflammation in livers.

Funding This work was supported by National Research Foundation of Korea (No. NRF-2017R1A6A3A11032024, 2017R1D1A3B03030521) and China Scholarship Council (No. 201708260022).

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Acknowledgements The study was conceived and designed by J-WK, JQ, ZZ, and BK; JQ and ZZ performed the experiments, and

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JQ wrote the manuscript; CWL, J-WK, and BK contributed to the critical review of the final manuscript.

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Conflict of interest

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References

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The authors declare that there are no conflicts of interest.

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Araujo AM, Antunes MM, Mattos MS, Diniz AB, Alvarenga DM, Nakagaki BN, Carvalho E, Lacerda VAS, Carvalho-Gontijo R, Goulart J et al. 2018. Liver immune cells release type 1 interferon due to DNA sensing and amplify liver injury from acetaminophen overdose. Cells. 7(8). Bachmann M, Waibler Z, Pleli T, Pfeilschifter J, Muhl H. 2017. Type i interferon supports inducible nitric oxide synthase in murine hepatoma cells and hepatocytes and during experimental acetaminophen-induced liver damage. Frontiers in immunology. 8:890. Clement JF, Meloche S, Servant MJ. 2008. The ikk-related kinases: From innate immunity to oncogenesis. Cell research. 18(9):889-899. Cover C, Liu J, Farhood A, Malle E, Waalkes MP, Bajt ML, Jaeschke H. 2006. Pathophysiological role of the acute inflammatory response during acetaminophen hepatotoxicity. Toxicol Appl Pharm. 216(1):98-107. Durand JK, Zhang Q, Baldwin AS. 2018. Roles for the ikk-related kinases tbk1 and ikkepsilon in cancer. Cells. 7(9). Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O'Connor T, Harada T, Yamamoto M. 2001. High sensitivity of nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of areregulated drug metabolizing enzymes and antioxidant genes. Toxicological sciences : an official journal of the Society of Toxicology. 59(1):169-177. Ferner RE, Dear JW, Bateman DN. 2011. Management of paracetamol poisoning. BMJ (Clinical research ed). 342:d2218. 15

Journal Pre-proof Ghaffari AA, Chow EK, Iyer SS, Deng JC, Cheng G. 2011. Polyinosinic-polycytidylic acid suppresses acetaminophen-induced hepatotoxicity independent of type i interferons and toll-like receptor 3. Hepatology (Baltimore, Md). 53(6):2042-2052. Ghosh S, Hayden MS. 2008. New regulators of nf-kappab in inflammation. Nature reviews Immunology. 8(11):837-848. Han X, Tai H, Wang X, Wang Z, Zhou J, Wei X, Ding Y, Gong H, Mo C, Zhang J et al. 2016. Ampk activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular nad(+) elevation. Aging cell. 15(3):416-427. He Y, Feng D, Li M, Gao Y, Ramirez T, Cao H, Kim SJ, Yang Y, Cai Y, Ju C et al. 2017. Hepatic mitochondrial DNA/toll-like receptor 9/microrna-223 forms a negative feedback loop to limit neutrophil overactivation and acetaminophen hepatotoxicity in mice. Hepatology (Baltimore, Md). 66(1):220-234.

of

Hiscott J. 2007. Triggering the innate antiviral response through irf-3 activation. The Journal of biological chemistry. 282(21):15325-15329.

ro

Holt MP, Cheng L, Ju C. 2008. Identification and characterization of infiltrating macrophages in acetaminophen-induced liver injury. Journal of leukocyte biology. 84(6):1410-1421.

-p

Hu K, Gong XQ, Ai Q, Lin L, Dai J, Cai L, Jiang R, Ge P, Zhang L. 2017. Endogenous ampk acts as a detrimental factor in fulminant hepatitis via potentiating jnk-dependent hepatocyte apoptosis. Cell Death Dis. 8.

re

Hwang JH, Kim YH, Noh JR, Choi DH, Kim KS, Lee CH. 2015. Enhanced production of adenosine triphosphate by pharmacological activation of adenosine monophosphate-activated protein kinase ameliorates acetaminophen-induced liver injury. Molecules and cells. 38(10):843-850.

lP

Imaeda AB, Watanabe A, Sohail MA, Mahmood S, Mohamadnejad M, Sutterwala FS, Flavell RA, Mehal WZ. 2009. Acetaminophen-induced hepatotoxicity in mice is dependent on tlr9 and the nalp3 inflammasome. The Journal of clinical investigation. 119(2):305-314.

na

Jaeschke H, Williams CD, Ramachandran A, Bajt ML. 2012. Acetaminophen hepatotoxicity and repair: The role of sterile inflammation and innate immunity. Liver international : official journal of the International Association for the Study of the Liver. 32(1):8-20.

Jo ur

Joo MS, Kim WD, Lee KY, Kim JH, Koo JH, Kim SG. 2016. Ampk facilitates nuclear accumulation of nrf2 by phosphorylating at serine 550. Molecular and cellular biology. 36(14):1931-1942. Kang SW, Haydar G, Taniane C, Farrell G, Arias IM, Lippincott-Schwartz J, Fu D. 2016. Ampk activation prevents and reverses drug-induced mitochondrial and hepatocyte injury by promoting mitochondrial fusion and function. PloS one. 11(10):e0165638. Kay HY, Kim YW, Ryu DH, Sung SH, Hwang SJ, Kim SG. 2011. Nrf2-mediated liver protection by sauchinone, an antioxidant lignan, from acetaminophen toxicity through the pkcdelta-gsk3beta pathway. British journal of pharmacology. 163(8):1653-1665. Kim JW, Roh YS, Jeong H, Yi HK, Lee MH, Lim CW, Kim B. 2018. Spliceosome-associated protein 130 exacerbates alcohol-induced liver injury by inducing nlrp3 inflammasome-mediated il-1beta in mice. The American journal of pathology. 188(4):967-980. Lafont E, Draber P, Rieser E, Reichert M, Kupka S, de Miguel D, Draberova H, von Massenhausen A, Bhamra A, Henderson S et al. 2018. Tbk1 and ikkepsilon prevent tnf-induced cell death by ripk1 phosphorylation. Nature cell biology. 20(12):1389-1399. Long YC, Zierath JR. 2006. Amp-activated protein kinase signaling in metabolic regulation. The Journal of clinical investigation. 116(7):1776-1783. Martin-Murphy BV, Kominsky DJ, Orlicky DJ, Donohue TM, Jr., Ju C. 2013. Increased susceptibility of natural killer t-cell-deficient mice to acetaminophen-induced liver injury. Hepatology (Baltimore, Md). 57(4):157516

Journal Pre-proof 1584. McCullough LD, Zeng ZY, Li H, Landree LE, McFadden J, Ronnett GV. 2005. Pharmacological inhibition of amp-activated protein kinase provides neuroprotection in stroke. J Biol Chem. 280(21):20493-20502. Meylan E, Tschopp J, Karin M. 2006. Intracellular pattern recognition receptors in the host response. Nature. 442(7098):39-44. Muvaffak A, Pan Q, Yan H, Fernandez R, Lim J, Dolinski B, Nguyen TT, Strack P, Wu S, Chung R et al. 2014. Evaluating tbk1 as a therapeutic target in cancers with activated irf3. Molecular cancer research : MCR. 12(7):1055-1066. Oral EA, Reilly SM, Gomez AV, Meral R, Butz L, Ajluni N, Chenevert TL, Korytnaya E, Neidert AH, Hench R et al. 2017. Inhibition of ikkvarepsilon and tbk1 improves glucose control in a subset of patients with type 2 diabetes. Cell metabolism. 26(1):157-170.e157.

of

Perkins ND. 2007. Integrating cell-signalling pathways with nf-kappab and ikk function. Nature reviews Molecular cell biology. 8(1):49-62.

ro

Reilly SM, Chiang SH, Decker SJ, Chang L, Uhm M, Larsen MJ, Rubin JR, Mowers J, White NM, Hochberg I et al. 2013. An inhibitor of the protein kinases tbk1 and ikk-varepsilon improves obesity-related metabolic dysfunctions in mice. Nature medicine. 19(3):313-321.

re

-p

Saberi B, Ybanez MD, Johnson HS, Gaarde WA, Han D, Kaplowitz N. 2014. Protein kinase c (pkc) participates in acetaminophen hepatotoxicity through c-jun-n-terminal kinase (jnk)-dependent and -independent signaling pathways. Hepatology. 59(4):1543-1554. Schneider AT, Gautheron J, Tacke F, Vucur M, Luedde T. 2016. Receptor interacting protein kinase 1 (ripk1) in hepatocytes does not mediate murine acetaminophen toxicity. Hepatology (Baltimore, Md). 64(1):306-308.

lP

Shen RR, Hahn WC. 2011. Emerging roles for the non-canonical ikks in cancer. Oncogene. 30(6):631-641.

na

Shirwany NA, Zou MH. 2014. Ampk: A cellular metabolic and redox sensor. A minireview. Frontiers in bioscience (Landmark edition). 19:447-474.

Jo ur

Streeter AJ, Dahlin DC, Nelson SD, Baillie TA. 1984. The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro. Chemico-biological interactions. 48(3):349366. Teratani T, Tomita K, Suzuki T, Furuhashi H, Irie R, Hida S, Okada Y, Kurihara C, Ebinuma H, Nakamoto N et al. 2017. Free cholesterol accumulation in liver sinusoidal endothelial cells exacerbates acetaminophen hepatotoxicity via tlr9 signaling. Journal of hepatology. 67(4):780-790. Tujios S, Fontana RJ. 2011. Mechanisms of drug-induced liver injury: From bedside to bench. Nature reviews Gastroenterology & hepatology. 8(4):202-211. Verhelst K, Verstrepen L, Carpentier I, Beyaert R. 2013. Ikappab kinase epsilon (ikkepsilon): A therapeutic target in inflammation and cancer. Biochemical pharmacology. 85(7):873-880. Wang LD, Zhang SL, Cheng H, Lv HM, Cheng GH, Ci XX. 2016. Nrf2-mediated liver protection by esculentoside a against acetaminophen toxicity through the ampk/akt/gsk3 beta pathway. Free Radical Bio Med. 101:401-412. Wendel A, Feuerstein S. 1981. Drug-induced lipid peroxidation in mice--i. Modulation by monooxygenase activity, glutathione and selenium status. Biochemical pharmacology. 30(18):2513-2520. Williams CD, Bajt ML, Sharpe MR, McGill MR, Farhood A, Jaeschke H. 2014. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans. Toxicol Appl Pharmacol. 275(2):122-133. Woolbright BL, Jaeschke H. 2015. Xenobiotic and endobiotic mediated interactions between the cytochrome 17

Journal Pre-proof p450 system and the inflammatory response in the liver. Advances in pharmacology (San Diego, Calif). 74:131161. Xiahou Z, Wang X, Shen J, Zhu X, Xu F, Hu R, Guo D, Li H, Tian Y, Liu Y et al. 2017. Nmi and ifp35 serve as proinflammatory damps during cellular infection and injury. Nature communications. 8(1):950. Xu J, Jia YF, Tapadar S, Weaver JD, Raji IO, Pithadia DJ, Javeed N, Garcia AJ, Choi DS, Matveyenko AV et al. 2018. Inhibition of tbk1/ikkepsilon promotes regeneration of pancreatic beta-cells. Scientific reports. 8(1):15587. Yan M, Huo Y, Yin S, Hu H. 2018. Mechanisms of acetaminophen-induced liver injury and its implications for therapeutic interventions. Redox biology. 17:274-283. Yang R, Tonnesseen TI. 2019. Damps and sterile inflammation in drug hepatotoxicity. Hepatology international. 13(1):42-50.

of

Yin H, Cheng L, Holt M, Hail N, Jr., Maclaren R, Ju C. 2010. Lactoferrin protects against acetaminopheninduced liver injury in mice. Hepatology (Baltimore, Md). 51(3):1007-1016.

ro

Yoon E, Babar A, Choudhary M, Kutner M, Pyrsopoulos N. 2016. Acetaminophen-induced hepatotoxicity: A comprehensive update. Journal of clinical and translational hepatology. 4(2):131-142.

-p

Zhang Y, Guan H, Li J, Fang Z, Chen W, Li F. 2015. Amlexanox suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss. Scientific reports. 5:13575.

re

Zhao P, Wong KI, Sun X, Reilly SM, Uhm M, Liao Z, Skorobogatko Y, Saltiel AR. 2018. Tbk1 at the crossroads of inflammation and energy homeostasis in adipose tissue. Cell. 172(4):731-743.e712.

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Figure legends

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Zimmermann K, Baldinger J, Mayerhofer B, Atanasov AG, Dirsch VM, Heiss EH. 2015. Activated ampk boosts the nrf2/ho-1 signaling axis--a role for the unfolded protein response. Free radical biology & medicine. 88(Pt B):417-426.

Fig. 1. APAP increases the expression and activation of IKKε and TBK1 in injured livers or hepatocytes.

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Mice were injected with APAP (300 mg/kg, i.p.) treated with or without amlexanox (50 mg/kg). Primary hepatocytes were treated with 10 mM APAP or PBS for 24 hours. (A) mRNA expression levels of IKKε and TBK1 in livers were measured by qRT-PCR. (B, C and D) Phosphorylation of IKKε and TBK1 were determined by Western blot analysis with relative quantification. Data are expressed as means ± SEM per group. *P < 0.05 and **P < 0.01 versus vehicle treated mice. n=12-13 mice (APAP); 8 mice (PBS).

Fig. 2. Amlexanox attenuates APAP-induced ALI. Mice were orally administered 25, 50 and 100 mg/kg amlexanox or vehicle 1 hour after APAP treatment; after 12 hours, serum and liver tissues were collected for analysis. (A) Serum levels of ALT and AST were measured. (B) Liver sections were stained with H&E and TUNEL to assess hepatocellular damage. (C) TUNEL-positive area was assessed. (D) Mice were treated with 18

Journal Pre-proof APAP alone or with amlexanox of a single dose (50 mg/kg). Protein levels of apoptosis-related Bax and Bcl2 were measured by Western blot analysis with relative quantification. Data are expressed as means ± SEM per group. *P < 0.05 and **P < 0.01 versus vehicle treated mice; # P < 0.05 and ## P < 0.01 versus APAP-treated mice. Scale bar = 100 um. n=12 mice per group (APAP); 4-5 mice per group (PBS).

Fig. 3. Amlexanox suppresses inflammation in the livers of mice with ALI. (A) The expression levels of tumor necrosis factor α, interleukin-1β, and interleukin-6 in injured livers were measured with qRT-PCR. (B)

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The expression levels of chemokines including CCL2, CXCL 1, and CXCL 2 in injured livers were measured with qRT-PCR. (C) Mice were treated with APAP alone or with amlexanox of a single dose (50 mg/kg). IHC

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was performed to detect Ly6G-positive cells, and the positive area was quantified. (D) Protein levels of pNF-

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κBp65 and NF-κBp65 were determined by Western blot analysis, and the relative protein intensity was quantified. Data are expressed as means ± SEM per group. *P < 0.05 and **P < 0.01 versus vehicle treated

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mice; # P < 0.05 and ## P < 0.01 versus APAP-treated mice. Scale bar = 100 um. n=12 mice per group (APAP);

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4-5 mice per group (PBS).

Fig. 4. IKKε and TBK1 inhibition with amlexanox attenuates oxidative stress via the AMPK/Nrf2

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pathway. (A) Hepatic GSH was measured. (B) Oxidative stress was determined by measuring hepatic MDA. (C) Mice were treated with APAP alone or with amlexanox of a single dose (50 mg/kg). Hepatic protein levels of pAMPKα1, nuclear-Nrf2 and HO-1 were measured by Western blot analysis with relative quantification. (D) HO-1, NQO1, GCLM, and GCLC expression were determined by qRT-PCR. Data are expressed as means ± SEM per group. *P < 0.05 and **P < 0.01 versus vehicle treated mice; # P < 0.05 and ## P < 0.01 versus APAP-treated mice. n=12 mice per group (APAP); 4-5 mice per group (PBS).

Fig. 5. Amlexanox attenuates APAP-induced hepatotoxicity through reducing oxidative stress in vitro. (A) Primary hepatocytes were treated with 10 mM APAP plus vehicle or 12.5, 25 and 50 uM amlexanox for 24 hours. Cell viability was assessed by MTT assay. (B) Cells were cultured with 10 mM APAP with or without 50 uM amlexanox for 24 hours. Hepatocellular MDA contents were measured. (C) Hepatic GSH was measured. 19

Journal Pre-proof (D) Protein levels of pAMPKα1 and nuclear-Nrf2 were immunoblotted, and the relative protein intensity was quantified. (E) HO-1, NQO1, GCLM, and GCLC expression were determined by qRT-PCR. Data are expressed as means ± SEM per group. *P < 0.05, **P < 0.01.

Fig. 6. The antioxidative effects of amlexanox against APAP-induced ALI are abolished by inhibiting AMPK. Compound C (25 mg/kg, i.p) was treated 1 hour before APAP treatment; one hour after APAP administration, mice were treated with vehicle or amlexanox (50 mg/kg). Serum and liver tissues were collected

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12 hours after APAP treatment. (A) Serum alanine and aspartate were measured. (B) Hepatic MDA contents were measured. (C) Protein levels of pAMPKα1, nuclear-Nrf2, and HO-1 in livers were determined by Western

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blot analysis with relative quantification. (D) HO-1, NQO1, GCLM, and GCLC expression were determined by

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qRT-PCR. Data are expressed as means ± SEM per group. *P < 0.05, **P < 0.01. n=10-11 mice per group

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(APAP); 9 mice (PBS).

Fig. 7. Amlexanox alleviates in vitro APAP-induced hepatotoxicity in an AMPK-dependent manner.

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Primary hepatocytes were treated with 10 mM APAP to induce hepatotoxicity; they were co-cultured with amlexanox (50 μM) and compound C (2.5 μM) for 24 hours. (A) Cell viability and cytotoxicity were measured

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using MTT and LDH assay, respectively. (B) Protein levels of pAMPKα1 and nuclear-Nrf2 in livers were determined by Western blot analysis with relative quantification for these proteins. (C) HO-1, NQO1, GCLM, and GCLC expression were determined by qRT-PCR. Data are expressed as means ± SEM per group. *P < 0.05, **P < 0.01

Fig. 8. Amlexanox is involved in the protection against APAP-induced ALI via the AMPK/Nrf2 pathway. Amlexanox could inhibit the activity of IKKε and TBK1 in livers, which contributes to the phosphorylation of AMPK and nuclear translocation of Nrf2. Nuclear accumulation of Nrf2 reduces hepatic oxidative stress induced by APAP. Consequently, amlexanox suppresses the severity of APAP-induced hepatotoxicity in mice.

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Journal Pre-proof Supplemental figure captions Supplemental Fig. 1. Mice were orally given 50 mg/kg amlexanox or equal volume of vehicle 1 hour after APAP administration. Liver tissues were harvested 12 or 24 hours after APAP. Representative liver histological sections were stained with H&E or TUNEL. Representative graphs show the quantification of TUNEL-positive area. Data are expressed as means ± SEM per group. **P < 0.01. Scale bar = 100 um. n=6 mice per group (APAP); 4-5 mice

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per group (PBS).

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Supplemental Fig. 2.

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Primary hepatocytes were treated with various concentrations of amlexanox or compound C for 24 hours. (A)

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Cell viability of hepatocytes treated with different concentrations of amlexanox was determined by MTT assay. (B) Protein levels of pAMPKα1 and AMPKα1 were measured and quantified. (C) Cell viability of hepatocytes

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treated with different concentrations of compound C was determined by MTT assay. Data are expressed as

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between groups at p<0.05.

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means ± SEM per group. Experimental groups marked by different letters represent significant differences

Supplemental Fig. 3.

Mice were orally administered with 50 mg/kg amlexanox or equal volume of vehicle 1 hour after APAP administration. Liver tissues were collected 12 hours after APAP treatment. (A) Hepatic GSH contents were measured. (B) Hepatic GSSG levels were determined. (C) The ratio of GSSG to GSH in livers was determined. Data are expressed as means ± SEM per group. *P < 0.05 and **P < 0.01 versus vehicle treated mice; # P < 0.05 and ## P < 0.01 versus APAP-treated mice. n=12 mice per group (APAP); 4-5 mice per group (PBS).

Supplemental Fig. 4. Mice were i.p. injected with 25mg/kg compound C or vehicle 1 hour before APAP treatment. Samples were 21

Journal Pre-proof collected 12 hours after APAP treatment. (A) Serum levels of ALT and AST were measured. (B) Hepatic MDA was measured. (C) Hepatic protein levels of pAMPKα1 and AMPKα1 were measured by Western blot analysis with relative quantification. (D) HO-1, NQO1, GCLM, and GCLC expression levels were determined by qRTPCR. Data are expressed as means ± SEM per group. *P < 0.05, **P < 0.01. n=6 mice per group (APAP); 4 mice per group (PBS).

Supplemental Fig. 5.

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Mice were treated with PBS or APAP with or without amlexanox administration. Hepatocytes and NPCs were

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Data are expressed as means ± SEM per group. **P < 0.01.

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isolated, and then total RNA of each type of cell was extracted. IFNβ expression was measured by qRT-PCR.

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Supplemental Fig. 6.

Mice were orally administered 50 mg/kg amlexanox or vehicle 1 hour after APAP treatment. Hepatic mRNA

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expression of CYP2E1, CYP1A2, and CYP3A11 were evaluated by qRT-PCR. Data are expressed as means ±

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SEM per group. *P < 0.05, **P < 0.01.

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Table 1 Primer sequence of qRT-PCR Gene

Forward

Reverse

5’-GGAGTGTGTGCAGACGTATCAGG-3’

5’-AATGAGATGCAGGTGGTTCTGG-3’

TBK1

5’-AAGTTGATGAAGGTCAACCTGGAAG-3’

5’-CCTGCTGCTGATGTCCTGAAG-3’

TNFα

5’- AGGGTCTGGGCCATAGAACT-3’

5’- CCACCACGCTCTTCTGTCTAC-3’

IL-1β

5’- GGTCAAAGGTTTGGAAGCAG-3’

IL-6

5’-ACCAGAGGAAATTTTCAATAGGC-3’

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5’- TGTGAAATGCCACCTTTTGA-3’

5’-AGCAGCAGGTGTCCCAAAGA-3’

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CCL2

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IKKε

5’-TGATGCACTTGCAGAAAACA-3’ 5’-GTGCTGAAGACCTTAGGGCAGA-3’

5’-GCCAAGGGTTGACTTCAAGAACA-3’

5’-AGGCTCCTCCTTTCCAGGTCA-3’

CXCL1

5’-TGCACCCAAACCGAAGTC-3’

5’-GTCAGAAGCCAGCGTTCACC-3’

HO-1

5’-TGCAGGTGATGCTGACAGAGG-3’

5’-GGGATGAGCTAGTGCTGATCTGG-3’

NQO1

5’-CAGCCAATCAGCGTTCGGTA-3’

5’-CTTCATGGCGTAGTTGAATGATGTC-3’

GCLM

5’-AGTTGGAGCAGCTGTATCAGTGG-3’

5’-TTTAGCAAAGGCAGTCAAATCTGG-3’

GCLC

5’-AATGACTGTTGCCAGGTGGATG-3’

5’-GGTTGCACTTCCAAATGAGGCTA-3’

IFNβ

5’-AGCTCCAAGAAAGGACGAACAT-3’

5’-GCCCTGTAGGTGAGGGTTGATCT-3’

CYP2E1

5’-AAGCGCTTCGGGCCAG-3’

5’-TAGCCATGCAGGACCACGA-3’

CYP1A2

5’-GGTCAGAAAGCCGTGGTTG-3’

5’-GACATGGCCTAACGTGCAG-3’

CYP3A11

5’-CGCCTCTCCTTGCTGTCACA-3’

5’-CTTTGCCTTCTGCCTCAAGT-3’

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CXCL2

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5’-ACGGCAAATTCAACGGCACAG-3’

5’-GAAGACTCCACGACATACTCAGCAC-3’

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IKKε, IkB kinases-ε; TBK1, TANK-binding kinase 1; TNFα, tumor necrosis factor α; IL-1β, interleukin-1β; CCL2, C-C Motif Chemokine Ligand 2; CXCL2, C-X-C motif ligand 2; CXCL1, C-X-C motif ligand 1; HO-1, heme oxygenase-1; NQO1, NAD(P)H Quinone Oxidoreductase 1; GCLM, glutamate-cysteine ligase modifier subunit; GCLC, glutamate-cysteine ligase catalytic subunit.

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Highlights: • Phosphorylation of IKKε and TBK1 increases significantly in the mouse livers after APAP treatment • Inhibition of IKKε and TBK1 by amlexanox attenuates the severity of APAP-

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induced ALI

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• Amlexanox attenuates oxidative stress via AMPK/Nrf2 pathway

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• AMPK is a key mediator by which amlexanox increases Nrf2 nuclear t

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ranslocation in APAP-induced hepatotoxicity

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