Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol

Oct 20, 2016 - It has emerged that hepatocyte necroptosis plays a critical role in chronic alcoholic liver disease (ALD). Our previous study has ident...
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Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol-Induced Hepatocyte Necroptosis Chunfeng Lu, Wenxuan Xu, Feng Zhang, Jiangjuan Shao, and Shizhong Zheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00562 • Publication Date (Web): 20 Oct 2016 Downloaded from http://pubs.acs.org on October 22, 2016

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Title

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Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol-Induced Hepatocyte

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Necroptosis

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Running title

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Curcumin prevents hepatocyte necroptosis

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Authors

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Chunfeng Lu,a Wenxuan Xu,b Feng Zhang,a,b Jiangjuan Shao,a Shizhong Zheng a,b,*

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Affiliations

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a

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Jiangsu, China

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b

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University of Chinese Medicine, Nanjing, Jiangsu, China

Department of Pharmacology, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing,

Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing

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Correspondence

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*

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Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, Jiangsu, China. Tel.: +86 25 85811246; Fax:

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+86 25 86798188. E-mail address: [email protected].

Address correspondence to: Department of Pharmacology, School of Pharmacy, Nanjing University of

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Abbreviations

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ALD, alcoholic liver disease; ALT, alanine aminotransferase; AST, aspartate aminotransferase;

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DAMPs, damage-associated molecular patterns; DMEM, Dulbecco’s modified eagle medium; DMSO,

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dimethylsulfoxide; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GAPDH,

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glyceraldehyde phosphate dehydrogenase; H&E, Haematoxylin-eosin; HMGB1, high-mobility group

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box 1; ISH, in situ hybridization; JNK, c-jun N-terminal kinase; MLKL, mixed lineage kinase

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domain-like; NQO1, NAD(P)H: quinone oxidoreductase 1; Nrf2, nuclear factor (erythroid-derived

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2)-like 2; NS, normal saline; RIP1, receptor-interacting protein 1; RIP3, receptor-interacting protein 3

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Abstract

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It has emerged that hepatocyte necroptosis plays a critical role in chronic alcoholic liver disease (ALD).

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Our previous study has identified that the beneficial therapeutic effect of curcumin on alcohol-caused

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liver injury might be attributed to activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2),

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whereas the role of curcumin in regulating necroptosis and the underlying mechanism remain to be

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determined. We firstly found that chronic alcohol consumption triggered obvious hepatocyte

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necroptosis, leading to increased expression of receptor-interacting protein 1, receptor-interacting

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protein 3, high-mobility group box 1, and phosphorylated mixed lineage kinase domain-like in murine

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livers. Curcumin dose-dependently ameliorated hepatocyte necroptosis and alleviated alcohol-caused

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decrease in hepatic Nrf2 expression in alcoholic mice. Then Nrf2 shRNA lentivirus was introduced to

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generate Nrf2-knockdown mice. Our results indicated that Nrf2 knockdown aggravated the effects of

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alcohol on liver injury and necroptosis and even abrogated the inhibitory effect of curcumin on

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necroptosis. Further, activated Nrf2 by curcumin inhibited p53 expression in both livers and cultured

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hepatocytes under alcohol stimulation. The next in vitro experiments, similar to in vivo ones, revealed

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that although Nrf2 knockdown abolished the suppression of curcumin on necroptosis of hepatocytes

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exposed to ethanol, p53 siRNA could clearly rescued the relative effect of curcumin. In summary, for

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the first time, we concluded that curcumin attenuated alcohol-induced hepatocyte necroptosis in a

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Nrf2/p53-dependent mechanism. These findings make curcumin an excellent candidate for ALD

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treatment and advance the understanding of ALD mechanisms associated with hepatocyte necroptosis.

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Keywords

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alcoholic liver disease; curcumin; necroptosis; Nrf2; p53

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INTRODUCTION

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Alcoholic liver disease (ALD) seriously threatens public health worldwide.1 In Asia, it carries a high

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incidence among diverse liver diseases, ranking just behind virus hepatitis.2 Chronic alcohol

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consumption damages hepatocytes and ultimately causes cell death. For long, only apoptosis was

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considered as a sole model of programmed cell death in alcohol-exposed hepatocytes.3, 4 Recently

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published studies highlighted an emerging role of hepatocyte necroptosis in facilitating ALD.5, 6 When

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apoptosis was inhibited, cellular necroptosis would be enlarged which failed classical therapies to halt

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cell loss and improve alcoholic liver injury.7

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Necroptotic cells would exhibit typical morphological changes, such as augmented cell volume,

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disruption of plasma membrane, and cellular collapse.8 Necroptotic cells would release massive

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intracellular contents named as damage-associated molecular patterns (DAMPs), including IL-1α and

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high-mobility group box 1 (HMGB1), triggering inflammatory response.9-12 HMGB1 is a nuclear

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protein secreted exclusively from necroptotic cells, which is generally used to distinguish necroptosis

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from apoptosis. Necroptosis is a highly regulated process, and several unique signal molecules are

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involved in executing necroptosis. Receptor-interacting protein 1 (RIP1) and receptor-interacting

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protein 3 (RIP3) are defined as central contributors for initiating necroptosis. Activated RIP1 binds to

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RIP3 and forms the necrosome complex.13 Formed necrosome recruits and activates mixed lineage

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kinase domain-like (MLKL).14, 15 Phosphorylated MLKL then oligomerizes and binds to membrane

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phospholipids, forming pores that cause necroptotic cell death. C-jun N-terminal kinase (JNK) is also a

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downstream target gene of RIP3 which when phosphorylated induces mitochondrial oxidative stress

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and fission and finally promotes necroptosis.16 Basic and clinical researches highlighted the execution

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of necroptosis in alcohol-related pathologies. Serum HMGB1 level, hepatic RIP3 expression and JNK

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phosphorylation were significantly induced in alcohol-exposed small rodents and large mammals,

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which resulted in severe liver inflammation and steatosis.17-19 RIP3 deficiency protected mice from

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alcohol-caused liver injury, inflammation, and steatosis.12, 20, 21 Thus negative regulation of necroptosis

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is anticipated to not only rescue hepatocyte fate but also improve inflammation and hepatic steatosis

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induced by alcohol. Developing drugs targeting at necroptosis inhibition is urgently required.

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Researches on pharmacological activities of curcumin have always been actively pursued.22 In our

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previous study, we found that curcumin prevented ALD in rats by suppressing liver inflammation and

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steatosis. Mechanistically, activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) was required

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for curcumin to protect hepatocytes from ethanol-induced cell injury and lipid accumulation.23 Recently,

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an original observation proposed that curcumin attenuated neuron necroptosis induced by iron overload

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via inhibiting RIP1, laying the foundation for curcumin to modulate necroptosis.24 Nrf2 which when

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activated could reduce HMGB1 release, implying that Nrf2 might serve as a modulator of

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necroptosis.25-27 In present study, we hypothesized that curcumin could suppress alcohol-induced

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hepatocyte necroptosis, in which activation of Nrf2 in hepatocytes could be a molecular basis. Both in

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vivo and in vitro systems were introduced to confirm this hypothesis.

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MATERIALS AND METHODS

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Reagents and Antibodies

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Curcumin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in

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dimethylsulfoxide (DMSO; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) in in vitro

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experiments. Lentivirus vectors encoding negative control shRNA (NC shRNA) and Nrf2 shRNA were

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constructed by Nanjing di rui biological technology Co., Ltd. (Nanjing, Jiangsu, China). Control

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siRNA, Nrf2 siRNA, and p53 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA). Primary antibody against RIP1 (17519-1-AP) was purchased from Proteintech Group, Inc.

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(Rosemont, IL, USA). Primary antibodies against Nrf2 (sc-722), NAD(P)H: quinone oxidoreductase 1

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(NQO1, sc-16464), RIP3 (sc-374639), p-JNK (sc-81502), JNK (sc-7345), and β-actin (sc-47778) were

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purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against

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HMGB1 (3935), p-MLKL (91689), MLKL (28640), and p53 (2524) as well as horseradish

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peroxidase-conjugated secondary antibodies (7076 and 7074) were purchased from Cell Signaling

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Technology (Danvers, MA, USA). The primers used in quantitative real-time polymerase chain

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reaction (qRT-PCR) analyses were purchased from GenScript Co. Ltd. (Nanjing, Jiangsu, China).

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Animal Models

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Animals

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All experimental procedures were approved by the institutional and local committee on the care and

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use of animals of Nanjing University of Chinese Medicine (Nanjing, Jiangsu, China), and all animals

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received human care in strict accordance with the National Institutes of Health guidelines. All animals

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were kept in the specific pathogen free clean room under a controlled condition of 21-25 °C and a

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12-h light/dark cycle and had free access to standard chow diet and water. This study was performed in

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male ICR mice purchased from Nantong University (Nantong, Jiangsu, China), weighing 20-25 g.

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Experimental Procedures

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Study I

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In this experimental protocol, sixty mice were randomly divided into five groups (12 mice/group).

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Group 1 was the vehicle control in which mice were not administrated with alcohol or curcumin but

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normal saline (NS) orally and twice daily for four weeks. Group 2 was the model group in which mice

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were orderly administrated with alcohol (56%, v/v, 10 mL/kg body weight) and NS without curcumin

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by gavage every day for four weeks. Groups 3-5 were treatment groups in which mice were all

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administrated with alcohol and respectively treated with curcumin that was suspended in sterile NS at

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100, 200, and 400 mg/kg every day for four weeks.

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Study II

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In a second experimental protocol, sixty mice were randomly divided into five groups (12 mice/group).

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Mice in groups 1-5 received corresponding treatments as follows: group 1, negative control (NC)

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shRNA lentivirus and NS; group 2, NC shRNA lentivirus, alcohol, and NS; group 3, Nrf2 shRNA

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lentivirus, alcohol, and NS; group 4, NC shRNA lentivirus, alcohol, and curcumin; group 5, Nrf2

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shRNA lentivirus, alcohol, and curcumin. At day 1 of the four-week experiment, lentivirus with a titer

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of 1 × 108 TU/mice was injected into caudal vein of mice.28-31 Alcohol (56%, v/v, 10 mL/kg body

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weight) and curcumin (200 mg/kg body weight) were given every day by gavage.

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Forty-eight hours after last administration, all mice were anesthetized by intraperitoneal injection with

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pentobarbital (50 mg/kg). Blood was collected and livers were harvested. A small portion of each liver

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was fixed in 10% neutral buffered formalin solution and embedded in paraffin for immunofluorescence

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staining. The remaining liver was stored at liquid nitrogen for extraction of total proteins and RNA.

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Cell Culture

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Human hepatocyte LO2 cells were purchased from Cell Bank of Chinese Academy of Sciences

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(Shanghai, China). Cells were cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen,

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Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen,

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Merelbeke, Belgium), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified

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atmosphere of 95% air and 5% CO2.

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SiRNA Transfection

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100 pmol Nrf2 siRNA or p53 siRNA was mixed with 150 µL medium without serum and antibiotics. 7

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µL lipofectamine 2000 reagent (life technologies, New York, NY, USA) was mixed with 150 µL

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medium. After incubation for 5 min at room temperature, both mixtures were gently mixed and

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incubated for 10 min at room temperature. Then 700 µL medium without FBS and antibiotics were

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added in and 1,000 µL transfection solution was prepared. Cells in 6-well plates were incubated with

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transfection solution (1,000 µL/well) for 24 h at 37 °C. Control siRNA was used as a negative control.

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Western Blot Analyses

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Total proteins were extracted from liver tissues and hepatocytes using radioimmunoprecipitation assay

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buffer supplemented with phenylmethylsufonyl fluoride and phosphatase inhibitor. Protein

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concentrations were detected using a bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL,

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USA) according to the protocol from manufacturer. Total proteins (50 µg/sample) were separated by

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sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene

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fluoride membranes. Then membranes were blocked with 5% skim milk in Tris-buffered saline

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containing 0.1% Tween 20 for 2 h. Target proteins were detected using corresponding primary

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antibodies and secondary antibodies conjugated with horseradish peroxidase. Protein bands were

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visualized using chemiluminescence reagent (Millipore, Burlington, MA, USA) and densitometrically

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detected using Quantity Ones 4.4.1 (Bio-Rad Laboratories, Berkeley, CA, USA). β-Actin was probed

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as an internal control. The relative abundance of target proteins was expressed as fold changes

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compared with the control after normalization to β-actin or total protein.

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RNA Extraction and QRT-PCR

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Total RNA was extracted from liver tissues and hepatocytes using Trizol reagent according to the

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protocol provided by manufacturer (Sigma-Aldrich, St. Louis, MO, USA). QRT-PCR was performed

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according to our previous description.32 Glyceraldehyde phosphate dehydrogenase (GAPDH) was used

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as an invariant control and mRNA levels were expressed as fold changes after normalization to

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GAPDH. Primers used were listed in table 1 and 2.

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Liver Histopathology and Immunofluorescence Staining

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Transmission electron microscopy, H&E and immunofluorescence staining were performed as

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previously described.23, 33

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In situ hybridization (ISH)

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ISH assays were performed with 5’digoxigenin (DIG)-labeled probe for Nrf2. The primer was used:

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Nrf2, 5’-GCTGTGCTTTAGGTCCATTCTGTTTGACACTTCC-3’. Hybridization was carried out

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according to the previous report.34

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Biochemical Analyses

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Serum was isolated from whole blood after centrifugation at 3600 rpm for 20 min and stored at −80 °C

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for further analyses. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT)

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levels were detected using corresponding commercial assay kits according to the protocols from

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manufacturer (Nanjing Jiancheng Bioengeering Institute, Nanjing, Jiangsu, China). Absorbance values

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were determined using a SPECTRAmaxTM microplate spectrophotometer (Molecular Devices,

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Sunnyvale, CA, USA).

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Enzyme-linked Immunosorbent Assay (ELISA)

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The levels of IL-1α and HMGB1 in serum and cell culture supernatant were detected using

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corresponding ELISA kits (Nanjing SenBeiJia Biological Technology Co., Ltd., Nanjing, Jiangsu,

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China) under the direction of protocols from manufacturer.

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MTT Assay

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LO2 cells were seed in 96-well plates and cultured in complete medium for 24 h followed by

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corresponding treatments. Cell viability was detected by MTT assay according to our precious

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description.23

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Statistical Analyses

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All data were presented as mean ± SD, and results were analyzed using GraphPad Prism Software

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Version 6.0 (GraphPad Software, La Jolla, CA). The significance of difference was determined by

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one-way analysis of variance with the post hoc Dunnett’s test. Values of P < 0.05 were considered

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statistically significant.

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RESULTS

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Curcumin Prevents Necroptosis in Alcohol-Fed Mice. To investigate the action of curcumin on

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alcohol-induced necroptosis, changes in DAMPs release to circular system were firstly measured for

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their sensitive capacities of indicating necroptosis. Our results showed a clear increase in serum IL-1α

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and HMGB1 levels in alcoholic mice but a dose-dependent decrease in curcumin-treated mice (Figure

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1A,B). The mRNA and protein abundance of necroptosis markers in liver tissues, HMGB1, RIP1, and

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RIP3, were upregulated under alcohol exposure but dose-dependently downregulated under curcumin

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treatment. Alcohol stimulated the phosphorylation of MLKL and JNK while curcumin

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dephosphorylated p-MLKL and p-JNK in a dose-dependent manner (Figure 1C,D). Taken together,

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these data suggested that curcumin inhibited hepatic necroptosis in alcohol-preferring mice.

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Curcumin Attenuates Alcoholic Liver Injury Dependent on Nrf2 Activation. Except for the

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preferable anti-necroptosis effects, curcumin also abrogated alcohol-induced decrease in hepatic Nrf2

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and NQO1 expression (Figure 1E). To explore the molecular basis underlying the anti-necroptosis

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capacity of curcumin, Nrf2 was probed as a target for that Nrf2 was a vital modulator of necroptosis

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and could be activated by curcumin as we previously reported. Nrf2-deficient animal model was

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established on ICR mice using RNA interference technology. ISH analysis showed that Nrf2 shRNA

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effectively silenced the mRNA expression of Nrf2 in mouse hepatocytes while no conspicuous change

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in Nrf2 mRNA abundance was shown in NC shRNA-injected control mice. Curcumin rescued the

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mRNA expression of Nrf2 in alcohol-treated mice, which was abrogated by Nrf2 shRNA (Figure 2A).

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The protein abundance of hepatic Nrf2 and NQO1 in mice was consistently decreased by Nrf2 shRNA

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but increased by curcumin (Figure 2B,C). Alcohol caused an increase in serum AST and ALT levels

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and severe hepatohistological alternations. Curcumin alleviated alcohol-induced a sequence of

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pathological changes. Notably, Nrf2 shRNA enhanced the toxic actions of alcohol, deteriorated hepatic

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damage, and impaired improvement on alcohol-caused hepatotoxicity by curcumin (Figure 2D,E). In

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summary, these data demonstrated that curcumin could activate hepatic Nrf2 which was a basis for

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curcumin to prevent mice from chronic alcoholic liver injury.

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Nrf2 Deficiency Blocks the Effective Improvement of Curcumin on Alcohol-Caused Hepatocyte

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Necroptosis. Nrf2 function in the hepatoprotection against alcoholic necroptosis provided by curcumin

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was further investigated. Curcumin treatment restrained alcohol-induced increase in release of IL-1α

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and HMGB1. However, Nrf2 shRNA deteriorated alcohol effect and further repressed curcumin

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function (Figure 3A,B). Transmission electron microscopy vividly concretized and visualized

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hepatocyte necroptosis. Normal hepatocytes displayed regular cellular morphology with intact

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cytoplasmic membranes, while necroptotic hepatocytes in alcohol-exposed wild-type or

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Nrf2-knockdown mice exhibited discontinuous cytoplasmic membranes, edema cytoplasm, and

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swollen mitochondria. Notably, hepatocytes undergoing necroptosis displayed unique ultrastructural

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modifications of nucleus, including dilatation of nuclear membranes, condensation of chromatin into

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small, irregular, and circumscribed patches, and chromatin margination. Curcumin transformed cellular

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morphology and inhibited cell necroptosis in alcohol-administrated wild-type mice but not

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Nrf2-knockdown mice (Figure 3C). Markers and signaling transduction of necroptosis were also

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measured. Results showed that the Nrf2 shRNA strengthened alcohol-induced increase in the

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expression of hepatic HMGB1, RIP1, and RIP3 at both transcriptional and translational levels and

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further enhanced phosphorylation of MLKL and JNK. Although curcumin had significant inhibitory

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effects on these necroptosis-related markers, Nrf2 shRNA obviously abolished the effects of curcumin

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(Figure 3D,E). Immunofluorescence staining for RIP1, RIP3, and p-JNK further validated the results of

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western blot (Figure 3F-H). Collectively, these data implied that inhibition of alcohol-induced

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hepatocyte necroptosis by curcumin was attributed to activation of Nrf2.

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Curcumin Suppresses p53 Expression Mediated by Activation of Nrf2. Given that Nrf2-mediated

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control of necroptosis might be attributed to regulating p53, we next investigated whether curcumin

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regulated p53 through activating Nrf2. Results from in vivo experiments showed that compared with

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control, alcohol administration induced hepatic p53 expression, which was further strengthened by

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Nrf2 shRNA. Curcumin inhibited hepatic p53 expression in alcohol-fed wild-type mice but not

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Nrf2-knockdown mice (Figure 4A-C). Then human hepatocyte LO2 cells treated with 100 mM

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ethanol for 24 hours were applied to establish an in vitro model of alcoholic liver injury. Nrf2 siRNA

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efficiently inhibited cellular Nrf2 and downstream gene NQO1 expression, suggesting that the

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transfection was successful (Figure 4D). Results from in vitro investigations exhibited parallel changes

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in p53 expression (Figure 4E-G). Altogether, these findings implied that inhibition of ethanol-induced

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necroptosis by curcumin might be associated with its modulation on Nrf2/p53 signaling pathway.

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Curcumin Inhibits Ethanol-Induced Hepatocyte Necroptosis through Nrf2/p53 Pathway. To

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provide a deeper mechanistic insight into the protection of curcumin against ethanol-induced

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hepatocyte necroptosis, the role of Nrf2/p53 pathway was explored. P53 siRNA obviously reduced the

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expression of p53 in LO2 cells, suggesting a successful gene transfection (Figure 5A). Results of MTT

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assay showed that hepatocyte viability was inhibited by ethanol exposure but regained by curcumin.

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The cytoprotective effect of curcumin was cancelled in Nrf2-knockdown hepatocytes but rescued in

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p53 siRNA-administrated Nrf2-knockdown hepatocytes (Figure 5B). Hepatocytes incubated with

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ethanol underwent necroptosis, released a large amount of HMGB1 into culture medium. Curcumin not

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only decreased HMGB1 release but also diminished the expression of intracellular HMGB1, RIP1,

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RIP3, p-MLKL, and p-JNK. Nrf2 siRNA abrogated the effects of curcumin. However, p53 siRNA

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neutralized the impact of Nrf2 siRNA and restored the pharmacological activity of curcumin, resulting

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in reduction of necroptosis-related markers (Figure 5C-E). Immunofluorescence staining for RIP1,

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RIP3, and p-JNK further enhanced the results above (Figure 5F-H). In brief, these data consistently

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indicated that curucmin inhibited ethanol-triggered hepatocyte necroptosis in a Nrf2/p53

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pathway-dependent mechanism.

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DISCUSSION

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Many studies and ours have highlighted the curative effect of curcumin on ALD and preliminarily

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revealed the foundational mechanisms. We observed that curcumin alleviated alcohol-induced lipid

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accumulation in both rat livers and human hepatocytes, wherein Nrf2 might be a molecular target for

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curcumin to regulate lipid metabolism.23 In this work, we for the first time provided compelling

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evidence that curcumin inhibited alcohol-triggered hepatocyte necroptosis. Mechanistically, Nrf2/p53

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pathway might be an important mediator for curcumin to alleviate hepatocyte necroptosis.

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We initially tested whether curcumin affected alcohol-induced hepatocyte necroptosis in mouse livers.

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ALD model was established in rodents as our previous description.23, 35, 36 Alcohol would cause

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hepatocyte injury via facilitating necroptosis characterized by released HMGB1 and increased RIP3.20,

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37, 38

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alcohol, suggesting that alcohol stimulated HMGB1 secretion. The mRNA and protein expression of

313

HMGB1 in mouse livers were also upregulated under alcohol exposure, suggesting that HMGB1 was

314

activated as a nuclear factor. RIP3 expression was obviously increased in alcoholic mouse livers, which

315

was accordant to a previous study.20 These results were consistent with prior evidence that HMGB1 and

316

RIP3 were involved in alcohol-induced liver injury.39, 40 Of importance was the fact that curcumin

317

simultaneously reduced HMGB1 release and decreased the expression of HMGB1 and RIP3. Notably,

318

hepatic RIP1 expression was found to be induced by alcohol, which was inconsistent with a prior study

319

that RIP1 level was not significantly altered by alcohol.20 We proposed that varieties of feeding patterns,

320

mouse strains, duration of alcohol exposure, and animal gender among experiments caused diverse

321

degrees of liver damage, which could explain this difference.41 Phosphorylation of MLKL is the

322

necrosome core machinery which leads to the formation of autologous oligomers that is essential and

323

sufficient for triggering necroptosis.42-44 Obviously, alcohol enhanced phosphorylation of MLKL,

324

which could be inhibited by curcumin. A previous study established a close correlation between JNK

325

activation and alcohol-induced hepatic steatosis and oxidative stress.45 Activation of JNK signaling is

326

an alternative way for RIP3 to execute necroptosis.20 Thus the activated state of JNK was investigated.

327

Results showed that curcumin could dose-dependently suppress alcohol-induced phosphorylation of

328

JNK. The present study highlighted the potential of curcumin to target necroptosis to intervene

329

alcoholic liver injury.

In this study, in analog to others, we observed an increase in serum HMGB1 level caused by

330

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To further explore whether Nrf2 activation was implicated in pharmacological function of curcumin,

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Nrf2 shRNA-mediated knockdown mice was generated according to our previous study with

333

modification.31 Hepatic Nrf2 expression was significantly suppressed by Nrf2 shRNA. NQO1, a Nrf2

334

downstream-regulated gene, was also inhibited, revealing an impaired Nrf2 activity in

335

Nrf2-knockdown mice. Our previous work indicated that curcumin significantly relieved inflammatory

336

response in rats exposed to chronic plus binge alcohol.23 In this study, serum biomarkers of liver injury

337

and hepatic microstructure were firstly measured to evaluate the model and drug efficacy. Results

338

showed that alcohol-induced liver injury was established in mice and aggravated by Nrf2 shRNA.

339

Curcumin could notably alleviate liver injury, which could be abolished by Nrf2 shRNA. These data

340

suggested that Nrf2 activation was required for curcumin to prevent alcohol-caused liver injury, further

341

expanding our previous in vivo findings.

342 343

We subsequently investigated whether activated Nrf2 mediated the inhibitory effect of curcumin on

344

necroptosis. Previous studies revealed that HMGB1 release could be inhibited by active compounds via

345

a Nrf2-dependent manner, which could support the current speculation that Nrf2 was a critical regulator

346

of necroptosis.25, 46-49 We found that Nrf2 shRNA stimulated the alcohol action and enlarged its

347

induction of HMGB1. Curcumin suppressed HMGB1 expression and secretion in a Nrf2-dependent

348

mechanism. Further, the inhibitory effect of curcumin on necroptosis-related regulatory factors was

349

abolished by Nrf2 shRNA. These discoveries uncovered the pivotal role of Nrf2 in regulation of

350

necroptosis and Nrf2 could be a target molecular for developing agents to treat necroptosis in alcoholic

351

liver injury.

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Next investigations were focused on exploring which factor mediated the Nrf2-dependent regulation of

354

necroptosis by curcumin. For a long time, it has been pursued by researchers that whether Nrf2 and p53

355

cooperatively controlled cell survival and death.50-52 Nuclear transcription factor p53 used to be

356

recognized as a routine controller of apoptosis and necrosis which would increase when liver was

357

damaged by alcohol consumption.53, 54 Newly researches uncovered its rising role in triggering

358

necroptosis. Indirect evidence suggested that p53 activation stimulated HMGB1 release.55-58 Direct

359

evidence confirmed that p53-cathepsin axis cooperated with ROS to activate programmed necrotic

360

death upon DNA damage, while p53 stable knockdown alleviated salinomycin-induced programmed

361

necrosis in glioma cells.55, 59 Based on these findings, we speculated that Nrf2/p53 pathway could be

362

involved in curcumin-based inhibition of hepatocyte necroptosis. By using RNA interference

363

technology in in vivo and in vitro systems, we observed that curcumin negatively regulated p53

364

expression via modulating Nrf2, suggesting a modulation by curcumin of Nrf2/p53 pathway. Herein,

365

immortalized human hepatocyte LO2 cells were incubated with 100 mM ethanol for 24 hours to

366

establish an in vitro model of ALD as described in our previous study.23 LO2 cells were derived from

367

primary normal human hepatocytes and maintained the biological features and ultrastructures of

368

normal adult hepatocytes. After stable transfection with human telomerase reverse transcriptase gene,

369

LO2 cells were immortalized and widely used as an in vitro model of liver tissues for studying the

370

pathophysiology of hepatocytes.60 And our findings showed a consolidated correlation between Nrf2

371

and p53, although how it worked was still unclear and needed exploration. Whether the pathway was

372

involved in the regulation of ethanol-stimulated hepatocyte necroptosis by curcumin was further

373

explored. We noticed that p53 siRNA exerted antagonistic action against Nrf2 siRNA, resulting in a

374

restoration of hepatocyte viability against alcoholic hepatotoxicity. P53 siRNA also restored the

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inhibitory effect of curcumin on necroptosis in ethanol-stimulated Nrf2-knockdown hepatocytes. All

376

these discoveries, including ours, consolidated the central impact of p53 in the execution of necroptosis.

377

Our study further demonstrated that p53 suppression contributed to Nrf2 activation-mediated control of

378

hepatocyte necroptosis by curcumin.

379 380

In summary, our work demonstrated that curcumin improved ethanol-induced hepatocyte necroptosis in

381

vivo and in vitro. Mechanistically, modulation of Nrf2/p53 pathway was a molecular basis for curcumin

382

to inhibit hepatocyte necroptosis. For the first time, our current discoveries not only indicated that

383

inhibition of hepoatocyte necroptosis could be a promising target for curcumin to attenuate

384

alcohol-induced liver injury but also provided insightful views of molecular mechanisms involved,

385

which pushed forward the progress of developing curcumin into a candidate agent for ALD treatment.

386 387

CONFLICT OF INTEREST

388

The authors declare that there are no conflicts of interest.

389 390

ACKNOWLEDGMENTS

391

This work was supported by the National Natural Science Foundation of China (81270514, 31401210,

392

31571455), A Project Funded by the Priority Academic Program Development of Jiangsu Higher

393

Education Institutions, the Youth Natural Science Foundation of Jiangsu Province (BK20140955), 2013

394

Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, the

395

Youth Natural Science Foundation of Nanjing University of Chinese Medicine (13XZR20), the Natural

396

Science Research General Program of Jiangsu Higher Education Institutions (14KJB310011), and 2015

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Program for Graduate Scientific Innovation of Jiangsu Higher Education Institutions (KYLX15_0999).

398 399

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the International Association for the Study of the Liver 2010, 30, 319-326.

The p53-cathepsin axis cooperates with ROS to activate programmed necrotic death

Interferon-beta-armed oncolytic adenovirus

p53 promotes inflammation-associated hepatocarcinogenesis by inducing HMGB1 release.

p53-dependent release of Alarmin HMGB1 is a central

ROS-p53-cyclophilin-D signaling mediates

Hepatitis B viral X protein alters the biological

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Table 1. Primers used for determination of mRNA expression levels in frozen mice liver tissues Gene

Forward primer

Reverse primer

SREBP-1c

5’-ACGGAGCCATGGATTGCACA-3’

5’-AAGGGTGCAGGTGTCACCTT-3’

PPAR-α

5’-TACGGTGTGTATGAAGCCATCTT-3’

5’-GCCGTACGCGATCAGCAT-3’

HMGB1

5’-CCATTGGTGATGTTGCAAAG-3’

5’-CTTTTTCGCTGCATCAGGTT-3’

RIP1

5’-CAGCCAAATCAAAGTGC-3’

5’-GGTGTTAGCGAAGACGG-3’

RIP3

5’-GGGACCTCAAGCCCTCTAAC-3’

5’-CTGGGTCCAAGTACGCTAGG-3’

p53

5’-GTAGGAAGGCGCGTGGTAG-3’

5’-CAGTTACAGGAACCCCGAG-3’

GAPDH

5’-CTATGACCACAGTCCATGC-3’

5’-CACATTGGGGGTAGGAACAC-3’

575 576 577 578 579 580 581 582 583 584 585 586 587

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Table 2. Primers used for determination of mRNA expression levels in LO2 hepatocytes Gene

Forward primer

Reverse primer

p53

5’-CAGCACATGACGGAGGTTGT-3’

5’-TCATCCAAATACTCCACACGC-3’

HMGB1

5’-AAGTGAGAGCCAGACGGG-3’

5’-TCCTTTGCCCATGTTTAATTATTTTC-3’

RIP1

5’-CTCCTTGCCACCAACAGATG-3’

5’-TCCGTCAGACTAGTGGTATTATCAAAG-3’

RIP3

5’-CTCTCTGCGAAAGGACCAAG-3’

5’-TCGTAGCCCCACTTCCTATG-3’

GAPDH

5’-CCAACCGCGAGAAGATGA-3’

5’-CCAGAGGCGTACAGGGATAG-3’

589

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Figure 1. Curcumin prevents necroptosis in alcohol-fed mice. (A and B) Levels of serum IL-1α and HMGB1, n=10. (C) The mRNA expression of HMGB1, RIP1, and RIP3 in liver tissues, n=3. (D and E) The protein expression of HMGB1, RIP1, RIP3, p-MLKL, MLKL, p-JNK, JNK, Nrf2, and NQO1 in liver tissues, n=3. For the statistics of each panel in this figure, data are expressed as mean ± SD, ##P < 0.01 and ###P < 0.001 compared with group 1, *P < 0.05, **P < 0.01, and ***P < 0.001 compared with group 2. 75x47mm (600 x 600 DPI)

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Figure 2. Curcumin attenuates alcoholic liver injury dependent on Nrf2 activation. (A) ISH of Nrf2 in murine livers (original magnification, 20×), n=6. (B) The protein expression of Nrf2 and NQO1 in liver tissues, n=3. (C) Immunofluorescence staining for Nrf2 in liver sections (original magnification, 20×), n=6. (D) Serum AST and ALT levels, n=10. (E) H&E staining of liver sections (original magnification, 20×), n=6. For the statistics of each panel in this figure, data are expressed as mean ± SD, ##P < 0.01 and ###P < 0.001 compared with group 1, **P < 0.01 compared with group 2, $P < 0.05 and $$P < 0.01 compared with group 4. 85x62mm (600 x 600 DPI)

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Figure 3. Nrf2 deficiency blocks the effective improvement of curcumin on alcohol-caused hepatocyte necroptosis. (A and B) Levels of serum IL-1α and HMGB1, n=10. (C) Transmission electron microscopy of livers, n=6. In hepatocytes (H), swollen mitochondria (M) are indicated by black arrows. Condensed and marginated chromatins in nuclei (N) are indicated by white arrows. (D) The mRNA expression of HMGB1, RIP1, RIP3 in liver tissues, n=3. (E) The protein expression of HMGB1, RIP1, RIP3, p-MLKL, MLKL, p-JNK, and JNK in liver tissues, n=3. (F-H) Immunofluorescence staining for RIP1, RIP3, and p-JNK in livers (original magnification, 40×) , n=6. For the statistics of each panel in this figure, data are expressed as mean ± SD, ##P < 0.01 and ###P < 0.001 compared with group 1, *P < 0.05 and **P < 0.01 compared with group 2, $P < 0.05 and $$P < 0.01 versus group 4. 131x136mm (600 x 600 DPI)

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Figure 4. Curcumin suppresses p53 expression mediated by activation of Nrf2. (A) The mRNA expression of hepatic p53, n=3. (B) The protein expression of hepatic p53, n=3. (C) Immunofluorescence staining for p53 in livers (original magnification, 40×), n=6. For the statistics of the panels above, data are expressed as mean ± SD. ##P < 0.01 and ###P < 0.001 compared with group 1, **P < 0.01 compared with group 2, $$P < 0.01 compared with group 4. (D) Human hepatocyte LO2 cells were treated with control siRNA or Nrf2 siRNA for 24 h. Western blot analyses of Nrf2 and NQO1 expression in hepatocytes. For this panel, data are expressed as mean ± SD, ***P < 0.001 versus blank control. LO2 cells were treated with DMSO (0.02%, w/v) and/or ethanol and/or curcumin and/or Nrf2 siRNA at the indicated doses for 24 h. (E) The mRNA expression of p53 in hepatocytes. (F) The protein expression of p53 in hepatocytes. (G) Immunofluorescence staining for p53 in hepatocytes (original magnification, 40×). All the in vitro experiments were repeated at least three times independently. For the statistics of these panels, data are expressed as mean ± SD, ##P < 0.01 and ###P < 0.001 compared with DMSO, *P < 0.05 compared with DMSO plus ethanol, $P < 0.05 compared with ethanol plus curcumin. 139x142mm (600 x 600 DPI)

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Figure 5. Curcumin inhibits ethanol-induced hepatocyte necroptosis through Nrf2/p53 pathway. (A) LO2 cells were treated with control siRNA or p53 siRNA for 24 h. Western blot analyses of p53 expression in hepatocytes. **P < 0.01 versus blank control. LO2 cells were treated with DMSO (0.02%, w/v) and/or ethanol and/or curcumin and/or Nrf2 siRNA and/or p53 siRNA at the indicated doses for 24 h. (B) MTT assay for evaluating the viability of LO2 cells. (C) HMGB1 levels in hepatocyte culture medium. (D) The mRNA expression of HMGB1, RIP1, and RIP3 in LO2 cells. (E) The protein expression of HMGB1, RIP1, RIP3, pMLKL, MLKL, p-JNK, and JNK in hepatocytes. (F-H) Immunofluorescence staining for RIP1, RIP3, and p-JNK in hepatocytes (original magnification, 40×). All the in vitro experiments were repeated at least three times independently. For the statistics of the panels above, data are expressed as mean ± SD, ##P < 0.01 and ### P < 0.001 compared with DMSO, **P < 0.01 and ***P < 0.001 compared with DMSO plus ethanol, $P < 0.05, $$P < 0.01, and $$$P < 0.001 compared with ethanol plus curcumin, &P < 0.05, &&P < 0.01, and &&&P < 0.001 compared with ethanol, curcumin plus Nrf2 siRNA. 183x195mm (600 x 600 DPI)

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Abstract graphic 28x20mm (600 x 600 DPI)

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