Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol

Subscriber access provided by RYERSON UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1

Title

2

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

3

Necroptosis

4 5

Running title

6

Curcumin prevents hepatocyte necroptosis

7 8

Authors

9

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

10 11

Affiliations

12

a

13

Jiangsu, China

14

b

15

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

16 17

Correspondence

18

*

19

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

20

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

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

21 22

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 34

23

Abbreviations

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31 32 33 34 35 36 37 38 39 40 41 42 43 44

2


Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


45

Abstract

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64 65

Keywords

66

alcoholic liver disease; curcumin; necroptosis; Nrf2; p53

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

67

INTRODUCTION

68

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

69

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

70

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

71

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

72

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

73

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

74

cell loss and improve alcoholic liver injury.7

Page 4 of 34

75 76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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

88

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

4


Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


89

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

90

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

91

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

92

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

93

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

94

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

95 96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106 107

MATERIALS AND METHODS

108

Reagents and Antibodies

109

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

110

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

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 34

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

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

122 123

Animal Models

124

Animals

125

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

126

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

127

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

128

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

129

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

130

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

131 132

Experimental Procedures

6


Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


133

Study I

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141 142

Study II

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150 151

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

152

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

153

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

154

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

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

155

Cell Culture

156

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

157

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

158

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

159

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

160

atmosphere of 95% air and 5% CO2.

Page 8 of 34

161 162

SiRNA Transfection

163

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

164

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

165

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

166

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

167

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

168

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

169 170

Western Blot Analyses

171

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

172

buffer supplemented with phenylmethylsufonyl fluoride and phosphatase inhibitor. Protein

173

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

174

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

175

sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene

176

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

8


Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


177

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

178

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

179

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

180

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

181

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

182

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

183 184

RNA Extraction and QRT-PCR

185

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

186

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

187

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

188

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

189

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

190 191

Liver Histopathology and Immunofluorescence Staining

192

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

193

previously described.23, 33

194 195

In situ hybridization (ISH)

196

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

197

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

198

according to the previous report.34

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 34

199

Biochemical Analyses

200

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

201

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

202

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

203

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

204

were determined using a SPECTRAmaxTM microplate spectrophotometer (Molecular Devices,

205

Sunnyvale, CA, USA).

206 207

Enzyme-linked Immunosorbent Assay (ELISA)

208

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

209

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

210

China) under the direction of protocols from manufacturer.

211 212

MTT Assay

213

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

214

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

215

description.23

216 217

Statistical Analyses

218

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

219

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

220

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

10


Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


221

statistically significant.

222 223

RESULTS

224

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

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233 234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 34

243

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

244

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

245

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

246

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

247

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

248

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

249

curcumin to prevent mice from chronic alcoholic liver injury.

250 251

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

252

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

253

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

254

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

255

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

256

hepatocyte necroptosis. Normal hepatocytes displayed regular cellular morphology with intact

257

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

258

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

259

swollen mitochondria. Notably, hepatocytes undergoing necroptosis displayed unique ultrastructural

260

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

261

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

262

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

263

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

264

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

12


Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


265

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

266

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

267

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

268

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

269

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

270

hepatocyte necroptosis by curcumin was attributed to activation of Nrf2.

271 272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283 284

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

285

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

286

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

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 34

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

pathway-dependent mechanism.

299 300

DISCUSSION

301

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

302

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

303

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

304

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

305

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

306

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

307 308

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

14


Page 15 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


309

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

310

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

311

37, 38

312

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

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 34

331

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

332

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.

352

16


Page 17 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


353

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

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 34

375

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

18


Page 19 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


397

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

398 399

REFERENCES

400

(1) Sugimoto, K.; Takei, Y.

401

official journal of the Japan Society of Hepatology 2016.

402

(2) Schwartz, J. M.; Reinus, J. F.

403

Liver Dis. 2012, 16, 659-666.

404

(3) Nagata, K.; Suzuki, H.; Sakaguchi, S.

405

progression of liver injury caused by non-alcoholic or alcoholic steatohepatitis. J. Toxicol. Sci. 2007, 32,

406

453-468.

407

(4) Malhi, H.; Guicciardi, M. E.; Gores, G. J.

408

Rev. 2010, 90, 1165-1194.

409

(5) Shulga, N.; Pastorino, J. G.

410

exposure to fructose and ethanol. J. Cell Sci. 2014, 127, 896-907.

411

(6) Barnes, M. A.; Roychowdhury, S.; Nagy, L. E.

412

disease: role of cytochrome P4502E1. Redox Biol 2014, 2, 929-935.

413

(7) Roychowdhury, S.; Chiang, D. J.; Mandal, P.; McMullen, M. R.; Liu, X.; Cohen, J. I.; Pollard, J.;

414

Feldstein, A. E.; Nagy, L. E.

415

of early markers of CCl4 -induced fibrosis but not steatosis or inflammation. Alcohol. Clin. Exp. Res.

416

2012, 36, 1139-1147.

417

(8) Vanden Berghe, T.; Vanlangenakker, N.; Parthoens, E.; Deckers, W.; Devos, M.; Festjens, N.;

418

Guerin, C. J.; Brunk, U. T.; Declercq, W.; Vandenabeele, P.

Pathogenesis of alcoholic liver disease. Hepatology research : the

Prevalence and natural history of alcoholic liver disease. Clin.

Common pathogenic mechanism in development

Hepatocyte death: a clear and present danger. Physiol.

Mitoneet mediates TNFalpha-induced necroptosis promoted by

Innate immunity and cell death in alcoholic liver

Inhibition of apoptosis protects mice from ethanol-mediated acceleration

Necroptosis, necrosis and secondary

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 34

419

necrosis converge on similar cellular disintegration features. Cell Death Differ. 2010, 17, 922-930.

420

(9) Krysko, D. V.; Agostinis, P.; Krysko, O.; Garg, A. D.; Bachert, C.; Lambrecht, B. N.;

421

Vandenabeele, P.

422

in inflammation. Trends in immunology 2011, 32, 157-164.

423

(10) Kaczmarek, A.; Vandenabeele, P.; Krysko, D. V.

424

molecular patterns and its physiological relevance. Immunity 2013, 38, 209-223.

425

(11) Kang, T. B.; Yang, S. H.; Toth, B.; Kovalenko, A.; Wallach, D.

426

RIPK3-mediated activation of the NLRP3 inflammasome. Immunity 2013, 38, 27-40.

427

(12) Kang, T. B.; Yang, S. H.; Toth, B.; Kovalenko, A.; Wallach, D.

428

inflammasome by proteins that signal for necroptosis. Methods in enzymology 2014, 545, 67-81.

429

(13) Saeed, W. K.; Jun, D. W.

430

journal of gastroenterology 2014, 20, 12526-12532.

431

(14) Sun, L.; Wang, H.; Wang, Z.; He, S.; Chen, S.; Liao, D.; Wang, L.; Yan, J.; Liu, W.; Lei, X.; Wang,

432

X.

433

Cell 2012, 148, 213-227.

434

(15) Zhao, J.; Jitkaew, S.; Cai, Z.; Choksi, S.; Li, Q.; Luo, J.; Liu, Z. G.

435

domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis.

436

Proceedings of the National Academy of Sciences of the United States of America 2012, 109,

437

5322-5327.

438

(16) Ramachandran, A.; McGill, M. R.; Xie, Y.; Ni, H. M.; Ding, W. X.; Jaeschke, H.

439

interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis

440

in mice. Hepatology 2013, 58, 2099-2108.

Emerging role of damage-associated molecular patterns derived from mitochondria

Necroptosis: the release of damage-associated

Caspase-8 blocks kinase

Activation of the NLRP3

Necroptosis: an emerging type of cell death in liver diseases. World

Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.

Mixed lineage kinase

Receptor

20


Page 21 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


441

(17) Nishitani, Y.; Matsumoto, H.

Ethanol rapidly causes activation of JNK associated with ER stress

442

under inhibition of ADH. FEBS letters 2006, 580, 9-14.

443

(18) Yang, L.; Wu, D.; Wang, X.; Cederbaum, A. I.

444

autophagy in acute alcohol-induced fatty liver. Free radical biology & medicine 2012, 53, 1170-1180.

445

(19) Wang, X.; Bu, H. F.; Zhong, W.; Asai, A.; Zhou, Z.; Tan, X. D.

446

involved in the mechanism underlying alcohol-induced impairment of macrophage efferocytosis. Mol

447

Med 2013, 19, 170-182.

448

(20) Roychowdhury, S.; McMullen, M. R.; Pisano, S. G.; Liu, X.; Nagy, L. E.

449

interacting protein kinase 3 prevents ethanol-induced liver injury. Hepatology 2013, 57, 1773-1783.

450

(21) Zhang, J.; Zhang, Y.; Xiao, F.; Liu, Y.; Wang, J.; Gao, H.; Rong, S.; Yao, Y.; Li, J.; Xu, G.

451

peroxisome proliferator-activated receptor gamma agonist pioglitazone prevents NF-kappaB activation

452

in cisplatin nephrotoxicity through the reduction of p65 acetylation via the AMPK-SIRT1/p300

453

pathway. Biochemical pharmacology 2016, 101, 100-111.

454

(22) Pulido-Moran, M.; Moreno-Fernandez, J.; Ramirez-Tortosa, C.; Ramirez-Tortosa, M.

455

and Health. Molecules 2016, 21.

456

(23) Lu, C.; Zhang, F.; Xu, W.; Wu, X.; Lian, N.; Jin, H.; Chen, Q.; Chen, L.; Shao, J.; Wu, L.; Lu, Y.;

457

Zheng, S.

458

signaling in hepatocytes. IUBMB life 2015, 67, 645-658.

459

(24) Dai, M. C.; Zhong, Z. H.; Sun, Y. H.; Sun, Q. F.; Wang, Y. T.; Yang, G. Y.; Bian, L. G.

460

protects against iron induced neurotoxicity in primary cortical neurons by attenuating necroptosis.

461

Neurosci. Lett. 2013, 536, 41-46.

462

(25) Wang, J.; Hu, X.; Xie, J.; Xu, W.; Jiang, H.

Cytochrome P4502E1, oxidative stress, JNK, and

MFG-E8 and HMGB1 are

Absence of receptor

The

Curcumin

Curcumin attenuates ethanol-induced hepatic steatosis through modulating Nrf2/FXR

Curcumin

Beta-1-adrenergic receptors mediate

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 34

463

Nrf2-HO-1-HMGB1 axis regulation to attenuate hypoxia/reoxygenation-induced cardiomyocytes

464

injury in vitro. Cell. Physiol. Biochem. 2015, 35, 767-777.

465

(26) Kim, S. R.; Ha, Y. M.; Kim, Y. M.; Park, E. J.; Kim, J. W.; Park, S. W.; Kim, H. J.; Chung, H. T.;

466

Chang, K. C.

467

cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals. Biochem.

468

Pharmacol. 2015, 95, 279-289.

469

(27) Tan, Y.; Wang, Q.; She, Y.; Bi, X.; Zhao, B.

470

activation of the Nrf2/HO-1 pathway and NF-kappaB suppression. The journal of trauma and acute

471

care surgery 2015, 78, 784-792.

472

(28) Liu, Z.; Wang, J.; Huang, X.; Li, Z.; Liu, P.

473

dysfunction and atherosclerosis in apolipoprotein E-deficient mice. Translational research : the journal

474

of laboratory and clinical medicine 2016.

475

(29) Zhang, W.; Niu, M.; Yan, K.; Zhai, X.; Zhou, Q.; Zhang, L.; Zhou, Y.

476

with the roles of leptin in mouse liver fibrosis and sterol regulatory element binding protein-1c

477

expression of rat hepatic stellate cells. The international journal of biochemistry & cell biology 2013,

478

45, 736-744.

479

(30) Zhai, X.; Yan, K.; Fan, J.; Niu, M.; Zhou, Q.; Zhou, Y.; Chen, H.

480

contributes to the effects of leptin on SREBP-1c expression in rat hepatic stellate cells and liver fibrosis.

481

British journal of pharmacology 2013, 169, 197-212.

482

(31) Lu, C.; Xu, W.; Zhang, F.; Shao, J.; Zheng, S.

483

effects of ligustrazine on hepatic fibrosis by targeting hepatic stellate cell transdifferentiation.

484

Toxicology 2016, 365, 35-47.

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7

Ketamine reduces LPS-induced HMGB1 via

Deletion of sirtuin 6 accelerates endothelial

Stat3 pathway correlates

The beta-catenin pathway

Nrf2 knockdown attenuates the ameliorative

22


Page 23 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


485

(32) Zhang, F.; Zhang, Z.; Chen, L.; Kong, D.; Zhang, X.; Lu, C.; Lu, Y.; Zheng, S.

Curcumin

486

attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells.

487

Journal of cellular and molecular medicine 2014, 18, 1392-1406.

488

(33) Bao, C.; Shao, Y.; Li, X.

489

Ultrastructural pathology 2014, 38, 217-223.

490

(34) Zhang, L.; Xiang, Z. L.; Zeng, Z. C.; Fan, J.; Tang, Z. Y.; Zhao, X. M.

491

prediction model for lymph node metastasis in hepatocellular carcinoma. Oncotarget 2016, 7,

492

3587-3598.

493

(35) Lu, C.; Xu, W.; Zhang, F.; Jin, H.; Chen, Q.; Chen, L.; Shao, J.; Wu, L.; Lu, Y.; Zheng, S.

494

Ligustrazine prevents alcohol-induced liver injury by attenuating hepatic steatosis and oxidative stress.

495

Int. Immunopharmacol. 2015, 29, 613-621.

496

(36) Chen, L. Y.; Chen, Q.; Cheng, Y. F.; Jin, H. H.; Kong, D. S.; Zhang, F.; Wu, L.; Shao, J. J.; Zheng,

497

S. Z.

498

apoptosis. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2016, 79, 35-43.

499

(37) Shulga, N.; Pastorino, J. G.

500

exposure to fructose and ethanol. Journal of cell science 2014, 127, 896-907.

501

(38) Ge, X.; Antoine, D. J.; Lu, Y.; Arriazu, E.; Leung, T. M.; Klepper, A. L.; Branch, A. D.; Fiel, M. I.;

502

Nieto, N.

503

disease (ALD). The Journal of biological chemistry 2014, 289, 22672-22691.

504

(39) Wang, X.; Bu, H. F.; Zhong, W.; Asai, A.; Zhou, Z.; Tan, X. D.

505

involved in the mechanism underlying alcohol-induced impairment of macrophage efferocytosis. Mol.

506

Med. 2013, 19, 170-182.

Hepatocyte necroptosis induced by ischemic acute kidney injury in rats.

A microRNA-based

Diallyl trisulfide attenuates ethanol-induced hepatic steatosis by inhibiting oxidative stress and

Mitoneet mediates TNFalpha-induced necroptosis promoted by

High mobility group box-1 (HMGB1) participates in the pathogenesis of alcoholic liver

MFG-E8 and HMGB1 are

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 34

507

(40) Ge, X.; Antoine, D. J.; Lu, Y.; Arriazu, E.; Leung, T. M.; Klepper, A. L.; Branch, A. D.; Fiel, M. I.;

508

Nieto, N.

509

disease (ALD). J. Biol. Chem. 2014, 289, 22672-22691.

510

(41) Eagon, P. K.

511

gastroenterology 2010, 16, 1377-1384.

512

(42) Cai, Z.; Jitkaew, S.; Zhao, J.; Chiang, H. C.; Choksi, S.; Liu, J.; Ward, Y.; Wu, L. G.; Liu, Z. G.

513

Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis.

514

Nat. Cell Biol. 2014, 16, 55-65.

515

(43) Chen, X.; Li, W.; Ren, J.; Huang, D.; He, W. T.; Song, Y.; Yang, C.; Zheng, X.; Chen, P.; Han, J.

516

Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell

517

death. Cell Res. 2014, 24, 105-121.

518

(44) Wang, H.; Sun, L.; Su, L.; Rizo, J.; Liu, L.; Wang, L. F.; Wang, F. S.; Wang, X.

519

kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.

520

Mol. Cell 2014, 54, 133-146.

521

(45) Yang, L.; Wu, D.; Wang, X.; Cederbaum, A. I.

522

autophagy in acute alcohol-induced fatty liver. Free Radic. Biol. Med. 2012, 53, 1170-1180.

523

(46) Bray, K.; Mathew, R.; Lau, A.; Kamphorst, J. J.; Fan, J.; Chen, J.; Chen, H. Y.; Ghavami, A.; Stein,

524

M.; DiPaola, R. S.; Zhang, D.; Rabinowitz, J. D.; White, E.

525

kinase-dependent necrosis enabling survival to mTOR inhibition. PloS one 2012, 7, e41831.

526

(47) Dong, W. W.; Liu, Y. J.; Lv, Z.; Mao, Y. F.; Wang, Y. W.; Zhu, X. Y.; Jiang, L.

527

barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced

528

mitochondrial oxidative damage via an Nrf2-dependent mechanism. Free radical biology & medicine

High mobility group box-1 (HMGB1) participates in the pathogenesis of alcoholic liver

Alcoholic liver injury: influence of gender and hormones. World journal of

Mixed lineage

Cytochrome P4502E1, oxidative stress, JNK, and

Autophagy suppresses RIP

Lung endothelial

24


Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


529

2015, 88, 404-416.

530

(48) Kim, S. R.; Ha, Y. M.; Kim, Y. M.; Park, E. J.; Kim, J. W.; Park, S. W.; Kim, H. J.; Chung, H. T.;

531

Chang, K. C.

532

cells and improves survival rate in septic mice by activation of Nrf2/HO-1 signals. Biochemical

533

pharmacology 2015, 95, 279-289.

534

(49) Kim, Y. M.; Kim, H. J.; Chang, K. C.

535

lipopolysaccharide-activated RAW 264.7 cells and endotoxemic mice by p38/Nrf2-dependent induction

536

of HO-1. International immunopharmacology 2015, 26, 112-118.

537

(50) Bui, C. B.; Shin, J.

538

p53-dependent mitotic catastrophe. Biochem. Biophys. Res. Commun. 2011, 412, 347-352.

539

(51) You, A.; Nam, C. W.; Wakabayashi, N.; Yamamoto, M.; Kensler, T. W.; Kwak, M. K.

540

Transcription factor Nrf2 maintains the basal expression of Mdm2: An implication of the regulation of

541

p53 signaling by Nrf2. Arch. Biochem. Biophys. 2011, 507, 356-364.

542

(52) Chen, W.; Jiang, T.; Wang, H.; Tao, S.; Lau, A.; Fang, D.; Zhang, D. D.

543

p53-mediated control of cell survival and death? Antioxidants & redox signaling 2012, 17, 1670-1675.

544

(53) Yokoyama, A.; Omori, T.; Tanaka, Y.; Yokoyama, T.; Sugiura, H.; Mizukami, T.; Matsushita, S.;

545

Higuchi, S.; Maruyama, K.; Ishii, H.; Hibi, T.

546

aldehyde dehydrogenase-2 genotype in Japanese alcoholic men with early esophageal squamous cell

547

carcinoma. Cancer letters 2007, 247, 243-252.

548

(54) Yokoyama, A.; Tanaka, Y.; Yokoyama, T.; Mizukami, T.; Matsui, T.; Maruyama, K.; Omori, T.

549

p53 protein accumulation, iodine-unstained lesions, and alcohol dehydrogenase-1B and aldehyde

550

dehydrogenase-2 genotypes in Japanese alcoholic men with esophageal dysplasia. Cancer letters 2011,

Ascorbic acid reduces HMGB1 secretion in lipopolysaccharide-activated RAW 264.7

Glycyrrhizin reduces HMGB1 secretion in

Persistent expression of Nqo1 by p62-mediated Nrf2 activation facilitates

Does Nrf2 contribute to

p53 Protein accumulation, cancer multiplicity, and

25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 34

551

308, 112-117.

552

(55) Tu, H. C.; Ren, D.; Wang, G. X.; Chen, D. Y.; Westergard, T. D.; Kim, H.; Sasagawa, S.; Hsieh, J.

553

J.; Cheng, E. H.

554

upon DNA damage. Proceedings of the National Academy of Sciences of the United States of America

555

2009, 106, 1093-1098.

556

(56) Huang, H.; Xiao, T.; He, L.; Ji, H.; Liu, X. Y.

557

induces both apoptosis and necroptosis in cancer cells. Acta biochimica et biophysica Sinica 2012, 44,

558

737-745.

559

(57) Yan, H. X.; Wu, H. P.; Zhang, H. L.; Ashton, C.; Tong, C.; Wu, H.; Qian, Q. J.; Wang, H. Y.; Ying,

560

Q. L.

561

Journal of hepatology 2013, 59, 762-768.

562

(58) Davalos, A. R.; Kawahara, M.; Malhotra, G. K.; Schaum, N.; Huang, J.; Ved, U.; Beausejour, C.

563

M.; Coppe, J. P.; Rodier, F.; Campisi, J.

564

mediator of senescent phenotypes. The Journal of cell biology 2013, 201, 613-629.

565

(59) Qin, L. S.; Jia, P. F.; Zhang, Z. Q.; Zhang, S. M.

566

salinomycin-induced glioma cell necrosis. Journal of experimental & clinical cancer research : CR

567

2015, 34, 57.

568

(60) Cheng, B.; Zheng, Y.; Guo, X.; Wang, Y.; Liu, C.

569

features and expressions of DNA repair enzymes in LO2 cells. Liver international : official journal of

570

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

571 572 573 26


Page 27 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


574

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

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

588

Page 28 of 34

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

28


Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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)


Page 30 of 34

Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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)


Page 32 of 34

Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract graphic 28x20mm (600 x 600 DPI)


Page 34 of 34

Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol

Recommend Documents