Modulation of AMPK-dependent lipogenesis mediated by P2x7R

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Bioactive Constituents, Metabolites, and Functions

Modulation of AMPK-dependent lipogenesis mediated by P2x7R-NLRP3 inflammasome activation contributes to the amelioration of alcoholic liver steatosis by dihydroquercetin Yu Zhang, Quan Jin, Xia Li, Jiang Min, Ben-Wen Cui, Kai-Li Xia, Yan-Ling Wu, Li-Hua Lian, and ji-xing nan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00944 • Publication Date (Web): 28 Apr 2018 Downloaded from http://pubs.acs.org on April 28, 2018

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Journal of Agricultural and Food Chemistry

Graphical abstract 220x225mm (300 x 300 DPI)

ACS Paragon Plus Environment


of

AMPK-dependent

lipogenesis

mediated

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1

Modulation

by

P2x7R-NLRP3

2

inflammasome activation contributes to the amelioration of alcoholic liver steatosis

3

by dihydroquercetin

4

5

Running title: Dihydroquercetin alleviates alcoholic liver steatosis

6

7

Yu Zhanga†, Quan Jin†a, Xia Lia, Min Jianga, Ben-Wen Cuia, Kai-Li Xiaa, Yan-Ling Wua,

8

Li-Hua Liana*, Ji-Xing Nan*a,b

9

10

a

Key Laboratory for Natural Resource of Changbai Mountain & Functional

11

Molecules, Ministry of Education, College of Pharmacy, Yanbi

12

Jilin Province 133002, China

13 14

15

b

an University, Yanji,

Clinical Research Center, Yanbian University Hospital, Yanji, Jilin Province

133002, China

†These authors contributed equally to this work.

16

*Corresponding to

17

Li-Hua Lian, Ji-Xing Nan, Key Laboratory for Natural Resource of Changbai Mountain

18

& Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian 1


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University, Yanji 133002 Jilin Province, China, Tel.: 86-433-2435061, fax:

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86-433-2435072. E-mail address: [email protected] (L.-H. Lian), [email protected]

21

(J.-X. Nan).

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ABSTRACT

23

Dihydroquercetin (TAX) is the most abundant dihydroflavone found in onions, milk

24

thistle and Douglas fir bark. We investigated whether TAX could inhibit the lipid

25

accumulation in alcoholic liver steatosis in vivo and in vitro. An in vivo model was

26

established by intragastrically treating mice with ethanol, and an in vitro model was

27

created by treating HepG2 cells with ethanol. TAX regulated SREBP1 and ACC

28

expression via elevating LKB1/AMPK phosphorylation. Also, TAX upregulated SIRT1

29

expression, which suppressed by ethanal intake. Decreased expression of P2x7R and

30

NLRP3 and suppressed cleavage of caspase-1 by TAX resulted in the inhibition of

31

IL-1β production and release. Additionally, TAX reduced lipogenesis and promoted

32

lipid oxidation via the regulation of AMPK and ACC in ethanol-treated steatotic

33

HepG2 cell. TAX downregulated IL-1β cleavage response to LPS plus ATP stimulation

34

in HepG2 cells. P2x7R deficiency attenuated lipid accumulation characterized by the

35

increased AMPK activity and decreased SREBP1 expression in ethanol-treated HepG2

36

cells. Our data showed that TAX exhibited the inhibitory properties on lipogenesis

37

and hepatoprotective capacity, indicating that TAX has therapeutic potential for

38

preventing alcoholic liver steatosis.

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Keywords: alcoholic liver steatosis; AMPK; dihydroquercetin; NLRP3; P2x7R

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INTRODUCTION

42

Acute alcoholic fatty liver refers to alcoholic liver disease (ALD) caused by

43

excessive intake of alcohol. Liver steatosis, liver cell damage and inflammatory cell

44

infiltration are frequently associated with acute alcoholic fatty liver. If drinking

45

continues, acute alcoholic fatty liver will develop to liver fibrosis, cirrhosis, or even

46

cancer. Severe drinking can cause extensive liver cell necrosis and even hepatic

47

failure 1. ALD brings huge damage to the human health and gives rise to significant

48

morbidity and mortality 2. Unfortunately, there is still no effective medical treatment

49

for any stage of ALD in the past few decades.

50

Acute alcoholic liver steatosis is characterized by lipid accumulation in

51

hepatocytes 3. Alcohol mediated liver steatosis involves many regulatory factors, such

52

as AMP dependent AMP-activated kinase (AMPK). AMPK, as a lipid regulating kinase,

53

plays an important regulatory role in the pathogenesis of alcoholic liver steatosis 4.

54

Activated AMPK promotes the phosphorylation of acetyl-CoA carboxylase (ACC),

55

inhibits the activity of ACC and results in the regulation of the fatty acids synthesis

56

and fatty acid oxidation. As an upstream kinase of AMPK, liver kinase B1 (LKB1) can

57

activate AMPK. Direct phosphorylation of AMPK also inhibits the activity of Sterol

58

regulatory element-binding protein 1 (SREBP1) in hepatocytes and thereby reduces

59

lipid deposition 5. Alcohol exposure can upregulate SREBP1 and increase fatty acid

60

synthesis in hepatocytes 6. Sirtuin 1 (SIRT1) plays an important role in the regulation

61

of lipid metabolism and inflammation. Alcohol can affect the SIRT1 signaling pathway

4



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in the pathogenesis of alcoholic liver steatosis 7, 8 . Alcohol downregulates SIRT1 level

63

leading to liver lipid accumulation and inflammatory response.

64

Inflammation plays a crucial role in the underlying pathogenesis of ALD 9.

65

Alcohol-induced increased gut permeability lead to increased lipopolysaccharides

66

(LPS) level in the circulation 9. Gut microflora-derived LPS is recognized by toll-like

67

receptor 4 (TLR4) and provides the first signal for inflammasome activation

68

Ethanal exposure increased circulating and liver adenosine triphosphate (ATP) levels,

69

indicating ATP signaling is involved in liver inflammation in ALD 11. An ATP-gated ion

70

channel, purinergic 2X7 receptor (P2x7R) is critical for inflammasome activation. High

71

levels of ATP, as a second signal in LPS-driven inflammation, results in the

72

downregulation of nod-like receptor pyrin containing 3 (NLRP3) inflammasome

73

activation and inflammatory mediators release. NLRP3 inflammasome activation

74

leads to the release of proinflammatory cytokines, such as interleukin (IL)-1β.

75

Regulation of P2x7R-NLRP3 inflammasome activation by small molecule is a

76

promising target to treat alcoholic liver steatosis 12. Although many researchers have

77

made extensive and profound studies on ALD, the precise pathogenesis responsible

78

for ALD are still not clear. Worse yet, there are no effective treatments for ALD.

79

Therefore, a safe and effective approach for therapy of ALD is urgently needed.

10

.

80

Flavonoids are a large family of components widely existed in most traditional

81

Chinese herbal medicines. Dihydroquercetin (also called taxifolin, TAX, Fig 1A), is the

82

most abundant dihydroflavone found in onions 13, milk thistle 14 and Douglas fir bark

5


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15

83

(Pseudotsuga taxifolia)

. TAX has been widely used in in food and healthcare

84

industry as food additives. Several studies showed that TAX possesses various

85

pharmacological activities, such as anti-virus 16, anti-inflammatory 17, anti-oxidant 18,

86

19

87

anti-oxidative capacities of TAX. TAX showed potent inhibitory properties on

88

lipogenesis in prostate and breast cancer cells by inhibiting fatty acid synthase

89

enzymes

90

silymarin (Legalon®) used in the phytotherapy and dietary supplement for chronic

91

hepatitis and alcoholics fatty liver

92

hepatoprotective mechanisms of the hepatoprotective flavonolignans from the

93

silymarin compound, including TAX. However, research on TAX in the

94

hepatoprotective properties is minimal. Considering the inhibitory properties on

95

lipogenesis and hepatoprotective capacity, we anticipate the possible beneficial

96

effects of TAX on liver lipid accumulation and inflammation induced by alcohol.

and anti-fibrosis 20. By far accumulating evidence has been surrounding the potent

21

. TAX is the only flavonoid found in the licensed hepatoprotective drug

15

. Polyak et al

16

97

6


aimed to identify potential


98

MATERIALS AND METHOD

99

Reagents and materials. TAX (Pubchem CID: 439533; purity ≥ 99.00%) was purchased

100

from Chengdu Push Bio-technology Co., Ltd. Anti-AMPKα, anti-phospho-AMPKα,

101

anti-LKB1, anti-phospho-LKB1, anti-ACC, and anti-phospho-ACC antibodies were

102

purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-SIRT1,

103

anti-SREBP1,

104

dehydrogenase (GAPDH) antibodies and A438079 (PubChem CID: 11673921) were

105

purchased from Abcam (Cambridge, MA, USA). Anti-Caspase-1 antibody and

106

Caspase-1 inhibitor VI (Z-YVAD-FMK, PubChem CID: 16760349) were obtained from

107

Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-IL-1β antibody was

108

purchased from R&D Systems (Minneapolis, MN, USA). Ultrapure lipopolysaccharides

109

from E. coli O111:B4 and TLR4 inhibitor (CLI-095) were purchased from InvivoGen

110

(San Diego, CA, USA), and adenosine 5′-triphosphate disodium salt was purchased

111

from Sigma Chemical Co. (St. Louis, MO, USA). Metformin, a LKB1/AMPK activator 22,

112

23

113

Biotechnology (Haiman, Jiangsu, China).

114

Animals experimental protocols. Male C57BL/6 mice (8-10 weeks old, 20-22g) were

115

purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd ([SPF, SCXK (JI)

116

2011-0004], Changchun, Jilin, China). All animals were housed in a 12/12 h light/dark

117

environment with a consist temperature 22 ± 2°C and humidity 55±5%. All

118

procedures throughout the animal experiments were carried out strictly to follow

Anti-P2x7R,

Anti-NLRP3

and

anti-glyceraldehyde-3-phosphate

, used as positive control and was purchased from Beyotime Institute of

7


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119

Guide for the Care and Use of Laboratory Animals (National Research Council, 1996)

120

and approved by Animal Research Committee of Yanbian University.

121

After a week of acclimatization, all animals were randomly assigned to six

122

groups, as follows: normal group, ethanol group, ethanol plus TAX groups (1, 5, 25

123

mg/kg, body weight) and TAX only group. Mice in normal groups were treated with

124

an isocalorical maltose solution. Mice were exposed to acute alcohol treatment

125

according to the previously published protocols

126

were gavaged with ethanol at a dose of 5 g/Kg (body weight) every 12 h for 3 times.

127

And TAX was intragastrically treated simultaneously with ethanol to the mice. Four

128

hours after the last administration of ethanol, all mice were sacrificed under

129

anesthetization by isoflurane. Blood was collected by direct cardiac puncture. The

130

mice liver was removed, and a portion of liver was fixed in 10% neutral buffered

131

formalin for histological analysis. The rest tissues were stored at -80 °C until

132

analyzed.

133

Cell culture. The Human Hepatoma Cell Line HepG2 were kindly provided by Dr. Jung

134

Joon Lee (Korea Institute of biological sciences and Biotechnology, Daejeon, South

135

Korea). HepG2 were cultured in DMEM supplemented with 10% fetal bovine serum

136

(FBS), 100 μg/ml streptomycin and 100 units/ml penicillin, in a 37 ℃ humidified

137

incubator with 5% CO2. HepG2 cells were exposed with ethanol (50mM) for 24 h or

138

primed with LPS (1 μg/ml) for 4 h then continuously stimulated ATP (3 mM) for

139

additional 30 min. TAX or appropriate inhibitors was pretreated 1 h prior to LPS

24, 25

. All mice except normal group

8



140

treatment or ethanol exposure.

141

Liver histological analysis. For HE and Oil Red O staining, liver tissues fixed with 10%

142

neutral buffered formalin were embedded in paraffin. 5-μm-thick sections were

143

stained with HE or oil red O.

144

Measurement of biochemical parameters. Blood samples were centrifuged at 3000

145

rpm and 4℃ for 30 min to collect serum and then stored at -80℃ for further

146

measurement. The levels of serum alanine aminotransferase (ALT) and aspartate

147

aminotransferase (AST) were detected by an Automatic biochemical analyzer

148

(Spotchem SP4430, Arkray, Kyoto, Japan). Serum and Hepatic triglycerides (TG) levels

149

were determined using commercial quantification assay kits (Nanjing Jiancheng

150

Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s

151

protocols.

152

Enzyme-linked immunosorbent assay (ELISA). Mouse IL-1β and lipopolysaccharide

153

(LPS)-Binding Protein (LBP) protein levels in serum were measured using murine

154

IL-1β Standard ABTS ELISA Development Kit (PeproTech, Rocky Hill, NJ, USA) or

155

mouse lipopolysaccharide-binding protein (LBP) ELISA kit (Cusabio Biotech Co., Ltd,

156

Wuhan, Hubei, China) according to the manufacturers' instruction.

157

ATP assay

158

Serum ATP levels were determined using Enhanced ATP Assay Kit (Beyotime Institute

159

of Biotechnology, Haiman, Jiangsu, China) with luminometer. 9


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Immunohistochemistry and immunocytochemistry staining. Paraffin sections of

161

mice liver were deparaffinized in xylene and passed through sequential decreasing

162

concentrations of ethanol. Sections were microwaved in 10 mM sodium citrate buffer

163

(pH 6.0), and then allowed to cool back to room temperature. Slides were treated

164

with 3% hydrogen peroxide, followed by blocking with 5% normal goat serum and

165

Avidin/Biotin Blocking solution (Vector Laboratories, Inc., Burlingame, CA, USA).

166

Immunohistochemical staining was performed using prediluted mouse anti-SREBP1,

167

anti-SIRT1, anti-NLRP3 or anti-P2x7R antibodies. The sections were then incubated

168

with appropriate secondary antibodies. Bound antibodies were visualized with Lab

169

Vision™ DAB Plus Substrate Staining System (Thermo Fisher Scientific, Fremont, CA,

170

USA) and then images were acquired by a light microscopy (Nikon TI-E, Nikon, Tokyo,

171

Japan). HepG2 cells were grown on coverslips in 6-well plates and then treated with

172

TAX. Cells were fixed with 4% paraformaldehyde and then performed as previously

173

described 26. Stained cells were visualized by a Nikon TI-E fluorescence microscope.

174

All these examinations were carried out in a blinded manner.

175

Small interference RNA (siRNA) transfection. HepG2 cells were transfected with

176

scrambled control siRNA or P2x7R-siRNA (Bioneer, Shanghai, China) using

177

Lipofectamine® RNAiMAX reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA).

178

The transfection efficiency of FAM-labeled negative control siRNA was >90% after 24

179

h

180

5′-AGAGCAAAGUGACCUGGUU-3′; antisense, 5′-AACCAGGUCACUUUGCUCU-3′.

transfection.

Sequences

for

human

P2x7R-siRNA

10


were:

sense,


181

Western blot analysis. Total protein or nuclear protein was extracted from livers or

182

HepG2 cells with lysis buffer or Nuclear and Cytoplasmic Protein Extraction Kit

183

(Beyotime Institute of Biotechnology). Equal amounts of protein were separated by

184

8–12% sodium dodecyl sulphase-polyacrylamide gel electrophoresis (SDS-PAGE), and

185

then transferred to PVDF membranes. And membranes were blocked 5% skim milk in

186

PBS containing 0.05% Tween 20, and then incubated with specific primary antibody,

187

followed by the incubation with an HRP-conjugated secondary antibody. Finally

188

target protein was visualized using Clarity™ ECL Western Blotting Substrate (Bio-Rad,

189

Hercules, CA, USA), and quantified densitometry with Bio-Rad Quantity One

190

software.

191

Statistical analysis. Results are expressed as the mean ± SD. All data were followed a

192

Gaussian distribution (p>0.1) analyzed by Kolmogorov–Smirnov normality tests. For

193

animal and cell studies, statistical significance was determined using one-way

194

analysis of variance (ANOVA) and Tukey multiple comparisons. A statistically

195

significant between groups (P value) is less than 0.05. Statistical analyses were

196

performed using the GraphPad Prism v6 (Graphpad Software Inc., San Diego, CA,

197

USA).

198

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RESULTS

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TAX attenuates ethanol-induced lipid accumulation in mice liver. After alcohol

201

exposure,

202

alcohol-administrated group than in normal group (Fig 1B). The TG contents in serum

203

and liver were determined by biochemical analysis. The increase of serum and

204

hepatic TG accumulation was observed in alcohol-administrated group, compared

205

with normal group (Fig 1C). TAX co-administration led to a significant decrease of

206

serum ALT and AST in alcohol-administrated group. A remarkable reduction of TG

207

level in serum and liver was also observed with TAX co-administration in

208

alcohol-administrated mice. Decreased serum aminotransferase and TG level hinted

209

that TAX has a potent hepatoprotective capacity against alcohol treatment.

serum

AST

and

ALT

levels

were

significantly

higher

in

210

To further evaluate liver histological changes, the liver sections were stained by

211

H&E or Oil-red O. Hepatocellular ballooning and accumulation of lipid droplets were

212

observed in mice livers after alcohol intake (Fig 1D and 1E). TAX treatment notably

213

reduced the degree of lipid accumulation, suggesting TAX could regulate

214

alcohol-induced liver steatosis.

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In alcohol-fed mice, serum ATP level was elevated, indicating high levels of ATP

216

in ALD progression might be acted as a danger signal, which was consistent with

217

previous studies 27. And TAX pretreatment suppressed circulating ATP level (Fig 1F).

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LBP forms a complex with LPS and then that complex bound to membrane

219

TLR4/CD14 28. Because LBP is released into circulation response to LPS, LBP level in 12



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circulation can be considered as a surrogate biomarker of LPS-induced response29.

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The serum LBP level in alcohol-exposed mice notably elevated compared with normal

222

mice and this increase was significantly abolished by TAX (Fig 1G).

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TAX regulates lipid metabolism by activating AMPK in alcohol-induced liver

224

steatosis. To clarify the underlying mechanism of reduced lipid accumulation in

225

TAX-pretreated mice exposed to ethanol, we determined the expression of proteins

226

involved in lipid metabolism, including lipid synthesis and β-oxidation. AMPK and its

227

upstream kinase-LKB1 regulates lipid metabolism. As shown in Fig 2A, the protein

228

levels of total and phosphorylated LKB1 and AMPK were downregulated under

229

ethanol exposure. Inhibitory effect of AMPK and LKB1 phosphorylation caused by

230

alcohol was improved by TAX treatment more than 176% and 144% compared with

231

that of the mice liver treated ethanol. The SREBP1, which regulates lipid biosynthesis,

232

are synthesized as precursors of 125 kDa and active mature nuclear forms (~68 kDa)

233

30

234

increased in nuclear fraction protein of mice liver (Fig 2A). And the increase of

235

SREBP1

236

immunohistological staining for SREBP1 (Fig 2C). With TAX pretreatment SREBP1

237

expression was descended in alcoholic fatty mice liver (Fig 2A and 2C). ACC is a key

238

enzyme, regulating synthesis and β-oxidation of lipid 31. And the protein expression

239

of phosphorylated ACC was decreased, accompanied with increased ACC expression,

240

while TAX abolished the inhibition of phosphorylated ACC induced by ethanol (Fig

241

2A). SIRT1 functions as upstream regulators of LKB1-AMPK axis 32. Ethanol exposure

. Also consumed with binge alcohol, mature forms of SREBP1 were significantly

in

the

alcohol-induced

steatotic

liver

13


was

also

detected

by

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242

downregulated protein expression of SIRT1 confirmed by western blot and

243

immunohistochemistry staining for SIRT1 (Fig 2A and 2D). As expected, TAX

244

successfully evoked SIRT1 activity by 1.26-fold in alcoholic steatotic mice livers,

245

compared with that of the mice liver treated ethanol. It indicated that TAX might

246

suppress lipid accumulation in alcoholic steatotic liver likely through inhibiting lipid

247

synthesis and promoting lipid oxidation.

248

TAX

249

inflammasome in mice liver. IL-1β signaling mediates steatosis, and is associated

250

with NLRP3 inflammasome activation

251

ethanol exposure was suppressed after TAX administration, with 261% decrease than

252

alcoholic steatotic mice (Fig 3A). TAX treatment also showed decreased tendency of

253

NLRP3 protein expression and caspase-1 cleavage, determined by western blotting

254

and immunohistochemistry stain (Fig 3B and 3D). Our data indicated that TAX could

255

inhibit the inflammasome activation after ethanol exposure. There results prompted

256

us to investigate whether P2x7R activation will be blocked by TAX. We observed that

257

protein expression of P2x7R was remarkably decreased by TAX in mice livers with

258

alcoholic liver steatosis (Fig 3B and 3E).

259

TAX alleviates lipid accumulation induced by ethanol in hepatocytes. In order to

260

focus on the lipid accumulation in hepatocytes, we applied HepG2 cells, which

261

express ADH4 metabolizing ethanol 34. Droplets in ethanol-treated HepG2 cells were

262

obviously red stained with oil red O, while TAX concentration-dependently

inhibits

ethanol-induced

inflammatory

33

response

via

P2x7R-NLRP3

. The increased secretion of IL-1β after

14



263

suppressed lipid droplets accumulation in HepG2 cells (Fig 4A). Exposure of HepG2

264

cells to ethanol let to a significant decrease of protein expression of phosphorylated-

265

and total-AMPKα (Fig 4B). With TAX pretreatment, AMPK phosphorylation was

266

restored in ethanol-treated HepG2 cells. In addition, TAX suppressed the protein

267

levels of ACC evoked by ethanol, and concomitantly activated ACC phosphorylation.

268

Our in vitro experiments results were consistent with results of in vivo experiments,

269

confirmed that TAX regulates lipid metabolism in ethanol-induced hepatocytes via

270

AMPK, and its AMPK-stimulating capability was superior to metformin to a certain

271

extent.

272

AMPK activation is linked with the inhibition of P2x7R in ethanol-treated steatotic

273

hepatocytes. We observed that ethanol treatment on HepG2 cells induce a

274

significant rise of mature IL-1β expression, which was abolished by TAX pretreatment

275

(Fig 4B). Taking into account the downregulation of IL-1β and P2x7R by TAX in

276

steatotic mice livers and hepatocytes, we were intrigued whether P2x7R activation is

277

involved in AMPK-mediated lipid accumulation in hepatocytes. HepG2 cells were

278

transfected with siRNA specific P2x7R or scramble siRNA and confirmed by western

279

blot (Fig 5A). Depletion of P2x7R results in the recovery of AMPK phosphorylation

280

despite ethanol exposure (Fig 5B). Application of P2x7R siRNA significantly reduced

281

protein expressions of SREBP1, which was increased in ethanol-treated HepG2 cells

282

(Fig 5D). Interestingly, with P2x7R deficiency, TAX pretreatment further enhanced

283

AMPK stimulation and suppressed SREBP1 expression. It indicated that

284

transcriptionally and pharmacologically inhibition of P2x7R will lead to alleviation of 15


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lipid accumulation in hepatocytes.

286

IL-1β cleavage initiated by LPS plus ATP is prevented by TAX in hepatocytes. Since

287

ATP is a second signal of P2x7R-dependent LPS-driven NLRP3 inflammasome

288

activation, we next investigated whether TAX will interfere IL-1β production

289

stimulated by LPS plus ATP. LPS provides the first signal for induction of pro-IL-1β,

290

and ATP present a second signal in inflammasome activation to mimic LPS-driven

291

inflammatory response in ALD 11. P2x7R expression was low in normal HepG2 cells,

292

but dramatically increased with stimulation of LPS plus ATP (Fig 6A). Concomitantly

293

pro-IL-1β production and cleavage was also induced by LPS plus ATP stimulation (Fig

294

6A). Pretreatment of TAX prior to LPS/ATP stimulation successfully inhibited P2x7R

295

expression, as well as pro-IL-1β production. P2x7R inhibitor (A438079), caspase-1

296

inhibitor IV and TLR4 inhibitor (CLI-095) also significantly suppressed the protein

297

level of P2x7R. Moreover, caspase-1 inhibitor IV and A438079 partially decreased

298

pro-IL-1β production, while CLI-095 completely suppressed pro-IL-1β production.

299

These results suggested that inhibition effects of TAX on IL-1β production works

300

through P2x7R-NLRP3 inflammasome.

16



301

DISCUSSION

302

Alcoholic liver disease (ALD) presents a broad spectrum of disorders, ranging

303

from steatosis, alcoholic hepatitis and fibrosis, even to hepatocellular caner.

304

Managing ALD is currently in a limited pharmacotherapy stage 35. The current Food

305

and Drug administration (FDA)-approved medication for ALD is naltrexone, disulfiram,

306

prednisolone and pentoxifylline

307

modest survival gains or only reserved for severe ALD, and abstinence and nutritional

308

support still remain the first line of therapeutic intervention 38.

36, 37

. However, these treatments only enhanced

309

Flavonoids are presented in common food plant-based functional compounds 39.

310

There is experimental evidence that the natural flavonoids show therapeutic

311

potential in the treatment of alcoholic liver steatosis through inhibition of lipid

312

accumulation and inflammation in liver

313

demonstrated that TAX protected alcoholic liver steatosis both in vitro and in vivo.

314

We provided preliminary evidence that TAX administration prevented the

315

imbalanced lipid metabolism induced by ethanol intake. TAX supplementation

316

significantly inhibited AMPK-mediated lipid metabolism in hepatocytes by inhibiting

317

P2x7R-dependent inflammatory response. With dose of TAX (25 mg/kg) we didn’t

318

observe adverse effect on hepatic lipid accumulation and inflammation. According to

319

Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical

320

Trials for Therapeutics in Adult Healthy Volunteers (issued by U.S. Department of

321

Health and Human Services, Food and Drug Administration), a human equivalent

40-42

. The data from our research

17


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322

dose of TAX for starting Phase I trials converted from 25 mg/kg in mice would be

323

approximately 121.8 mg daily for a 60 kg man, which are feasible daily doses for

324

humans through dietary sources.

325

AMPK regulates the balance of lipid metabolism and activated by its upstream 43, 44

326

kinase, LKB1

. AMPK activation raises the lipid β-oxidation and conversely,

327

downregulates lipid synthesis. Currently, we investigated the effects of TAX on the

328

phosphorylation of AMPK and LKB1. Our data demonstrated that TAX remarkably

329

increased the activity of LKB1 and AMPK, indicating that its inhibition of lipid

330

accumulation probably linked to liver-specific activation of LKB1-AMPK. AMPK

331

phosphorylates and inhibits ACC, which is involved in lipid metabolism, while AMPK

332

also regulates SREBP1 activity, which controls lipid synthesis

333

SREBP1 expression and stimulated phosphorylated ACC in alcohol-induced mice

334

steatosis liver, suggesting that TAX modulated lipid metabolism by inhibiting lipid

335

biosynethesis and promoting lipid β-oxidation.

45

. TAX suppressed

336

Ethanol and its metabolites in liver can induce inflammatory responses. Also

337

chronic binge alcohol intake sensitizes hepatocytes to inflammatory signals and

338

impairs the biological function of hepatocytes 9. As shown in Fig 3A and 4B, IL-1β

339

secretion and IL-1β production was abolished by TAX application in alcohol-induced

340

steatotic liver and steatotic hepatocytes, suggesting ethanol sensitizes hepatocytes

341

to IL-1 signaling in response to stimuli. IL-1β maturation and secretion in alcoholic

342

liver steatosis was caspase-1-dependently processed

18


27

. In addition, NLRP3


343

inflammasome activation leads to severe liver inflammation and hepatocytes

344

pyroptosis, characterize by caspase-1 activation

345

NLRP3 inflammasome activation in livers of alcohol-treated mice supported by

346

increased levels of the cleaved caspase-1 and NLRP3 expression, while TAX

347

suppressed those to normal level, indicating that TAX inhibited inflammasome

348

activation in alcoholic liver steatosis, consequently suppressed IL-1β secretion to

349

avoid accelerating the inflammation.

46

. Ethanol consumption increased

350

IL-1β release via NLRP3 activation needs two-step signals, including the

351

synthesis of pro-IL-1β via TLRs and the cleavage of pro-IL-1β to biologically active,

352

mature IL-1β then release to extracellular, induced by PAMP or DAMP 47. Among the

353

DAMP, high levels of ATP acts as a second danger signal and mediated by P2x7R in

354

inflammasome activation 48. Currently several groups reported P2x7R promotes IL-1β

355

release NLRP3 inflammasome signaling-dependently in macrophages and neutrophils

356

50, 51

357

might be a potential target for liver fibrosis. Although P2x7R also is known as a key

358

modulator in nonalcoholic steatohepatitis

359

will regulate alcoholic steatohepatitis 12. Therefore, we are intrigued in whether TAX

360

regulates alcoholic liver steatosis via P2x7R suppression, especially in hepatocytes. As

361

expected ethanol increased P2x7R protein expression, which was suppressed by TAX

362

administration accompanied by decreased IL-1β secretion and caspase-1 activity (Fig

363

3), suggesting that TAX might be involved in the inhibition of NLRP3 inflammasome

364

activation through regulating P2X7R signaling.

. We also reported the inhibition of P2x7R-NLRP3 inflammasome activation

52, 53

, we reported that blockade of P2x7R

19


Interestingly, P2x7R gene

Page 20 of 41

Page 21 of 41


365

knockdown in HepG2 cells could restore the activity of AMPK, and decreased SREBP1

366

expression (Fig 5). It confirmed P2x7R activation is involved in the regulation of lipid

367

metabolism in ethanol-induced steatotic hepatocyte. With TAX supplementation,

368

AMPK activity further recovered in P2x7R deficient cells, indicating the inhibition of

369

lipid accumulation effect by TAX is partially dependent P2x7R signaling.

370

Hepatocytes present in a microenvironment with high level of bacterial LPS and

371

ATP in the development of alcoholic liver disease. The increase of serum ATP was

372

observed in alcohol-fed mice (Fig 1F) and ALD patients

373

hepatocytes release ATP, and these sterile danger signal promotes inflammasome in

374

LPS-primed immune cells. And increased gut permeability resulted in the increased

375

translocation of LPS from gut to the liver through the portal circulation

376

successfully inhibited the increasing serum ATP and LBP level in alcohol-induced

377

steatotic mice, suggesting that TAX might regulate TLR4 and P2X7R signaling. To

378

evaluate how hepatocytes will response to two-step signals stimulus with TAX

379

interference, we applied LPS for the ligand of TLR4 as a first signal and ATP for ligand

380

of P2x7R as a second signal to stimulate heaptocytes. Our data demonstrated that

381

two-step signal significantly activated P2x7R and induced IL-1β cleavages. All above

382

increasing evoked by two-step signal was abolished by pretreatment of TAX, A438079

383

(P2x7R inhibitor), caspase-1 inhibitor or CLI-095 (TLR4 antagonist). These results

384

hinted us TAX might be a pharmacological inhibitor of P2x7R and could target P2x7R

385

activation in hepatocytes.

20


27

. Alcohol-damaged

49

. TAX


386

In conclusion, our research demonstrated that TAX affects lipid synthesis and

387

lipid oxidation through the activity of AMPK, thereby inhibiting alcohol-induced lipid

388

accumulation in mice liver. And TAX has certain inhibitory effect on the activation of

389

P2x7R-caspase-1-NLRP3 inflammasome induced by alcohol, indicating that TAX might

390

be a potential candidate to treat alcoholic liver steatosis.

21


Page 22 of 41

Page 23 of 41


391

AUTHOR INFORMATION

392

Author Contribution

393

Ms. Yu Zhang and Dr. Quan Jin are the primary investigators in this study. Ms. Xia

394

Li and Ms. Min Jiang participated in part of in vivo experiments. Mrs. Ben-Wen Cui

395

and Ms. Kai-Li Xia participated in part of in vitro experiments. Dr. Yan-Ling Wu

396

participated in part of statistical analysis. Dr. Li-Hua Lian and Prof. Ji-Xing Nan

397

designed the whole study and wrote the manuscript.

398

Funding

399

This study was supported by a grant from the National Natural Science

400

Foundation of China (81560597, 81660689and 81460564), and partially by Science

401

and Technology Planning Projects from the Science and Technology Department of

402

Jilin

403

20180519010JH).

404

Conflict of Interest Statement

Province

(20160101205JC,

20180414048GH,

20180201065YY

and

405

The authors declare that the research was conducted in the absence of any

406

commercial or financial relationships that could be construed as a potential conflict

407

of interest.

408

22



409

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578

579

30



Page 32 of 41

580

Figure captions

581

Figure 1. TAX attenuates ethanol-induced lipid accumulation in mice liver. Mice

582

were gavaged with ethanol (5 g/kg) every 12 h for three times. TAX (1, 5 and

583

25mg/kg) was gavaged simultaneously with ethanol intake. (A) Chemical structure of

584

TAX. (B) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

585

(C) Serum and hepatic TG contents. Each value is expressed as the mean ± SD (n = 6).

586

HE (D, 200 × original magnification) and Oil red O staining (E, 400 × original

587

magnification) were performed with samples obtained at 4 h after the last ethanol

588

administration. (F) ATP level in mice serum. (G) Lipopolysaccharide (LPS)-Binding

589

Protein (LBP) protein levels in mice serum. ## p < 0.01, ### p < 0.001significantly

590

different when compared with normal group; * p

Modulation of AMPK-dependent lipogenesis mediated by P2x7R

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