Lychee (Litchi chinensis Sonn.) Pulp Phenolic Extract Confers a

Article

Lychee (Litchi chinensis Sonn.) pulp phenolic extract confers a protective activity against alcoholic liver disease in mice by alleviating mitochondrial dysfunction Juan Xiao, Ruifen Zhang, Lei Liu, Fei Huang, Yuanyuan Deng, Yongxuan Ma, zhencheng Wei, Xiaojun Tang, Yan Zhang, and Mingwei Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01844 • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 7, 2017

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Journal of Agricultural and Food Chemistry

Lychee (Litchi chinensis Sonn.) pulp phenolic extract confers a protective activity against alcoholic liver disease in mice by alleviating mitochondrial dysfunction

Juan Xiao, Ruifen Zhang, Fei Huang, Lei Liu, Yuanyuan Deng, Yongxuan Ma, Zhencheng Wei, Xiaojun Tang, Yan Zhang, Mingwei Zhang*

Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture/Guangdong Key laboratory of Agricultural Products Processing, Guangzhou 510610, China

*

Corresponding author: Mingwei Zhang

Tel: +86-20-8723 7865; Fax: +86-20-8723 6354; E-mail: [email protected]

The authors declare no competing financial interest.

1

ACS Paragon Plus Environment


1

Abstract

2

Mitochondria play an important role in the initiation and development of alcoholic

3

liver disease (ALD). Our previous studies found lychee pulp phenolic extract (LPPE)

4

exerted protective effect against ALD partly by inhibiting fatty acid β-oxidation, and

5

phenolic-rich lychee pulp extract improved restraint stress-induced liver injury by

6

inhibiting mitochondrial dysfunction. The aim of this study was to investigate

7

whether LPPE exerted protective effect against ALD via modulating mitochondrial

8

function, The mice were treated with an ethanol-containing liquid diet alone or in

9

combination with LPPE for 8 weeks. LPPE supplementation significantly alleviated

10

hepatic steatosis, suppressed serum aspartate aminotransferase activity, and

11

decreased triglyceride levels in serum and liver. Based on lipid peroxidation and

12

antioxidant enzyme analyses, LPPE supplementation inhibited serum and hepatic

13

oxidative stress. Moreover, LPPE supplementation significantly suppressed

14

mitochondrial 8-hydroxy-2’-deoxyguanosine level, and increased mitochondrial

15

membrane potential, mitochondrial DNA content, activities of mitochondrial

16

complexes I and IV, and hepatic ATP level. Furthermore, LPPE supplementation

17

significantly inhibited cytoplasmic cytochrome c level and caspase-3 activity,

18

repressed Bax expression and Bax/Bcl-2 ratio, and increased Bcl-2 expression in

19

liver. In summary, LPPE exerts beneficial effects against alcoholic liver injury by

20

alleviating mitochondrial dysfunction.

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Keywords: Lychee pulp phenolic extract; Alcoholic liver disease; Mitochondrial

22

dysfunction; Oxidative stress;

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Introduction

24

Long-term excessive alcohol consumption inevitably results in different levels of

25

alcoholic liver disease (ALD), which is a major cause of morbidity and mortality

26

worldwide

27

development of fatty liver, a reversible and benign condition 1. Excessive fat

28

accumulation increases the risk of progression to alcoholic steatohepatitis, ﬁbrosis,

29

and cirrhosis 1, 3.

1-2

. In the early stages of ALD, chronic alcohol consumption leads to the

30

Given their primordial roles in energy production, intermediary metabolism and

31

cell death processes, mitochondria play an important role in the initiation and

32

development of ALD 3-4. Mitochondrial dysfunction has long been regarded as one of

33

the earliest manifestations of ethanol-induced liver injury 5. Chronic ethanol feeding

34

promotes the excessive formation of mitochondrial reactive oxygen species (ROS),

35

which is the most important contributory factor for oxidative stress. The excessive

36

production of ROS and decreased mitochondrial reduced glutathione (GSH) level

37

induced by ethanol make mitochondria more susceptible to oxidative damage

38

Mitochondrial DNA (mtDNA) is a significant target for ethanol-induced oxidative

39

stress. Oxidative modifications of mtDNA have been observed in alcohol-fed animals

40

8-9

41

electron transport chain, resulting in the decreased activities of mitochondrial electron

42

transport chain complexes I, III, IV and V

43

responsible for decreased mitochondrial membrane potential and the onset of

44

mitochondria permeability transition 8. Extensive mitochondria permeability transition

6-7

.

. Such alterations are responsible for reducing mitochondria-encoded subunits of the

4, 9

. Oxidative mitochondrial damage is

3



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induces the release of mitochondrial cytochrome c into the cytoplasm, which in turn

46

induces the activation of caspases pathway and subsequently initiates the hepatocyte

47

apoptotic process

48

effectively slows the progression of ALD in animals

49

mitochondrial dysfunction in streptozotocin-induced diabetic mice and diet-induced

50

obese mice, suggesting that phenolics may ameliorate ALD by inhibiting hepatic

51

mitochondrial dysfunction

52

phenolics on hepatic mitochondrial dysfunction in ALD animals, although phenolics

53

have been demonstrated to protect against ALD in many studies 14-15.

10

. The prevention of mitochondrial dysfunction in the liver 11

. Phenolics prevent hepatic

12-13

. However, there is no literatures about the effect of

54

Lychee (Litchi chinensis Sonn.) is a subtropical fruit grown cultivated throughout

55

Southeast Asia with an attractive appearance, delicious taste and good nutritional

56

value.16-18 Recent studies have revealed that lychee pulp contains an abundance of

57

phenolic compounds 19-23. In our previous study, lychee pulp phenolic extract (LPPE)

58

exerts protective effect against ethanol-induced liver injury partly by inhibiting

59

oxidative stress and fatty acid β-oxidation

60

mitochondria in anti-oxidant defense and fatty acid β-oxidation, LPPE may exert

61

beneficial effects on mitochondrial function. Additionally, our group have also found

62

that phenolic-rich lychee pulp extract exhibits antioxidant activity in vitro and

63

hepatoprotective activity against restraint stress-induced liver injury in mice by

64

inhibiting oxidative stress and mitochondrial dysfunction

65

findings, we hypothesized that LPPE exerts beneficial effects on ALD by modulating

66

mitochondrial function.

24

. Given the important roles of

21, 25

. Based on previous

4


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In the present study, we investigated the dose-dependently protective effect of

68

LPPE against ALD in C57BL/6 mice fed an ethanol-containing liquid diet. The

69

potential mechanism associated with mitochondrial function was studied.

70

Materials and Methods

71

Materials and reagents. Fresh lychee (cv. Feizixiao) was purchased from a local fruit

72

market in Guangzhou, Guangdong, China. Procyanidin B2, (-)-epicatechin, rutin and

73

isorhamnestin-3-O-rutinoside were purchased from Sigma-Aldrich (St. Louis, MO,

74

USA). Quercetin 3-O-rutinoside-7-O-a-L-rhamnosidase (purity > 98%) was separated

75

as described in our previous study 22. Assay kits for alanine aminotransferase (ALT),

76

aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC),

77

thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD),

78

glutathione peroxidase (GSH-Px), catalase (CAT), 8-hydroxy-2’-deoxyguanosine

79

(8-OHdG), GSH, oxidized glutathione (GSSH), mitochondrial electron transport chain

80

complexes I and IV and cytochrome c were all obtained from Nanjing Jiancheng

81

Bioengineering Institute (Nanjing, Jiangsu, China). Lieber–DeCarli control and

82

ethanol liquid diets were purchased from TROPHIC Animal Feed High-Tech Co.

83

LTD (Nantong, Jiangsu, China).

84

Preparation and analyses of LPPE. Phenolic-rich lychee pulp extract was prepared

85

as we previously described

86

and applied onto a Toyopearl HW-40s column (250 mm × 50 mm I.D., Tosoh

87

Chemical Co., Tokyo, Japan). The column was eluted using 1% methanol (900 mL) to

88

remove low-molecular-mass impurities, followed by elution with methanol (1500 mL).

25

. The extract (600 mg) was dissolved in 1% methanol

5



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89

The eluant was concentrated and subsequently lyophilized to recover LPPE (357 mg)

90

24

91

with a calibration curve of rutin by the AlCl3-NaNO2 method

92

compositions were identified by HPLC-MS in our previous studies 26. The contents of

93

phenolic compositions of LPPE were determined by HPLC-DAD as we previously

94

described

95

3-O-rutinoside-7-O-a-L-rhamnosidase, rutin and isorhamnestin-3-O-rutinoside were

96

quantified with their own standard curves (mg/g). Other procyanidins and flavanone

97

glycosides were calculated as (-)-epicatechin equivalent (mg EE/g) and rutin

98

equivalent (mg RE/g), respectively.

. The total flavonoid content of LPPE was determined on the basis of comparison

25

.

Procyanidin

B2,

23

(-)-epicatechin,

. The phenolic

quercetin

99

The total flavonoid content of LPPE was 85.60 ± 3.98%. The HPLC phenolic

100

profiles of LPPE at 280 nm were shown in Fig. 1. The phenolic compositions and

101

their contents were presented in Table 1.

102

Animals and experimental design. The use of animals were approved by Animal

103

Ethical and Welfare Committee of Sun Yat-Sen University (approval no.

104

IACUC-DB-16-0302) and followed the Guiding Principles in the Care and Use of

105

Animals. Ten-week-old specific pathogen-free male C57BL/6 mice (26 ± 2 g) were

106

purchased from the Center of Laboratory Animal Science Research of Sun Yat-Sen

107

University (Guangzhou, Guangdong, China). The mice were housed in a specific

108

pathogen-free, environmentally controlled room with constant temperature (22 ±

109

1 °C), humidity (55-60%) and a 12-h light/12-h dark cycle.

110

During the one-week acclimation period, the mice were supplied with rodent 6


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chow diet and water ad libitum. The mice were randomly divided into four groups (n

112

= 10 per group) and provided a control liquid diet (control group, CON), a 4% (w/v)

113

ethanol-containing liquid diet (ethanol group, EtOH), a 4% (w/v) ethanol-containing

114

liquid diet supplemented with 0.2 g/L LPPE (low-dose LPPE-supplemented group,

115

EtOH+L-LPPE), or a 4% (w/v) ethanol-containing liquid diet supplemented with 0.4

116

g/L LPPE (high-dose LPPE-supplemented group, EtOH+H-LPPE) for 8 weeks. The

117

animals were housed two per cage. The liquid diet provides 1 kcal/mL based on the

118

Lieber–DeCarli formulation, and 35% of the calories are derived from fat, 19% from

119

carbohydrate,

120

ethanol-containing liquid diet) or isocaloric maltose dextrin (control liquid diet). The

121

CON group was pair-fed with the EtOH group, and the other groups were fed ad

122

libitum. All diets were freshly prepared from powder and provided daily at 5:00 p.m.

123

Body weight and caloric intake were monitored weekly and daily, respectively.

18%

from

protein,

and

28%

from

ethanol

(4%

(w/v)

124

The mice were euthanized through inhalation with ether after fasting for 12 h.

125

Blood samples were collected and centrifuged at 3000g for 10 min at 4 ºC to obtain

126

serum. After the mice were sacrificed, the livers were immediately removed, washed,

127

weighed and cut into many portions. One portion was fixed in 4% paraformaldehyde

128

for hematoxylin and eosin (H&E) staining analysis. Additional portions were used to

129

prepare fresh mitochondria and cytoplasm. The remaining portions were flash-frozen

130

in liquid nitrogen and subsequently stored at -80 °C.

131

Measurement of ALT and AST activities in the serum. ALT and AST activities in

132

the serum were determined using commercial kits. All biochemical indices were 7



Page 8 of 40

133

measured using an Infinite® M200 PRO plate reader (Tecan Austria GmbH, Grödig,

134

Salzburg-Umgebung, Austria).

135

Measurement of TG and TC levels in the serum and liver. Serum TG and TC

136

levels were colorimetrically determined using commercial kits. Total lipids were

137

extracted from liver homogenates using a chloroform/methanol mixture (2:1, v/v), 27

138

and TG and TC levels in total lipids were measured using commercial kits.

139

Liver histopathology. Liver histopathology was assessed via H&E staining and

140

oil-red O staining following a standard procedure. Briefly, paraffin sections (5

141

µm-thick) were cut, deparaffinized in xylene, rehydrated in alcohol gradients, and

142

subsequently stained with H&E. Frozen sections (5 µm-thick) were cut, stained with

143

oil-red O, washed and counterstained with hematoxylin. Stained sections were

144

observed

145

Baden-Württemberg, Germany). Semi-quantification of oil-red O staining was

146

performed by Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

147

Determination of lipid peroxidation and antioxidant enzymes in the serum and

148

liver. Frozen liver samples were homogenized with chilled normal saline in an ice

149

bath. The 10% (w/v) homogenates were centrifuged at 3000g for 10 min at 4 °C. The

150

supernatant and serum were used for TBARS, SOD, GSH-Px and CAT tests. All

151

parameters were measured using commercial kits according to the manufacturer’s

152

instructions.

153

Preparation of fresh liver mitochondria and cytoplasm. Mitochondria were

154

immediately isolated from fresh liver samples as previously described using a

using

a

light

microscope

(Leica

DMI

4000B,

Heidelberger,

8


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155

commercial mitochondrial fractionation isolation kit (Beyotime Institute of

156

Biotechnology, Shanghai, China)

157

isolation buffer containing 1 mM PMSF using a dounce homogenizer (Kimble,

158

Chicago, Illinois, USA) in an ice bath. The homogenates were centrifugated at 600g

159

for 5 min at 4 °C, and subsequently the supernatants were centrifuged at 11000g for

160

10 min at 4 °C. The precipitates were used as the mitochondrial fractions, and the

161

supernatants were used to isolate the cytoplasm fractions by centrifuging at 15000g

162

for 10 min at 4 °C. The mitochondrial fractions were suspended in preserving

163

solution. Fresh mitochondrial fractions were used to assess the mitochondrial

164

membrane potential. The remaining mitochondrial fractions and the cytoplasmic

165

fractions were aliquoted and stored at -80 °C. Mitochondrial quantification was

166

performed by quantifying the protein content using a Bradford Protein Assay Kit

167

(Beyotime Institute of Biotechnology, Shanghai, China).

168

Measurement of hepatic mitochondrial oxidative stress. Mitochondrial 8-OHdG

169

level. mtDNA was isolated using a Mitochondrial DNA Isolation Kit (Abcam,

170

Cambridge, England, UK), and subsequently prepared using previously published

171

methods to obtain the nucleoside samples

172

determine 8-OHdG level using an 8-OHdG ELISA Kit according to the

173

manufacturer’s instructions. Mitochondrial 8-OHdG content was determined from a

174

standard curve, normalized to the total mtDNA concentration, and subsequently

175

expressed as pg per µg mtDNA (pg/µg mtDNA).

176

25

. Briefly, fresh livers were homogenized in

28

. The nucleoside samples were used to

Mitochondrial GSH and GSSH levels. Mitochondrial GSH and GSSH levels were 9



Page 10 of 40

177

measured using a GSH/GSSH kit. Briefly, the supernatant of liver mitochondrial

178

lysate was treated with trichloroacetic acid to extract total glutathione and GSSH, and

179

subsequently centrifuged to remove denatured protein. Total glutathione and GSSH

180

were determined in the supernatant using the previously described recycling

181

enzymatic method of Tietze

182

between the total glutathione content and two-fold GSSH content. The content was

183

expressed as nmol per mg protein (nmol/mg prot), and the GSH/GSSG ratio was

184

calculated.

185

Measurement of hepatic mitochondrial function. mtDNA content quantification.

186

mtDNA content quantification was quantified according to Ahn et al with some

187

modifications 30. Briefly, mtDNA and nuclear DNA were extracted from fresh livers

188

using a Mitochondrial DNA Isolation Kit (Abcam, Cambridge, England, UK) and a

189

DNeasy kit (QIAGEN, Hilden, Nordrhein-Westfalen, Germany), respectively. The

190

relative amounts of mtDNA and nuclear DNA were determined by quantitative

191

real-time PCR (qRT-PCR). Ct values were measured for the mtDNA-encoded ND1

192

and COXI genes and the nuclear DNA-encoded GAPDH gene. The relative

193

expression levels of the ND1 and COXI genes were calculated by the 2-△△CT method,

194

and the results were normalized to GAPDH. The mtDNA content was expressed as

195

the relative expression of the ND1 and COXI genes. The following primer sequences

196

were used: GAPDH, forward, 5’-GGAGAAACCTGCCAAGTATGATGAC-3’,

197

reverse,

198

GGTCCATACGGCATCCTACAACC-3’,

29

. GSH content was calculated from the difference

5’-GAGACAACCTGGTCCTCAGTGTA-3’;

ND1, reverse,

forward,

5’5’10


Page 11 of 40


199

AGTGTGAGTGATAGGGTGGGTGC-3’;

200

GCCCACTTCGCCATCATATTCGT

201

5’-CTGGGTAGTCTGAGTAGCGTCGT-3’.

and

COXI, -3’,

forward:

5’reverse,

202

Activities of mitochondrial electron transport chain complexes I and IV. The

203

activities of mitochondrial electron transport chain complexes I and IV were

204

determined for the supernatant of liver mitochondrial lysate using previously

205

published methods

206

changes during the oxidation of NADH to NAD+ at 340 nm using a commercial

207

Complex I Enzyme Activity Assay kit. Complex IV activity was quantified by

208

measuring the oxidation of reduced cytochrome c using a commercial Cytochrome c

209

Oxidase Assay Kit. Enzyme activities were expressed as nmol of substrate used per

210

minute per mg protein (nmol/min/mg prot).

31

. Complex I activity was determined based on the colorimetric

211

Mitochondrial Membrane Potential. Mitochondrial membrane potential was

212

measured using a commercial Mitochondrial Membrane Potential Assay Kit with

213

JC-1 (Beyotime Institute of Biotechnology, Shanghai, China) following the

214

manufacturer’s protocol. JC-1 is a cationic dye that accumulates in mitochondria with

215

high membrane potential. After accumulation in mitochondria, JC-1 forms

216

J-aggregates that emit red fluorescence. Briefly, the fresh mitochondrial fraction was

217

stained with JC-1 solution for 10 min at 37 °C and washed twice with the dyeing

218

buffer provided by the kit. Fluorescence was detected at 485/590 nm for J-aggregates

219

using an Infinite® M200 PRO plate reader in fluorescence detection mode (Tecan

220

Austria GmbH, Grödig, Salzburg-Umgebung, Austria). The results were expressed as 11



221 222

Page 12 of 40

the fold-change based on the fluorescence of the CON group. Hepatic ATP level. Hepatic ATP level was measured according to previously 32

223

published methods

224

Biotechnology, Shanghai, China). Briefly, liver tissues were homogenized in lysis

225

buffer using a potter type homogenizer in an ice bath. ATP level in the supernatant

226

was determined following the manufacturer’s instructions. Hepatic ATP level was

227

expressed as nmol per mg protein (nmol/mg prot).

228

Measurement of cytochrome c content in the liver cytoplasm. Cytochrome c

229

content was determined in the cytoplasmic fraction as previously described

230

Mouse Cytochrome c ELISA Assay Kit following the manufacturer’s protocol. The

231

content was expressed as ng per mg protein (ng/mg prot).

232

Measurement of hepatic caspase-3 activity. Caspase-3 activity was measured as

233

previously described

234

Institute of Biotechnology, Shanghai, China) following the manufacturer’s

235

instructions. Caspase-3 activity was evaluated by enzymatic cleavage of chromophore

236

p-nitroanilide from the substrate N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide at 405 nm.

237

The activity was expressed as pmol of substrate used per minute per mg protein

238

(pmol/min/mg prot).

239

qRT-PCR. Total RNA was isolated from liver samples using Trizol reagent

240

(Invitrogen, Carlsbad, CA, USA), and reverse-transcribed on a B960 real-time

241

thermocycler (Hangzhou Jingle Scientific Instruments Co., Ltd., Hangzhou, Zhejiang,

242

China) using a Reverse Transcriptase M-MLV (RNase H-) (Vazyme Biotech Co., Ltd.,

34

using a commercial ATP Assay Kit (Beyotime Institute of

33

using a

using a commercial Caspase-3 Activity Assay Kit (Beyotime

12


Page 13 of 40


243

Nanjing, Jiangsu, China). The synthesized cDNA was stored at –20 °C. qRT-PCR was

244

conducted on an ABI ViiA 7 Detection System (Applied Biosystems, Foster City,

245

CA,USA) using an AceQ® qPCR SYBR® Green Master Mix (Vazyme Biotech Co.,

246

Ltd., Nanjing, Jiangsu, China). Each sample was assessed in triplicate, and normalized

247

to GAPDH. The relative expression levels of the genes were calculated by the 2-△△CT

248

method as previously described, and presented as a ratio of the treatment group to the

249

CON group 33. The following primer sequences were used (Sangon Biotech (Shanghai)

250

Co.,

251

5’-GGAGAAACCTGCCAAGTATGATGAC-3’,

252

5’-GAGACAACCTGGTCCTCAGTGTA-3’;

253

CAGGATGCGTCCACCAAGAAGC

254

GTCCGTGTCCACGTCAGCAATC-3’;

255

CCTGAACTTGCGTGAAGGCTTGA

256

GCCACACCCAAACATCCAGAGAC -3’.

257

Western blot analysis. Total protein was extracted from frozen livers using a RIPA

258

buffer

259

phenylmethylsulfonyl fluoride. Protein concentrations were measured by the BCA

260

assay. Total protein was loaded onto a 10% SDS-polyacrylamide gel, transferred onto

261

a polyvinylidene difluoride membrane (0.45 µm, Merck Millipore, Darmstadt,

262

Hesse-Darmstadt, Germany), and blocked with 5% skim milk, followed by

263

immunostaining with primary antibodies against Bax or Bcl-2 (1:1000, Cell Signaling

264

Technology,

Ltd.,

Shanghai,

supplemented

Danvers,

with

MA,

China)

1%

USA).

:

GAPDH,

reverse, Bax,

-3’, and

After

forward,

5’-

reverse, Bcl-2,

-3’,

protease

forward,

inhibitor

incubation

5’-

forward: reverse,

cocktail

with

a

5’5’-

and

1%

horseradish 13



Page 14 of 40

265

peroxidase-conjugated secondary antibody (1:10000, Tianjin Sungene Biotech,

266

Tianjin, China), immunoreactive proteins were stained with ECL substrate from the

267

Fast Western Blot Kit (Pierce, Rockford, IL, USA) and subsequently exposed to a

268

film. The film was scanned using a Plustek SW500 scanner (Plustek, Taiwan, China),

269

and the band intensities were measured using Quantity One 1-D analysis software

270

(Bio-Rad, Hercules, CA, USA). β-actin (Tianjin Sungene Biotech, Tianjin, China)

271

was used as an internal standard.

272

Statistical analysis. The data were expressed as means ± standard deviation (SD). All

273

data were analyzed using one-way ANOVA, followed by Duncan post hoc test.

274

Statistical analyses were performed using SPSS 16.0 software, and p < 0.05 was

275

regarded as statistical significance.

276

Results

277

Effects of LPPE on general parameters. There were no significant differences in the

278

initial body weight, final body weight or total caloric intake among the four groups

279

(p > 0.05) (Table 2). Both L-LPPE and H-LPPE supplementation reversed the

280

ethanol-induced increase in the liver-to-body weight ratio, indicating that LPPE

281

supplementation alleviated the ethanol-induced liver swelling.

282

Effects of LPPE on liver histopathology. As shown in Fig. 2, ethanol feeding

283

resulted in liver damage characterized by the irregular arrangement of hepatocytes and

284

extensive fat droplets in the hepatocytes compared with the CON group. The vacuoles

285

in H&E-stained sections and aggregation of orange dye in oil-red O-stained sections

286

reflected the fat droplets in the hepatocytes. In contrast to the EtOH group, fewer and 14


Page 15 of 40


287

smaller hepatocytes fat droplets were observed in both LPPE-supplemented groups.

288

Semi-quantification of oil-red O staining showed that fat droplets were 15.73-fold

289

more in the liver of the EtOH group compared with the CON group (p < 0.05).

290

However, fat droplets of the L-LPPE and H-LPPE supplemented groups were

291

decreased by 73.81% and 92.84%, respectively, compared with that of the EtOH

292

group (p < 0.05). These results indicated that LPPE supplementation alleviated

293

ethanol-induced hepatic steatosis.

294

Effects of LPPE on serum biomarkers of hepatic function. Ethanol feeding

295

resulted in significant increases in serum ALT and AST activities (1.40-fold and

296

1.26-fold, respectively, p < 0.05) compared with the CON group (Fig. 3). However,

297

compared with the EtOH group, L-LPPE supplementation remarkably decreased

298

serum AST activity by 22.89% (p < 0.05), and had no obvious effects on serum ALT

299

activity. H-LPPE supplementation normalized both AST and ALT activities.

300

Moreover, serum ALT activity of EtOH+L-LPPE group was 1.42-fold higher than

301

that of EtOH+H-LPPE group (p < 0.05). Thus, LPPE supplementation alleviated

302

ethanol-induced liver injury in a dose-dependent manner.

303

Effects of LPPE on serum and liver lipid profiles. Compared with the CON group,

304

serum and hepatic TG levels of the EtOH group were remarkably increased by

305

25.79% and 39.23% (p < 0.05). However, compared with the EtOH group, L-LPPE

306

supplementation notably decreased serum and hepatic TG levels by 25.22% and

307

11.70%, respectively (p < 0.05) (Fig. 3). H-LPPE supplementation remarkably

308

decreased serum and hepatic TG levels by 24.02% and 26.56%, respectively (p < 15



Page 16 of 40

309

0.05). Hepatic TG level was decreased in a dose-dependent manner in the

310

LPPE-supplemented groups compared with the EtOH group (p < 0.05). There were

311

no significant differences in serum and hepatic TC levels among the four groups.

312

Effects of LPPE on ethanol-induced serum and hepatic oxidative stress. TBARS

313

level and SOD, GSH-Px and CAT activities in the serum and liver were measured to

314

investigate changes in oxidative stress. Additionally, as an index of oxidative stress,

315

the ratio of TBARS to SOD (TBARS/SOD ratio) was also calculated. Ethanol feeding

316

significantly increased TBARS level and the TBARS/SOD ratio, and significantly

317

decreased SOD, GSH-Px and CAT activities in the serum and liver compared with the

318

CON group (Table 3). However, compared with the EtOH group, L-LPPE

319

supplementation significantly decreased the TBARS/SOD ratio in the serum and liver,

320

increased SOD and CAT activities in the serum, and improved hepatic SOD and

321

GSH-Px

322

ethanol-induced changes in these indices. The EtOH+H-LPPE group exhibited a

323

significantly lower TBARS/SOD ratio and higher CAT activity in the liver than the

324

EtOH+L-LPPE group. Thus, LPPE supplementation alleviated ethanol-induced

325

oxidative stress in a dose-dependent manner.

326

Effects of LPPE on ethanol-induced mitochondrial oxidative damage.

327

Mitochondrial 8-OHdG, a marker of oxidatively damaged mtDNA, was 1.25-fold

328

higher in the liver of the EtOH group compared with the CON group (p < 0.05) (Fig.

329

4). In addition, significantly decreased mitochondrial GSH level (0.61-fold, p < 0.05)

330

and mitochondrial GSH/GSSG ratio (0.54-fold, p < 0.05) were observed in the liver of

activities.

H-LPPE

supplementation

significantly

reversed

the

16


Page 17 of 40


331

the EtOH group. However, both L-LPPE and H-LPPE supplementation normalized

332

ethanol-induced changes in mitochondrial 8-OHdG level, mitochondrial GSH level

333

and mitochondrial GSH/GSSG ratio in the liver, indicating the ameliorative effects of

334

LPPE supplementation on ethanol-induced mitochondrial oxidative stress.

335

Effects of LPPE on ethanol-induced mitochondrial dysfunction. mtDNA content

336

was expressed as the relative expression of the mtDNA-encoded ND1 (complex I

337

subunit) and COXI (complex IV subunit) genes by qRT-PCR. Consistent with

338

oxidative damage driving the exacerbation of mtDNA damage, the EtOH group,

339

which exhibited higher mitochondrial 8-OHdG level, had lower mtDNA content in

340

the liver compared with the CON group (p < 0.05) (Fig. 5). Both LPPE-supplemented

341

groups exhibited a significant increase in mtDNA content compared with the EtOH

342

group (p < 0.05).

343

The activities of mitochondrial electron transport chain complexs I and IV in the

344

liver of the EtOH group were decreased by 53.92% and 51.79%, respectively,

345

compared with those of the CON group (Fig. 5). Both L-LPPE and H-LPPE

346

supplementation significantly increased the complex I activity by 81.00% and

347

100.00%, respectively, compared with the EtOH group (p < 0.05). In addition,

348

complex IV activity was increased by LPPE supplementation in a dose-dependent

349

manner compared with the EtOH group (p < 0.05).

350

Moreover, the ethanol-induced decreases in mitochondrial membrane potential and

351

hepatic ATP level were significantly improved by both L-LPPE (1.59-fold and

352

2.58-fold, respectively) and H-LPPE supplementation (1.39-fold and 1.88-fold, 17



Page 18 of 40

353

respectively) compared with the EtOH group (p < 0.05).

354

Effects of LPPE on the ethanol-induced mitochondrial pathway of hepatocyte

355

apoptosis. The release of cytochrome c from mitochondria into the cytoplasm

356

initiates the mitochondrial pathway of hepatocyte apoptosis. Cytochrome c content in

357

the liver cytoplasm was 1.41-fold higher in the EtOH group compared with the CON

358

group (p < 0.05) (Fig. 6). Both L-LPPE and H-LPPE supplementation remarkably

359

decreased cytochrome c content in the liver cytoplasm by 31.56% and 42.96%,

360

respectively, compared with the EtOH group (p < 0.05).

361

Released cytochrome c is one of the upstream signals for caspase-3 activation.

362

Caspase-3 is the crucial initiating molecule in apoptosis. As shown in Fig. 6, the

363

EtOH group exhibited 3.30-fold higher caspase-3 activity in the liver compared with

364

the CON group (p < 0.05), indicating ethanol-induced caspase-3 activation. Hepatic

365

caspase-3 activity decreased with LPPE supplementation in a dose-dependent manner

366

compared with the EtOH group (p < 0.05).

367

Bcl-2 family proteins regulate mitochondrial outer membrane permeability through

368

mechanisms that are still not fully understood, resulting in the release of cytochrome c.

369

The Bax/Bcl-2 ratio is vital for regulating mitochondrial cytochrome c release, and is

370

typically used as an indicator of cell apoptosis. Bcl-2 and Bax expression was

371

measured by qRT-PCR and western blotting, and the Bax/Bcl-2 ratio was calculated.

372

Compared with the CON group, Bax mRNA (2.41-fold) and protein levels (1.86-fold)

373

were significantly increased in the liver of the EtOH group (p < 0.05). In contrast,

374

Bcl-2 mRNA (0.57-fold) and protein levels (0.65-fold) were significantly decreased in 18


Page 19 of 40


375

the liver of the EtOH group (p < 0.05). The Bax/Bcl-2 ratio was significantly elevated

376

at both the mRNA (4.21-fold) and protein levels (2.91-fold) following ethanol

377

exposure compared with the CON group (p < 0.05). However, LPPE supplementation

378

decreased Bax expression and the Bax/Bcl-2 ratio, and increased Bcl-2 expression at

379

both the mRNA and protein levels in a dose-dependent manner compared with the

380

EtOH group (p < 0.05).

381

Discussion and conclusions

382

Given the prevalence of alcohol consumption and alcohol-induced risks to health in

383

modern

society,

studies

have

focused

on

intervention

strategies

against

384

alcohol-induced injuries

385

consumption occurs in the liver, which is the major organ that metabolizes alcohol 5.

386

Abnormal hepatic function indices (AST and ALT) and lipid profiles have been

387

observed in alcohol-treated animals

388

increases in the liver-to-body weight ratio, serum AST activity and serum TG level

389

were ameliorated by LPPE supplementation. In addition, LPPE supplementation

390

decreased serum ALT activity and liver TG level in a dose-dependent manner. Thus,

391

LPPE alleviated ethanol-induced liver injury in a dose-dependent manner.

1-2

. The most important injury resulting from alcohol

11,14-15

. In the present study, ethanol-induced

392

Ethanol-induced oxidative stress is closely associated with the initiation and

393

progression of ALD 4. Excess ROS accumulation directly induces the oxidative

394

modifications of proteins, lipid and nucleic acids

395

mitochondrial dysfunction and cell apoptosis, which promote the development of

396

ALD. In the present study, L-LPPE and H-LPPE supplementation inhibited

36

. This oxidative damage causes

19



Page 20 of 40

397

ethanol-induced oxidative stress, evidenced by decreased TBARS level and

398

TBARS/SOD ratio, and increased antioxidant enzyme activity in the serum and liver

399

compared with the EtOH group. In a previous study, we demonstrated the ability of

400

phenolic-rich lychee pulp extract to alleviate restraint stress-induced liver injury in

401

mice via the inhibition of oxidative stress 25. The LPPE used in the present study was

402

a mixture of 11 types of phenolics, which were identified by HPLC-MS in our

403

previous studies

404

44.80% of the all of the quantified individual compounds, has been demonstrated to

405

possess potent antioxidant activity in vitro 23. The antioxidant activities of procyanidin

406

B2, rutin and (-)-epicatechin, another three main phenolic components in LPPE, have

407

been elucidated in animals

408

ethanol-induced oxidative stress in this study. Phenolic-rich extracts from mulberry

409

and coca also exert hepatoprotective effects in ethanol-fed animals by inhibiting

410

oxidative stress, consistent with our findings 14, 40.

26

. Quercetin 3-O-rutinoside-7-O-a-L-rhamnosidase, accounted for

37-39

. Thus, LPPE showed the inhibitive effects on

411

The mitochondrial electron transport chain is a major source of ROS in cells.

412

Therefore, mitochondria play a key role in alcohol-induced oxidative stress, and

413

mitochondria are the specific targets of oxidative stress

414

mtDNA to the source of ROS makes mtDNA more vulnerable to oxidative damage;

415

therefore, mtDNA is a sensitive marker of overall mitochondrial oxidative stress 4, 8, 30.

416

8-OHdG levels in mtDNA indicate the severity of oxidative mtDNA damage

417

Mitochondrial GSH level is also regulated by oxidative stress. The GSH/GSSG ratio

418

is frequently used as an indicator of oxidative stress

4,6-8

. The close proximity of

9, 31

.

7, 32

. In the present study, LPPE 20


Page 21 of 40


419

supplementation reversed ethanol-induced changes in mitochondrial 8-OHdG level,

420

mitochondrial GSH level and mitochondrial GSH/GSSG ratio in the liver. These

421

observations are consistent with our previous study in which treatment with

422

phenolic-rich lychee pulp extract reduced mitochondrial ROS generation in the liver

423

of restraint-stressed mice

424

oxidative stress may be a key mechanism involved in the protective effects of LPPE

425

against ethanol-induced liver injury.

25

. Based on these results, the inhibition of mitochondrial

426

Mitochondrial oxidative damage induces a decline in mitochondrial membrane

427

potential and the onset of mitochondria permeability transition, which are

428

characteristic markers of mitochondrial dysfunction 4, 6, 8. In addition to mitochondrial

429

membrane potential level, mtDNA content, mitochondrial electron transport chain

430

complexe activity and ATP level are frequently determined to assess the

431

mitochondrial function

432

transport chain complexes I, III, IV and V, is responsible for the activities of these

433

complexes 9. ATP is synthesized in the mitochondria, and its level reflects the

434

function of mitochondrial energy metabolism

435

by ethanol has been demonstrated in many studies

436

present study, ethanol feeding led to significant mitochondrial dysfunction, evidenced

437

by decreased mitochondrial membrane potential level, hepatic ATP level, mtDNA

438

content and the activities of complexes I and IV. Importantly, the abovementioned

439

indices of mitochondrial function were improved by LPPE supplementation. Thus,

440

LPPE exerted potent protective effects against ethanol-induced liver injury by

9, 11, 32, 41

. mtDNA, which encodes components of electron

42

. Mitochondrial dysfunction induced 9, 11, 32, 42

. As expected, in the

21



441

Page 22 of 40

alleviating mitochondrial dysfunction. Mitochondrial dysfunction is a central regulatory mechanism of ethanol-induced

442

4, 10

443

hepatocyte apoptosis

. The mitochondrial membrane potential plays a key role in

444

the normal mitochondrial function and control of mitochondria permeability transition

445

8

446

cytochrome c into the cytoplasm, which is a recognized landmark event in

447

mitochondrial pathway of hepatocyte apoptosis. The release of cytochrome c from

448

mitochondria into the cytoplasm activates caspases pathway and leads to hepatocyte

449

apoptosis 10. Caspase-3 is the crucial initiating molecule in apoptosis. The Bax/Bcl-2

450

ratio is vital for regulating mitochondrial cytochrome c release, and is typically used

451

as an used as the indicator of apoptosis

452

induced the release of cytochrome c, the activation of caspase-3 and an increase in the

453

Bax/Bcl-2 ratio, consistent with previous studies 8, 32. Importantly, LPPE treatment

454

ameliorated the ethanol-induced mitochondrial pathway of hepatocyte apoptosis,

455

indicated by decreased cytoplasmic cytochrome c level, caspase-3 activity and

456

Bax/Bcl-2 ratio. As we all known, released cytochrome c is one of the upstream

457

signals for caspase-3 activation. In this study, there was no distinct difference in the

458

content of cytoplasmic cytochrome c between CON and EtOH+L-LPPE groups, while

459

the activity of caspase-3 between CON and EtOH+L-LPPE groups was significantly

460

different. Besides the released cytochrome c, the released smac/DIABLO and

461

Omi/HtrA2 from mitochondria also regulate the activation of caspase-3

462

EtOH+L-LPPE group, the activity of caspase-3 may be resulted from not only

. Extensive mitochondria permeability transition induces the release of mitochondrial

43

. In the present study, ethanol feeding

44-46

. In

22


Page 23 of 40


463

released cytochrome c but also released smac/DIABLO and Omi/HtrA2. Thus,

464

inhibition of the mitochondrial pathway of hepatocyte apoptosis may be a key

465

mechanism underlying the protective effects of LPPE against ethanol-induced liver

466

injury.

467

Based on the above results, LPPE confers protection against ethanol-induced liver

468

injury in mice by inhibiting serum and hepatic oxidative stress, and suppressing

469

mitochondrial oxidative stress, mitochondrial dysfunction and subsequent hepatocyte

470

apoptosis in the liver. Phenolic-rich lychee pulp extract exerts antioxidant activities in

471

vitro and in vivo

472

3-O-rutinoside-7-O-a-L-rhamnosidase, procyanidin B2, rutin and (-)-epicatechin,

473

which exhibit significant antioxidant activities in vitro and in vivo 23, 37-39. LPPE may

474

scavenge excessive ROS and increase antioxidant defenses by increasing antioxidant

475

enzyme activity in the liver and throughout the entire body. Consequently, LPPE

476

treatment inhibited oxidative damage to the hepatic mitochondria and alleviated the

477

mitochondrial dysfunction and hepatocyte apoptosis. Thus, the protective effects of

478

LPPE on ethanol-induced liver injury are closely associated with its antioxidant

479

activities.

19, 21, 25

. The major phenolic components in LPPE are quercetin

480

In summary, LPPE exhibited the ameliorative effects on ALD in mice in a

481

dose-dependent manner. The potential mechanism involved was associated with the

482

inhibition of serum and hepatic oxidative stress, and the repression of hepatic

483

mitochondrial oxidative stress, mitochondrial dysfunction and hepatocyte apoptosis.

484

Based on these findings, the intake of LPPE or lychee pulp may be useful to prevent 23



Page 24 of 40

485

and control ALD.

486

Acknowledgements

487

This work was supported by a Joint Fund from the NSFC and Guangdong Provincial

488

Government (U1301211), the National Nature Science Foundation of China

489

(31501478, 31571828), the PhD Start-up Fund of the Natural Science Foundation of

490

Guangdong (2014A030310328), the China Postdoctoral Science Foundation

491

(2016M590764), and the Guangdong Provincial Science and Technology Project

492

(2016B070701012, 2016A050503034).

493

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Figure captions

Fig. 1 HPLC profile of lychee pulp phenolic extract (LPPE) at 280 nm. Peak 1, Procyanidin B2; Peak 2, (-)-epicatechin; Peak 3, A-type procyanidin trimer; Peak 4, B-type procyanidin trimer; Peak 5, Quercetin 3-O-rutinoside-7-O-a-L-rhamnosidase; Peak 6, B-type procyanidin dimer; Peak 7, Kaempferol rhamnosyl-rutinoside; Peak 8, Rhamnetin rhamnosyl-rutinosede; Peak 9, Isorhamnetin rhamnosyl-rutinosede; Peak 10, Rutin; Peak 11, Isorhamnestin-3-O-rutinoside.

Fig. 2 Effects of LPPE on liver histopathology in ethanol-induced liver injured mice. (A) H&E staining (200×, 400×); (B) oil-red O staining (200×). Data are presented as the mean ± SD (n = 9). The data with different superscripts indicate significantly different (p < 0.05). CON, mice fed a control liquid diet; EtOH, mice fed an ethanol-containing liquid diet; EtOH+L-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.2 g/L LPPE; and EtOH+H-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.4 g/L LPPE.

Fig. 3 Effects of LPPE on ethanol-induced liver injury in mice. (A) Enzymatic activity of AST and ALT in the serum; (B) Serum TC and TG content; (C) Liver TC and TG content. The data are presented as the mean ± SD (n = 10). The data with different superscripts indicate significant differences (p < 0.05). CON, mice fed a control liquid diet; EtOH, mice fed an ethanol-containing liquid diet; EtOH+L-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.2 g/L LPPE; and EtOH+H-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.4 g/L LPPE. AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglyceride; TC, total cholesterol. 29



Page 30 of 40

Fig. 4 LPPE supplementation ameliorates ethanol-induced mitochondrial oxidative stress in the livers of mice. (A) Mitochondrial 8-OHdG content normalized to the total mitochondrial DNA (mtDNA); (B) mitochondrial GSH content; (C) mitochondrial GSSH content; (D) mitochondrial GSH/GSSH ratio. The data are presented as the mean ± SD (n = 10). The data with different superscripts indicate significant differences (p < 0.05). 8-OHdG, 8-hydroxy-2’-deoxyguanosine level; GSH, reduced glutathione; GSSH, oxidized glutathione.

Fig. 5 LPPE supplementation ameliorates ethanol-induced mitochondrial dysfunction in the livers of mice. (A) mtDNA content was determined by measuring the expression levels of the mtDNA-encoded ND1 (complex I subunit) and COXI (complex IV subunit) genes relative to the expression level of the nuclear DNA gene (GAPDH) using qRT-PCR; (B) enzymatic activities of complexes I and IV of the mitochondrial electron transport chain; (C) mitochondrial membrane potential; (D) hepatic ATP content. The data are presented as the mean ± SD. The data with different superscripts indicate significant differences (p < 0.05).

Fig. 6 LPPE supplementation ameliorates ethanol-induced hepatocyte apoptosis in mice. (A) Cytoplasmic cytochrome c content; (B) caspase-3 activity; (C) qRT-PCR analysis of the mRNA levels of Bax and Bcl-2 in the liver (n = 4) relative to the expression of GAPDH; (D) representative western blot of Bax and Bcl-2 protein levels in the liver (n = 4). The data are presented as the mean ± SD. The data with different superscripts indicate significant differences (p < 0.05).

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Table 1 Contents of phenolic compositions in LPPE Peak no.

Retention time (min)

λ max (nm)

Compound 26

Content

Content of each peak/

total content of 11 peaks (%)

1

20.12

230, 279

Procyanidin B2

104.98 ± 3.11 a

2

22.47

232, 279

(-)-epicatechin

34.91 ± 1.20

3

23.99

230, 279

A-type procyanidin trimer

33.96 ± 0.96 b

5.84

4

24.75

230, 279

B-type procyanidin trimer

6.36 ± 0.21 b

1.09

5

25.77

255, 352

Quercetin 3-O-rutinoside-7-Oa-L-rhamnosidase

260.49 ± 9.21 a

44.80

6

27.53

279, 322

B-type procyanidin dimer

4.56 ± 0.13 b

0.78

7

28.57

265, 346

Kaempferol rhamnosyl-rutinoside

22.79 ± 0.45 c

3.92

8

28.88

268, 352

Rhamnetin rhamnosyl-rutinosede

16.04 ± 0.56 c

2.76

9

29.82

254, 352

Isorhamnetin rhamnosyl-rutinosede

26.76 ± 0.43 c

4.60

10

30.6

255, 352

Rutin

54.06 ± 1.52 a

9.30

11

35.29

254, 354

Isorhamnestin-3-O-rutinoside

16.49 ± 0.50 a

2.85

a

18.06 6.00

a

Procyanidin B2, epicatechin, quercetin 3-O-rutinoside-7-O-a-L-rhamnosidase, rutin and isorhamnestin-3-O-rutinoside were quantified with their own standard curves (mg/ g). b Peak 3, 4, 6 were calculated as epicatechin equivalent (mg EE/g) using the standard curve of epicatechin. c Peak 7-9 were calculated as rutin equivalent (mg RE/g) using the standard curve of rutin.

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Table 2 Effects of LPPE on general parameters CON

EtOH

EtOH+L-LPPE

EtOH+H-LPPE

Initial body weight (g)

26.18±1.68a

26.23±1.79a

26.24±1.81a

26.29±1.88a

Final body weight (g)

26.89±1.51a

27.28±1.39a

27.93±2.13a

26.90±1.81a

Total caloric intake (kcal/mice)

665.36±40.21a

675.95±36. 90a

683.18±52.25a

669.74±35.66a

Liver-to-body weight ratio (%)

3.16±0.24a

3.77±0.36b

3.38±0.17a

3.41±0.20a

The data are presented as the mean ± SD (n = 10). The data in the same row with different superscripts indicate significant differences (p < 0.05). CON, mice fed a control liquid diet; EtOH, mice fed an ethanol-containing liquid diet; EtOH+L-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.2 g/L LPPE; and EtOH+H-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.4 g/L LPPE.

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Table 3 Effects of LPPE on serum and hepatic oxidative stress in ethanol-induced liver injury in mice. CON

EtOH

EtOH+L-LPPE

EtOH+H-LPPE

TBARS (nmol/mL)

8.24±1.60a

10.96±1.42c

9.70±1.12bc

8.73±1.21ab

SOD (U/mL)

110.88±15 .55b 91.26±13.83a

114.94±16.88b

118.80±10.01b

TBARS/SOD ratio (%)

7.35±1.40a

12.00±2.76b

8.45±1.63a

7.30±1.22a

GSH-Px (U/mL)

634.86±24.26b

561.74±55.15a 610.02±44.64ab

621.00±48.42b

CAT (U/mL)

5.72±0.47b

3.83±0.64a

7.03±1.78bc

8.09±1.71c

TBARS (nmol/mg prot)

0.83±0.16a

1.14±0.18b

0.98±0.21ab

0.88±0.12a

SOD (U/mg prot)

616.12±59.88b

558.08±31.18a

662.56±48.60b

702.38±86.86b

TBARS/SOD ratio (%)

0.13±0.03ab

0.21±0.04c

0.15±0.04b

0.12±0.01a

GSH-Px (U/mg prot)

518.21±67.17b

413.46±54.74a

554.55 ±42.93b

541.86±76.52b

CAT (U/mg prot)

23.54±2.14b

16.77±3.24a

16.76±3.78a

20.88±2.33b

Serum

Liver

The data are presented as the mean ± SD (n = 10). The data in the same row with different superscripts indicate significant differences (p < 0.05). CON, mice fed a control liquid diet; EtOH, mice fed an ethanol-containing liquid diet; EtOH+L-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.2 g/L LPPE; and EtOH+H-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.4 g/L LPPE. TBARS, thiobarbituric acid reactive substances; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase.

33



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TOC Graphics

34


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Fig. 1 HPLC profile of lychee pulp phenolic extract (LPPE) at 280 nm. 189x89mm (300 x 300 DPI)



Fig. 2 Effects of LPPE on liver histopathology in ethanol-induced liver injured mice. (A) H&E staining (200×, 400×); (B) oil-red O staining (200×). Data are presented as the mean ± SD (n = 9). The data with different superscripts indicate significantly different (p < 0.05). CON, mice fed a control liquid diet; EtOH, mice fed an ethanol-containing liquid diet; EtOH+L-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.2 g/L LPPE; and EtOH+H-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.4 g/L LPPE. 104x198mm (300 x 300 DPI)


Page 36 of 40

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Fig. 3 Effects of LPPE on ethanol-induced liver injury in mice. (A) Enzymatic activity of AST and ALT in the serum; (B) Serum TC and TG content; (C) Liver TC and TG content. The data are presented as the mean ± SD (n = 10). The data with different superscripts indicate significant differences (p < 0.05). CON, mice fed a control liquid diet; EtOH, mice fed an ethanol-containing liquid diet; EtOH+L-LPPE, mice fed an ethanolcontaining liquid diet supplemented with 0.2 g/L LPPE; and EtOH+H-LPPE, mice fed an ethanol-containing liquid diet supplemented with 0.4 g/L LPPE. AST, aspartate aminotransferase; ALT, alanine aminotransferase; TG, triglyceride; TC, total cholesterol.

124x104mm (300 x 300 DPI)



Fig. 4 LPPE supplementation ameliorates ethanol-induced mitochondrial oxidative stress in the livers of mice. (A) Mitochondrial 8-OHdG content normalized to the total mitochondrial DNA (mtDNA); (B) mitochondrial GSH content; (C) mitochondrial GSSH content; (D) mitochondrial GSH/GSSH ratio. The data are presented as the mean ± SD (n = 10). The data with different superscripts indicate significant differences (p < 0.05). 8-OHdG, 8-hydroxy-2’-deoxyguanosine level; GSH, reduced glutathione; GSSH, oxidized glutathione.

135x113mm (300 x 300 DPI)


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Fig. 5 LPPE supplementation ameliorates ethanol-induced mitochondrial dysfunction in the livers of mice. (A) mtDNA content was determined by measuring the expression levels of the mtDNA-encoded ND1 (complex I subunit) and COXI (complex IV subunit) genes relative to the expression level of the nuclear DNA gene (GAPDH) using qRT-PCR; (B) enzymatic activities of complexes I and IV of the mitochondrial electron transport chain; (C) mitochondrial membrane potential; (D) hepatic ATP content. The data are presented as the mean ± SD. The data with different superscripts indicate significant differences (p < 0.05). 130x107mm (300 x 300 DPI)



Fig. 6 LPPE supplementation ameliorates ethanol-induced hepatocyte apoptosis in mice. (A) Cytoplasmic cytochrome c content; (B) caspase-3 activity; (C) qRT-PCR analysis of the mRNA levels of Bax and Bcl-2 in the liver (n = 4) relative to the expression of GAPDH; (D) representative western blot of Bax and Bcl-2 protein levels in the liver (n = 4). The data are presented as the mean ± SD. The data with different superscripts indicate significant differences (p < 0.05). 121x197mm (300 x 300 DPI)


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Lychee (Litchi chinensis Sonn.) Pulp Phenolic Extract Confers a

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