Solanum nigrum Polyphenol Extracts Inhibit Hepatic Inflammation

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Solanum nigrum polyphenol extracts inhibit hepatic inflammation, oxidative stress, and lipogenesis in high-fat-diet-treated mice Jia-Jen Chang, Dai-Jung Chung, Yi-Ju Li, Bo-Han Wen, and Chau-Jong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03578 • Publication Date (Web): 05 Oct 2017 Downloaded from http://pubs.acs.org on October 6, 2017

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.

Journal of Agricultural and Food Chemistry 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 32

Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


Page 2 of 32

1

Solanum nigrum polyphenol extracts inhibit hepatic inflammation, oxidative

2

stress, and lipogenesis in high-fat-diet-treated mice

3

Ja-Jen Chang†, Dai-Jung Chung†#, Yi-Ju Lee‡, Bo-Han Wen†, Hsing-Yu Jao†,

4

Chau-Jong Wang†§*

5

†

6

University, Number 110, Section 1, Jianguo North Road, Taichung 402, Taiwan.

7

‡

8

Jianguo North Road, Taichung 402, Taiwan.

9

§

10

Institute of Biochemistry, Microbiology and Immunology, Chung-Shan Medical

Institute of Medicine, Chung-Shan Medical University, Number 110, Section 1,

Department of Medical Research, Chung-Shan Medical University Hospital, Number

110, Section 1, Jianguo North Road Taichung 402, Taiwan

11 12

#Coauthor (These authors contributed equally to this work thus sharing the first

13

authorship)

14

*Correspondence:

15

Microbiology and Immunology, Chung-Shan Medical University, Number 110,

16

Section 1, Jianguo North Road, Taichung 402, Taiwan.

17

E-mail: [email protected]

18

Tel: +866-4-24730022 ext 11670.

19

Fax: +866-4-23248167.

Professor

Chau-Jong

Wang,

Institute

20 21 22 23


of

Biochemistry,

Page 3 of 32

24


Abstract

25

Patients with diabetes, obesity, and hyperlipidemia are all high-risk groups for

26

fatty liver; however, the mechanism of fatty liver formation is not completely

27

understood. Studies have indicated that abnormal fat metabolism, oxidative stress, and

28

insulin resistance are positively correlated with peroxidation and abnormal cytokine

29

production. Recent studies have revealed that Solanum nigrum extracts (SNE) possess

30

anti-inflammatory, antioxidation, antihyperlipidemia, and liver protection abilities.

31

Therefore, the present study investigated the in vivo and in vitro effects of an SNE on

32

nonalcoholic fatty liver (NAFL)-induced hepatitis. In vivo data demonstrated that the

33

SNE reduced blood triglyceride, sugar, and cholesterol levels, as well as fat

34

accumulation, oxidative stress, and lipid peroxidation in high-fat-diet-treated mice.

35

The results indicated that the SNE downregulated the expression of fatty acid

36

synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase),

37

and sterol regulatory element-binding proteins (SREBPs) through the AMP-activated

38

protein kinase (AMPK) pathway and upregulated the expression of carnitine

39

palmitoyltransferase 1 (CPT1) and peroxisome proliferator-activated receptor alpha.

40

Furthermore, we prepared an Solanum nigrum polyphenol extract (SNPE) from the

41

SNE; the SNPE reduced hepatic lipid (oleic acid) accumulation. Therefore, SNE have

42

the potential to alleviate NAFL-induced hepatitis, and polyphenolic compounds are

43

the main components of SNE. Moreover, SNE can be used to develop health food

44

products for preventing NAFL disease.

45

Keywords:

46

Solanum nigrum polyphenol extract, nonalcoholic fatty liver, anti-inflammatory,

47

antioxidation, antihyperlipidemia

48



49

Introduction

50

Overweight and obesity are associated with several chronic diseases including

51

fatty liver, metabolic syndrome, and cardiovascular disease1. Fatty liver disease is

52

related to increased abdominal or visceral fat accumulation; high triglyceride (TG),

53

low high-density lipoprotein (HDL) cholesterol, and high low-density lipoprotein

54

(LDL) cholesterol levels; and insulin resistance. All of these lead to impaired

55

metabolic regulation in adipose tissue2-3.

56

Many complicated biochemical reactions occur in the liver, including the

57

metabolic activities of multiple nutrients such as TG oxidation and the synthesis of

58

lipoproteins, cholesterol, and phospholipids. Abnormal lipid and lipoprotein

59

metabolism can result in dyslipidemia, including elevated plasma cholesterol and/or

60

TG levels and reduced HDL levels, which contribute to the increased prevalence of

61

morbid obesity. Several proteins associated with dyslipidemia have been reported to

62

play crucial roles in lipogenesis, including fatty acid synthase (FAS)4-5, sterol

63

regulatory element-binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl

64

-coenzyme A reductase (HMG-CoA reductase)6-8, and the expression levels of these

65

enzymes are dynamic in response to cellular energy status. Acetyl-CoA carboxylase

66

(ACC) catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA in a

67

biotin-dependent manner9. Malonyl-CoA is a substrate of FAS for fatty acid synthesis

68

and also a potent inhibitor of carnitine palmitoyltransferase 1 (CPT1) for fatty acid

69

β-oxidation. FAS is mainly involved in catalyzing the transformation of malonyl-CoA

70

into palmitate during the long-term process of long-chain fatty acid catalysis, which

71

catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA. FAS

72

regulated by multiple transcription factors. SREBPs, a transcription factor family,

73

regulate the expression of enzymes for the synthesis of fatty acids, cholesterol, TG,

74

and phospholipids, suggesting that this family plays a central role in energy


Page 4 of 32

Page 5 of 32


75

homeostasis through the modulation of glycolysis, lipogenesis, and adipogenesis. In

76

humans, the SREBP gene family mainly consists of SREBP-1 and SREBP-2,

77

specifically responding to lipogenic and cholesterogenic metabolism, respectively10-14.

78

In addition, this increase in lipid synthesis is caused by the increased activities of

79

HMG-CoA reductase, a key enzyme of cholesterol synthesis. HMG-CoA reductase is

80

a membrane protein located on the endoplasmic reticulum and plays a role in

81

cholesterol synthesis and mevalonate production from HMG-CoA15. In mammals,

82

hepatic lipolysis is mainly controlled by several enzymes, including peroxisome

83

proliferator-activated receptors (PPARs), CPT1, and AMP-activated protein kinase

84

(AMPK), in response to nutritional and hormonal conditions. PPARs are members of

85

the steroid hormone nuclear receptor superfamily, and they have three subtypes:

86

PPARα, PPARβ (PPARδ), and PPARγ; their respective tissues and regulators play

87

different roles. PPARα is highly expressed in the liver and promotes lipid uptake, fatty

88

acid β-oxidation, ketogenesis, and gluconeogenesis by regulating the expression of

89

genes involved in fatty acid catabolism. PPARα is thus a potential target for

90

alleviating lipid disorders, diabetes, and obesity16. CPT1 is localized in the

91

mitochondrial outer membrane and synthesizes acylcarnitine for fatty acid transport

92

across the inner mitochondrial membrane. AMPK is widely present in mammalian

93

tissues. It is a serine/threonine kinase comprising a heterotrimeric complex and is

94

regarded as an energy sensor in most tissues. AMPK regulates cellular lipid

95

metabolism through increased fatty acid oxidation and constitutive lipoprotein

96

exocytosis. In general, AMPK is a very important factor in intracellular energy

97

regulation, regulating metabolism and energy in the body17-18.

98

Solanum nigrum is traditionally used as food and has some pharmacological

99

effects such as fever reduction, antifever diuretics, liver protection, eyesight

100

improvement, blood pressure reduction, and glucose tolerance reduction19-20. Solanum



101

nigrum extract (SNE) contain high amounts of polyphenols exhibiting antioxidant and

102

antitumor properties. Many herbally derived polyphenolic compounds can prevent

103

obesity by inhibiting hypolipidemia and reducing adipose tissue mass, thus

104

suppressing the occurrence of metabolic, hepatic, and cardiovascular alterations

105

associated with obesity21. Sohrabipour et al demonstrated that administration of

106

Solanum nigrum reduced the blood glucose, cholesterol, and triglycerides to normal

107

level in streptozotocin-induced diabetic rats22.

108

On the basis of previous reports that an SNE has anti-atherogenic properties and

109

hepatoprotectivee effects, the present study was conducted to examine whether an

110

SNE can reduce hepatic lipid accumulation induced by a high-fat diet (HFD). In

111

addition, the hepatic regulation of lipid synthesis, oxidation, inflammation, and

112

clearance was investigated.

113 114

Experimental method

115

Preparation of SNE and Solanum nigrum polyphenol extract

116

In brief, Solanum nigrum was collected from the central part of Taiwan. Whole plants

117

were cut into small pieces, vacuum freeze-dried, and stored at room temperature until

118

further use. Dried Solanum nigrum was mixed with water for 30 min and subjected to

119

continuous heat extraction at 100°C for 40 min. The resulting water extract was

120

filtered and subsequently concentrated in a water bath at 90°C until it became creamy

121

and was dried in an oven at 70°C. For the preparation of the Solanum nigrum

122

polyphenol extract (SNPE), we mixed 100 g of dried Solanum nigrum powders in 300

123

mL of ethanol, followed by heating at 50°C for 3 h. The extracts were filtered,

124

followed by lyophilization under reduced pressure at room temperature. The powders

125

were resuspended in 500 mL distilled water, extracted with 180 mL of ethyl acetate

126

three times, resolved in 250 mL of distilled water, stored at -70°C overnight, and


Page 6 of 32

Page 7 of 32


127

lyophilized. Furthermore, the identities of the 18 components were established from

128

recorded mass spectra. The powders were resuspended in distilled water and passed

129

through a 0.22-μm filter for use in cell culture.

130 131

HPLC-UV analysis

132

The SNE was separated through HPLC-UV; this process was using a Waters system

133

(the in-line 4-channel Degasser AF, Waters 600 HPLC Controller and pump, 2998

134

PDA detector). Separating column was by a Mightysil RP-18 GP (5 μm particle size,

135

250 x 4.6 mm I.D) at room temperature. The mobile phase contained of formic acid in

136

water (solution A, pH = 2.5), acetonitrile (solution B) and methanol (solution C).

137

HPLC method was flowing with gradient elution program: 0 min, 100% A; 5 min,

138

90% A, 0% B; 15 min, 85% A, 3% B; 20 min, 85% A, 5% B; 30 min, 85% A, 8% B;

139

50 min, 85% A, 15% B; 65 min, 75% A, 25% B; 70 min, 70% A, 30% B; 80 min,

140

50% A, 50% B; 85 min, 100% B; 100 min, 100% B. Flow rate was 1.0 mL/min and

141

injection volume was 20 μL. The UV detection range was on 210 ~ 400 nm. Methanol

142

and acetonitrile (liquid chromatography grade, 99.9% and 99+ %, respectively) were

143

purchased from Merck Millipore (Germany). Formic acid (reagent grade, 96%) was

144

purchased from Tedia (USA). The standers of various phenolic compounds included

145

peltatoside, isoquercitrin and miquelianin (liquid chromatography grade, 99%, 95%

146

and 98%, respectively). These were obtained from Extrasynthese (French).

147 148

Animal model design

149

Male C57BL/6 mice (25–30 g; age, 42 days) were purchased from Lasco Laboratory

150

Animal Co. (Yilian County, Taiwan), housed in an environmentally controlled

151

laboratory, and acclimatized for 7 days. All mice housed under standard laboratory

152

conditions (18–23C, humidity 55–60%, and 12 h light/dark cycle) in agreement with



153

animal protocols approved by the Institutional Animal Care and Use Committee of the

154

Chung Shan Medical University (IACUC approval No. 740), Taichung, Taiwan. We

155

used an HFD to induce nonalcoholic fatty liver (NAFL) in the animal model. The feed

156

was pulverized to prepare high-lipid feed by using lard and cholesterol in the

157

preparation of high-lipid formulations. In the HFD group, the contents of lard and

158

cholesterol were 20% and 2%, respectively. Lard and cholesterol were mixed

159

uniformly after the addition of water. The formulation for the high-lipid feed was

160

based on a reference. The animals were divided into five dietary groups (n =

161

12/group) : (A) control diet group, (B) HFD group, and(C, D, E) diet containing HFD

162

group (0.5% SNE ;1% SNE, and 2% SNE group). After the completion of the

163

experiment, the mice were killed, and liver and blood samples were collected for

164

further examination. Plasma was separated by centrifugation at 5000 g for 10 min at

165

4°C and was then maintained at −20°C for additional analyses (Commission Permit

166

No. 1235).

167 168

Histological examination of the liver

169

After the mice were killed, the liver was immediately fixed in 10% buffered

170

formaldehyde for 1 day and then maintained in a decalcification solution. After 3 days,

171

the specimens were reimmersed in formaldehyde, and hematoxylin and eosin (H&E)

172

staining was performed for histological examination. The liver section was observed

173

under high-power fields (× 200).

174

Immunohistochemical staining

175

A formalin-fixed paraffin-embedded liver section was analyzed to determine the

176

expression of inflammation markers in the liver. The bone section was incubated in 10

177

mmol/L citrate-Tween buffer at 100°C and assessed using the Thermo-HRP system.


Page 8 of 32

Page 9 of 32


178

Finally, the results were developed by reacting the liver section with 3,3ʹ-

179

diaminobenzidine (DakoCytomation, Carpinteria, CA, USA) and subjecting it to H&E

180

staining and subsequent microscopic examination. Furthermore, the section was

181

conjugated with a mouse monoclonal antibody against interleukin (IL)-6 (1:200;

182

Abcam Co, Cambridge Science Park, Cambridge, UK) and tumor necrosis

183

factor-alpha (TNF-α; 1:200). The intensity of IL-6- and TNF-α-positive cells in a

184

cross section was observed by examining five randomly selected areas of the section

185

under high-power fields (×200). Image Pro Plus 4.0 was used to quantify the

186

integrated optical density within the positive areas.

187 188

Cell Line and Cell Culture

189

Human hepatoma (HepG2) cells were obtained from the American Type Culture

190

Collection and grown in Dulbecco’s modified Eagle’s medium supplemented with

191

10% fetal bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, and 2 mM

192

L-glutamine (HyClone R, Thermo Scientific, Logan, UT, USA). The cells were

193

cultured at 37°C in a humidified atmosphere of 95% air to 5% CO2.

194 195

Cytotoxicity assay

196

HepG2 cells were seeded at a density of 1 × 10 5 cells/well into a 24-well plate and

197

treated with oleic acid (OA) and the SNPE at various concentrations for 24 h. The

198

MTT reagent (0.5 mg/mL) was added to each well and incubated for 4 h. The medium

199

was removed, and isopropanol was added to dissolve the MTT–formazan complex.

200

The absorbance was measured at 563 nm with a spectrophotometer (Hatachi 3210,

201

Hitachi, Tokyo, Japan).

202



203

Oil red staining

204

HepG2 cells were seeded in a six-well plate (3 × 106 cells/well) and treated with 500

205

mM OA and indicated concentrations of the SNPE for 24 h. The cells were washed

206

two times with phosphate-buffered saline (PBS), fixed with 4% formaldehyde in PBS

207

for 1 h, and then stained with 1 μg/mL oil red for 30 min at room temperature. After

208

the staining process, the lipid distribution in cells was immediately analyzed using a

209

FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA). In addition,

210

lipid-bound Nile red fluorescence was detected through inverted fluorescence

211

microscopy.

212

Western blot analysis

213

HepG2 cells were seeded in a 10-cm dish (3 × 106 cells/well) and treated with 500

214

mM OA at indicated concentrations of the SNPE for 24 h. The proteins were

215

harvested from the cells in a cold radioimmunoprecipitation assay buffer (1% NP-40,

216

50 mM Tris-base, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholic acid, 150

217

mM NaCl, pH 7.5) containing leupeptin (1.7 μg/mL) and sodium orthovanadate (10

218

μg/mL). The cell mixture was vortexed at 4°C for 4 h. All mixtures were then

219

centrifuged at 12000 rpm and 4°C for 10 min, and the protein contents of the

220

supernatants were determined with the Coomassie Brilliant Blue total protein reagent

221

(Kenlor Industries, Costa Mesa, CA, USA) by using bovine serum albumin (BSA) as

222

the standard. Equal amounts of protein samples were subjected to SDS–

223

polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes

224

(Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk

225

powder with 0.1% Tween-20 in tris-buffered saline and then incubated with the first

226

antibody at 4°C overnight. Thereafter, the membranes were washed three times with

227

0.1% Tween-20 in PBS and incubated with the secondary antibody to antimouse

228

horseradish peroxidase (GE Healthcare, Little Chalfont, Buckinghamshire, UK).


Page 10 of 32

Page 11 of 32


229

Antibodies against AMPK and phospho-AMPK (AMPK-P) were purchased from Cell

230

Signaling Technology (Beverly, MA, USA). FAS, SREBP-1c, and CPT1 antibodies

231

were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody

232

against

233

(Charlottesville, VA, USA). Band detection was revealed through enhanced

234

chemiluminescence (ECL) by using ECL Western blotting detection reagents and

235

exposed ECL hyper film in FUJIFILM LAS-3000 (Tokyo, Japan). The proteins were

236

quantified through densitometry by using FUJIFILM-Multi Gauge V2.2 software

237

(FUJIFILM, Stockholm, Sweden).

238

Statistical analysis

239

Data are reported as the mean ± standard deviation of three independent experiments

240

and were evaluated using the Student t test. A P value of

Solanum nigrum Polyphenol Extracts Inhibit Hepatic Inflammation

Recommend Documents