Protective Effect of Camellia Oil (Camellia oleifera ... - ACS Publications

May 31, 2017 - Histological injury score and hemorrhage score in ethanol-induced gastric mucosal damage dramatically elevated from the control group (...
0 downloads 0 Views 2MB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Protective Effect of Camellia Oil (Camellia oleiferaAbel.) against Ethanol-induced Acute Oxidative Injury of the Gastric Mucosa in Mice Pang-Shuo Tu, Yu-Tang Tung, Wei-Ting Lee, and Gow-Chin Yen J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on June 1, 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 41

Journal of Agricultural and Food Chemistry

1

Protective Effect of Camellia Oil (Camellia oleifera Abel.) against

2

Ethanol-induced Acute Oxidative Injury of the Gastric Mucosa in

3

Mice

4 5 6 7

Pang-Shuo Tu,†,∇ Yu-Tang Tung,†,‡,∇ Wei-Ting Lee,† and Gow-Chin Yen†,§,*

8



Department of Food Science and Biotechnology, National Chung Hsing University,

9 10

145 Xingda Road, Taichung 40227, Taiwan ‡

School of Nutrition and Health Sciences, Taipei Medical University, 250 Wu-Hsing

11 12

Street, Taipei 110, Taiwan §

Agricultural Biotechnology Center, National Chung Hsing University, 145 Xingda

13

Road, Taichung 40227, Taiwan

14 15



16

*Author to whom correspondence should be addressed.

17

Tel: 886-4-2287-9755, Fax: 886-4-2285-4378,

18

E-Mail: [email protected]

These authors contributed equally to this work.

19 20

RUNNING TITLE: Camellia Oil Reduces Ethanol-induced Acute Gastric Mucosal

21

Injury

22 23

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

24

ABSTRACT

25

Camellia oil, a common edible oil in Taiwan and China, has health effects for the

26

gastrointestinal tract in folk medicine, and it contains abundant in unsaturated fatty

27

acids and phytochemicals. However, the preventive effect of camellia oil on

28

ethanol-induced gastric ulcers remains unclear. This study was aimed to evaluate the

29

preventive effect of camellia oil on ethanol-induced gastric injury in vitro and in vivo as

30

well as its mechanisms of action. In an in vitro study, our results showed that

31

pre-treatment of RGM-1 cells with camellia oil enhanced the migration ability as well

32

as increased heat shock protein expression and reduced apoptotic protein expression. In

33

animal experiments, mice pre-treated with camellia oil effectively showed improved

34

ethanol-induced acute injury of the gastric muscosa and oxidative damage through the

35

enhancement of antioxidant enzyme activities and heat shock protein and PGE2

36

production, as well as the suppression of lipid peroxidation, apoptosis-related proteins,

37

pro-inflammatory cytokines and NO production. Histological injury score and

38

hemorrhage score in ethanol-induced gastric mucosal damage dramatically elevated

39

from the control group (0.00±0.0) to 3.40 ± 0.7 and 2.60 ± 0.5, respectively. However,

40

treatments with camellia oil or olive oil (2 mL/kg b.w.), and lansoprazole (30 mg/kg

41

b.w.) showed the significant decreases in elevation of injury score and hemorrhage score

42

(p < 0.05). Therefore, camellia oil has the potential to ameliorate ethanol-induced acute

43

gastric mucosal injury through the inhibition of inflammation and oxidative stress.

44 45

KEYWORDS: gastric ulcer, camellia oil, ethanol, RGM-1, gastrointestinal health

46

2

ACS Paragon Plus Environment

Page 2 of 41

Page 3 of 41

Journal of Agricultural and Food Chemistry

47

INTRODUCTION

48

The gastric mucosa is the first guard that contacts exogenous toxic substances, possibly

49

leading to gastric bleeding, ulceration, and perforation generation.1 For many decades,

50

gastric ulcers were the most frequent cause of surgery with high morbidity and mortality

51

rates.2 Gastric ulcers are usually associated with an imbalance between mucosal

52

defensive and aggressive factors. The most common causes of gastric ulcers are

53

excessive alcohol consumption, pressure, smoking, hyperacidity, and hyper-secretion of

54

pepsin and bile. In addition, larger ulcers require vigorous and prolonged therapy.

55

Therefore, how to prevent or cure gastric ulcers is an urgent research issue.

56

Excessive alcohol consumption undoubtedly increases healthcare costs and

57

economic burden in individuals and society. Alcoholism plays an important role in

58

gastric bleeding, ulcer, or diseases.3, 4 Ethanol is metabolized to generate acetaldehyde

59

via microsomal oxidase. The intermediate substances of ethanol metabolism could

60

impair the functions of antioxidant enzymes. Alvarez-Suarez et al.5 demonstrated that

61

ethanol-induced lipid peroxidation and oxidative stress are involved in the pathogenesis

62

of acute gastric mucosal injury. Additionally, ethanol caused severe inflammation and

63

excessive reactive oxygen species (ROS) generation, which affect DNA and lipid

64

degradation, and direct resulted in irreversible damage to cells and tissues.6 Therefore, it

65

is a new trend of the modern diet to enhance the antioxidant and anti-inflammatory

66

properties of the gastric mucosa to ameliorate gastric damage.

67

Camellia oil (Camellia oleifera Abel.), a common edible oil in Taiwan and China,

68

is widely distributed in the tropical and subtropical regions of Asia and is used as a

69

traditional remedy to cure gastrointestinal, lung, and kidney diseases. Camellia oil is

70

rich in oleic acid (C18:1), linoleic acid (C18:2), palmitic acid (C16:0), squalene,

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

71

vitamin E, and flavonoid.7 Previous study showed that camellia oil could more

72

effectively prevent hypertension, hyperlipidemia, and hyperglycemia in the prevention

73

of cardiovascular diseases than ordinary edible oil.8 In addition, camellia oil has great

74

advantages in the prevention and control of skin diseases such as newborn dermatitis,

75

skin redness, pain, and swelling.8 Our previous study revealed that camellia oil has

76

hepatoprotective effect against CCl4-induced oxidative damage in rats, and this effect

77

might be related to its antioxidant properties.9 Cheng et al.10 showed that camellia oil

78

can inhibit COX-2 protein expression and the production of IL-6 and NO, decrease

79

oxidative damage, and thus alleviate the damage of ketoprofen to the gastrointestinal

80

mucosa. However, the protective effect of camellia oil against the gastric mucosal

81

damage induced by ethanol is still lacking relevant scientific literature support. Hence,

82

the objective of this study was to investigate the effect of camellia oil on

83

ethanol-induced gastric mucosal damage in RGM-1 cells and in mice, and its

84

mechanisms of action.

85 86

MATERIALS AND METHODS

87

Chemicals. Dulbecco's Modified Eagle’s Medium (DMEM), F12 nutrient mixture

88

culture medium, and fetal bovine serum (FBS) were purchased from Thermo Fisher

89

Scientific (Waltham, MA, USA). HEPES, glucose, penicillin-streptomycin antibiotics,

90

Triton X-100, TWEEN 20, BSA, sodium bicarbonate, trypsin, dimethyl sulfoxide

91

(DMSO), lansoprazole, and protein inhibitor cocktail were purchased from

92

Sigma-Aldrich Corporation (St. Louis, MO, USA). Potassium chloride (KCl), sodium

93

dihydrogen phosphate (NaH2PO4), and disodium hydrogen phosphate (Na2HPO4) were

94

purchased from Hayashi Corporation (Osaka, Japan). Tris and the protein assay kit were

4

ACS Paragon Plus Environment

Page 4 of 41

Page 5 of 41

Journal of Agricultural and Food Chemistry

95

purchased from BIO-RAD (Hercules, CA, USA). Dipotassium hydrogenphosphate

96

(K2HPO4), hydrogen peroxide (H2O2), and sodium chloride were purchased from Wako

97

(Tokyo, Japan). Ethylenediaminetetraacetic acid (EDTA) and magnesium chloride

98

(MgCl2·6H2O) were purchased from Showa (Tokyo, Japan). Methanol and n-butanol

99

were purchased from Baker Company (Chicago, USA). Anti-β-actin, anti-Bax,

100

anti-Bcl-2, anti-caspase-3, anti-cytochrome c, anti-HSP90, anti-HSP70, anti-HSP60,

101

and anti-iNOS antibodies were obtained from Cell Signaling Technology (Beverly, MA,

102

USA). The anti-COX-2 antibody, Prostaglandin E2 Express EIA Kit, TBARS analysis

103

kit, Glutathione analysis kit, Glutathione peroxidase analysis kit, and Glutathione

104

reductase analysis kit were obtained from Cayman Chemicals (Ann Arbor, MI, USA).

105

The anti-HO-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,

106

CA, USA). Peroxidase AffiniPure Goat Anti-Mouse IgG (H + L) and Peroxidase

107

AffiniPure Goat Anti-Rabbit IgG (H + L) antibodies were purchased from West

108

Baltimore Pike (West Grove, PA, USA). TNF-α ELISA Ready-SET-Go! was obtained

109

from eBioscience (San Diego, CA, USA). The anti-IL-6 antibody was obtained from

110

Abcam (Cambridge, UK). The SOD assay kit-WST was purchased from Dojindo

111

Molecular Technologies Inc. (Kumamoto, Japan). The prostaglandin E2 express ELISA

112

kit was purchased from Cayman Chemical Company (Ann Arbor, MI, USA).

113 114

Preparation of Camellia Oil. Commercial camellia oil, commercial 100% pure

115

olive oil from Italy, and commercial refined soybean oil were purchased from the HsinI

116

Country Farmer’s Association (Nantou, Taiwan), a local supermarket (Taichung,

117

Taiwan), and Sigma-Aldrich Corporation (St. Louis, MO, USA), respectively. All oil

118

samples were stored in an airtight container at 4°C until further use.

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

119 120

Chemical Characteristics and Antioxidant Activity of Camellia Oil. Fatty acid

121

compositions and the squalene were performed using Gas chromatography (GC) and

122

Ultra Performance Convergence Chromatography (UPCC), respectively. The total

123

phenolic content was measured as described by Yen et al.11 The α-tocopherol and

124

catechin contents were performed using HPLC as the method of Nakasato et al.12 and

125

Lee et al.9, respectively. Total antioxidant activity assay (Trolox equivalent antioxidant

126

capacity, TEAC assay) of camellia oil was determined using the TEAC assay as

127

described by Lee and Yen.13

128 129

Cell Culture and Treatments. The rat gastric mucosa RGM-1 cell line was

130

obtained from Riken cell bank (Tsukuba, Japan). The RGM-1 cells were cultured in

131

Dulbecco's modified Eagle’s medium (high glucose) and F-12 nutrient mixture at a ratio

132

of 1:1, supplemented with 20% FBS, 0.49% (w/v) NaHCO3, 0.357% (w/v) HEPES, and

133

1% PS antibiotic solution (100 units/mL penicillin and 100 µg/mL streptomycin), and

134

then the cells were incubated under 5% CO2 at 37°C.

135 136

Wound Healing Migration Assay. The wound healing migration assay was

137

determined as described by Liang et al.14 with slight modifications. RGM-1 cells were

138

grown to 90% confluence in a 24-well cell culture plate. The wound healing migration

139

assay was determined by scratching the wounds with a sterile pipette tip, removing

140

floating cells with PBS, and then adding the medium with 0-75 µg/mL camellia oil for 0,

141

12, and 18 h. For each image, distances between one side of scratch and the other can be

142

determined at certain intervals using Image Pro software (Media Cybernetics, Bethesda,

6

ACS Paragon Plus Environment

Page 6 of 41

Page 7 of 41

Journal of Agricultural and Food Chemistry

143

MD, USA).

144 145

Analysis of the Proteins in Ethanol-Induced RGM-1 Cells. The RGM-1 cells

146

were pretreated with 0, 25, 50 or 75 µg/mL camellia oil for 6 h and then were incubated

147

in fresh DMEM with or without 5% ethanol for 6 h. Cells were homogenized in lysis

148

solution and the homogenates were centrifuged at 10,000 x g for 15 min at 4°C. The

149

total protein concentration of RGM-1 cells was measured colorimetrically using a

150

commercial protein reagent kit (Bio-rad, Hercules, CA, USA). The expression of heat

151

shock proteins (HSP90, HSP70, HSP60, and HSP32) and apoptosis-related proteins

152

(Bax, Bcl 2, cytochrome c, and caspase-3) in cell protein extracts was analyzed using

153

western blot analysis, which was performed following the method of Cheng et al.15.

154 155

Cell Cycle Analysis by Propidium Iodide (PI) Staining. For cell cycle analysis,

156

RGM-1 cells were seeded 1 × 105 cells/well in 24-well plate and then were grown for

157

12 h for adherence. The cells were pretreated with 0-75 µg/mL camellia oil for 6 h and

158

then were incubated for 6 h in fresh DMEM with or without 5% ethanol for 6 h. The

159

cells were harvested and then were stained with 500 µL of PI solution for more than 1 h

160

at 4°C in the dark. Finally, the stained cells were analyzed using a FACScan flow

161

cytometer (Becton Dickson Immunocytometry System USA, San Jose, CA), and the

162

cell numbers in the sub-G1 phase were analyzed by CellQuest software.

163 164

Animal Treatment Procedures. Male BALB/c mice (aged 5 weeks and weighing

165

19 ± 1 g) were purchased from the Livestock Research Institute (Taipei, Taiwan). The

166

experimental animals were given 1 week to acclimatize to the environment and diet. All

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

167

mice were given a chow diet and distilled water ad libitum and were maintained at a

168

normal 12 h light-dark cycle at 60%~70% humidity and room temperature (22 ± 2°C).

169

The experimental protocols for all animals were approved by the Institutional Animal

170

Care and Use Committee (IACUC) of National Chung Hsing University, Taichung,

171

Taiwan (IACUC Approval No: 103-85).

172

For this study, ethanol was used to induce acute gastric mucosal injury according

173

to the methods of Li et al.16 and Liu et al.17 Seventy-six-week-old BALB/c mice were

174

randomly assigned to seven groups for treatment (n = 10 per group): (1) control group;

175

(2) EtOH group; (3) COL (0.5 mL/kg of camellia oil) + EtOH group; (4) COM (1

176

mL/kg of camellia oil) + EtOH group; (5) COH (2 mL/kg of camellia oil) + EtOH group;

177

(6) OOH (2 mL/kg of olive oil) + EtOH group; and (7) Lan (30 mg/kg of lansoprazole)

178

+ EtOH group. Mice were pretreated orally with camellia oil or olive oil once a day for

179

21 consecutive days. The mice of the Lan + EtOH group were pretreated orally with

180

soybean oil for 14 days and then were pretreated with Lan for 7 days as the methods of

181

Duran et al.18 and Batista et al.19. In addition, the control group or EtOH group received

182

soybean oil (2 mL/kg b.w.) once a day for 21 consecutive days. Briefly, mice were orally

183

gavaged with 5 mL/kg b.w. of absolute ethanol (for the groups of EtOH, COL + EtOH,

184

COM + EtOH, COH + EtOH, OOH + EtOH, and Lan + EtOH) or RO water (for the

185

control group) 1 h before sacrifice.

186 187

Pathological Histology. The gastric mucosa was fixed in 10% buffered

188

formaldehyde and was examined using hematoxylin and eosin (H&E) staining as

189

described previously.9 The histological injury score or hemorrhage score of the gastric

190

mucosa was scored, and the degrees of lesions were graded from one to five depending

8

ACS Paragon Plus Environment

Page 8 of 41

Page 9 of 41

Journal of Agricultural and Food Chemistry

191

on severity: 1 = minimal (< 1%); 2 = slight (1-25%); 3 = moderate (26-50%); 4 =

192

moderate/severe (51-75%); and 5 = severe/high (76-100%).

193 194

Preparation of Gastric Mucosal Homogenate. The gastric mucosa was extracted

195

according to the method of Cheng et al.10 with a slight modification. Finally, the

196

homogenates of gastric mucosa were collected and stored at -80°C for assay.

197 198

Analysis of the Total Protein Concentration. To determine the antioxidant

199

enzyme activities as U per milligram of protein or nanomoles per minute per milligram

200

of protein, the total protein concentration of gastric mucosal tissues was determined

201

colorimetrically using a commercial protein reagent kit (Bio-rad, Hercules, CA, USA).

202 203

Measurement of TBARS. The content of thiobarbituric acid reactive substances

204

(TBARS) in the gastric mucosa was measured using commercial kits for TBARS. The

205

absorbance at 535 nm was recorded, and the amounts of TBARS were expressed as

206

malondialdehyde (MDA) equivalents, i.e., nmol of MDA per mg protein.

207 208

Measurement of Antioxidant Enzymes. The activities of antioxidant enzymes,

209

including SOD, catalase, GSH, GPx, and GRd, in the gastric mucosa were assayed as

210

the previous method of Cheng et al.10

211 212

Analysis of the Proteins in the Gastric Mucosa. The expression levels of heat

213

shock proteins (HSP90, HSP70, HSP60, and HSP32), apoptosis-related proteins (Bax,

214

Bcl 2, cytochrome c, and caspase-3), and inflammatory proteins (COX-2, IL-6, and

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

215

iNOS) in the gastric mucosa were determined using western blot analysis.

216 217

Enzyme-Linked Immunosorbent Assay (ELISA). TNF-α and PGE2 in the

218

gastric mucosa were measured using the specific ELISA kits TNF-α ELISA

219

Ready-SET-Go! (eBioscience, San Diego, CA) and PGE2 express ELISA kit (Cayman

220

Chemical Company, Ann Arbor, MI, USA), respectively, and the protocols were

221

performed as stated by according to the manufacturer's instructions.

222 223

Determination of Nitric Oxide (NO). The content of NO was assayed according

224

to a previous study.9 Briefly, the nitrite concentration of the gastric mucosa homogenate

225

solution was determined as an indicator of NO production according to the Griess

226

reaction.

227 228

Statistical Analysis. Experimental data were expressed as the mean ± SD (n = 10).

229

ANOVA was employed to calculate differences among different groups with Duncan’s

230

test. P value < 0.05 was considered statistically significant.

231 232

RESULTS

233

Chemical Characteristics and Antioxidant Activity of Camellia Oil.

234

In this study, camellia oil had a fatty acid composition of oleic acid (764 mg/g of

235

camellia oil), linoleic acid (108 mg/g of camellia oil), and palmitic acid (96 mg/g of

236

camellia oil). In addition, camellia oil contained high content of antioxidants, including

237

total phenolic content (13.4 mg/g of camellia oil), α-tocopherol (209 µg/g of camellia

238

oil), catechin (1.4 µg/g of camellia oil), and squalene (322.3 µg/g of camellia oil).

10

ACS Paragon Plus Environment

Page 10 of 41

Page 11 of 41

Journal of Agricultural and Food Chemistry

239

Moreover, the TEAC value of the methanolic extract of camellia oil was the equal of

240

147.2 µmole Trolox per gram of methanolic extract.

241 242

Effect of Camellia Oil on Wound Healing in RGM-1 Cells. In the wound

243

healing migration assay, migration of RGM-1 cells was determined by the migration

244

area on culture plates. RGM-1 cells were incubated with 0, 25, 50, and 75 µg/mL of

245

camellia oil for 0, 12 and 18 h. Treatments with camellia oil enhanced wound healing in

246

a dose-dependent manner (Figure 1). After 12 or 18 h of incubation, 75 µg/mL camellia

247

oil showed the greatest effect on wound healing compared with the control group (P