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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

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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.

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Journal of Agricultural and Food Chemistry

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

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Ethanol-induced Acute Oxidative Injury of the Gastric Mucosa in

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Mice

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

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

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145 Xingda Road, Taichung 40227, Taiwan ‡

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

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Street, Taipei 110, Taiwan §

Agricultural Biotechnology Center, National Chung Hsing University, 145 Xingda

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Road, Taichung 40227, Taiwan

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*Author to whom correspondence should be addressed.

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

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E-Mail: [email protected]

These authors contributed equally to this work.

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

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Injury

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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enhancement of antioxidant enzyme activities and heat shock protein and PGE2

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

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

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hemorrhage score in ethanol-induced gastric mucosal damage dramatically elevated

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

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

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

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

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

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

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INTRODUCTION

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

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

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

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

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

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

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

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

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Excessive alcohol consumption undoubtedly increases healthcare costs and

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

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

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via microsomal oxidase. The intermediate substances of ethanol metabolism could

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

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

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

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

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

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

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properties of the gastric mucosa to ameliorate gastric damage.

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

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

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

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

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

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effectively prevent hypertension, hyperlipidemia, and hyperglycemia in the prevention

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

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

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

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

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

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

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

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

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

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

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

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mechanisms of action.

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MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Chemical Characteristics and Antioxidant Activity of Camellia Oil. Fatty acid

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

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

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

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

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

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

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described by Lee and Yen.13

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

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

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

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

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

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

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Wound Healing Migration Assay. The wound healing migration assay was

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

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

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

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

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

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

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MD, USA).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Taiwan (IACUC Approval No: 103-85).

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

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

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

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

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

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

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

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

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

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

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

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

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

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control group) 1 h before sacrifice.

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

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

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

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

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

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

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Preparation of Gastric Mucosal Homogenate. The gastric mucosa was extracted

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

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

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Analysis of the Total Protein Concentration. To determine the antioxidant

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

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

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

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Measurement of TBARS. The content of thiobarbituric acid reactive substances

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

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

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

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Measurement of Antioxidant Enzymes. The activities of antioxidant enzymes,

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

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the previous method of Cheng et al.10

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

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

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

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

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

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

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

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

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

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

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

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

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reaction.

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

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

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test. P value < 0.05 was considered statistically significant.

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RESULTS

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Chemical Characteristics and Antioxidant Activity of Camellia Oil.

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

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

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

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

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

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

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147.2 µmole Trolox per gram of methanolic extract.

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

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

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

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

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

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