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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

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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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∇

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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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ACS Paragon Plus Environment


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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

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

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