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Dec 30, 2013 - Beneficial Effects of Camellia Oil (Camellia oleifera Abel.) on ... effect of camellia oil on ketoprofen-induced acute gastrointestinal...
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Beneficial Effects of Camellia Oil (Camellia oleifera Abel.) on Ketoprofen-Induced Gastrointestinal Mucosal Damage through Upregulation of HO‑1 and VEGF Yu-Ting Cheng,† Shu-Li Wu,† Cheng-Ying Ho,† Shang-Ming Huang,† Chun-Lung Cheng,§ and Gow-Chin Yen*,†,‡ †

Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 402, Taiwan Agricultural Biotechnology Center, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan § Food Technology and Precessing Section, Animal Industry Department, Council of Agriculture, Executive Yuan, Taipei 10014, Taiwan ‡

ABSTRACT: Nonsteroidal anti-inflammatory drugs, such as ketoprofen, are generally used to treat pain and inflammation and as pyretic agents in clinical medicine. However, the usage of these drugs may lead to oxidative injury to the gastrointestinal mucosa. Camellia oil (Camellia oleifera Abel.) is commonly used in Taiwan and China as cooking oil. Traditional remedies containing this oil exert beneficial health effects on the bowel, stomach, liver, and lungs. However, the effects of camellia oil on ketoprofen-induced oxidative gastrointestinal mucosal lesions remain unknown. The objective of this study was to evaluate the effect of camellia oil on ketoprofen-induced acute gastrointestinal ulcers. The results showed that treatment of Int-407 cells with camellia oil (50−75 μg/mL) not only increased the levels of heme oxygenase-1 (HO-1), glutathione peroxidase (GPx), and superoxide dismutase (SOD) mRNA expression but also increased vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) protein secretion, which served as a mucosal barrier against gastrointestinal oxidative injury. Moreover, Sprague− Dawley (SD) rats treated with camellia oil (2 mL/kg/day) prior to the administration of ketoprofen (50 mg/kg/day) successfully inhibited COX-2 protein expression, inhibited the production of interleukin-6 (IL-6) and nitrite oxide (NO), reversed the impairment of the antioxidant system, and decreased oxidative damage in the gastrointestinal mucosa. More importantly, pretreatment of SD rats with camellia oil strongly inhibited gastrointestinal mucosal injury induced by ketoprofen, which was proved by the histopathological staining of gastrointestinal tissues. Our data suggest that camellia oil exerts potent antiulcer effects against oxidative damage in the stomach and intestine induced by ketoprofen. KEYWORDS: gastrointestinal ulcer, ketoprofen, camellia oil, antioxidant enzyme, VEGF, HO-1

1. INTRODUCTION Nonsteroidal anti-inflammatory drugs (NSAIDs), such as ketoprofen and indomethacin, are generally used to alleviate swelling and pain of rheumatoid arthritis and other inflammatory diseases in clinical medicine. Despite their benefits as antiinflammatory agents, these drugs may cause peptic ulcers.1,2 Currently, peptic ulcers caused by mucosal damage are a common digestive disease in the United States and it affects approximately 500000 individuals per year.3 The major causes of peptic ulcers include gastric acid, pepsin, bile salts, consumption of alcohol and tobacco, Helicobacter pylori infection, and NSAIDs. Musumba et al.4 indicated that NSAID users have a higher risk of peptic ulcers than those with Helicobacter pylori infection. Thus, it is imperative to search for novel compounds that may help to prevent ulceration of the gastrointestinal tract. Digestive diseases, including peptic ulcers, are associated with mucosal lipid peroxidation and oxidative damage.5 Proton pump inhibitors (PPIs), such as lansoprazole, are usually used to cure gastric and duodenal ulcers.6,7 Some reports have indicated that lansoprazole can also reverse the effects of diclofenac, indomethacin, ketoprofen, and piroxicam on the mucosal content of lipid peroxidation products (malondialdehyde, MDA), glutathione (GSH), and myeloperoxidase (MPO).6−8 Therefore, the up-regulated activity of antioxidant enzymes may © 2013 American Chemical Society

play an important role against oxidative stress in gastrointestinal mucosa.9,10 Recent evidence indicates that heme oxygenase-1 (HO-1) breaks down heme to biliverdin and carbon monoxide, which has antioxidative effects in addition to mitigating inflammation, controlling cell growth, and eliminating cell death.11 Guo et al.12 suggested that the upregulation of HO-1 expression may reduce inflammation and tissue injury caused by reactive oxygen species (ROS) in the gastrointestinal tract. We recently found that catechin effectively inhibits ketoprofeninduced oxidative damage by upregulating HO-1 in Int-407 cells, which might play a key role in the prevention of NSAIDs-induced gastrointestinal injury.13 These results suggest that HO-1 expression induced by pharmacological modulators may be a novel means of increasing gastrointestinal protection. Ketoprofen is one of the most common NSAIDs. Some reports have indicated that photodegradation products of ketoprofen by sunlight or UV-irradiated are associated with oxidative stress-induced peptic ulcer.2,14 de la Lastra et al.14 reported that the gastrointestinal toxicity of ketoprofen in rats Received: Revised: Accepted: Published: 642

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Chung Hsing University (approval no. 100-21). The compounds were orally administered as in the previous methods.13,20 Briefly, the animals received an oral administration of camellia oil (1, 2, and 4 mL/kg/day) for 3 weeks. The control group received soybean oil (Sigma, St. Louis, MO, USA) only. On the 21st day, all animals were orally administrated with ketoprofen (50 mg/kg) and were sacrificed using CO2 after 24 h. 2.5. Preparation of Gastrointestinal Mucosal Homogenate. Extraction of the gastrointestinal mucosa was carried out according to a previously reported method with some modifications.21 Briefly, gastrointestinal mucosa was homogenized in 1.15% potassium chloride (KCl) buffer. The tissue samples were centrifuged at 10000g for 10 min (4 °C) to obtain a homogenate of gastrointestinal mucosa. Finally, aliquots of the homogenate were collected and preserved at −80 °C for the subsequent assay. 2.6. Measurement of Antioxidant Enzymes. The activities of GSH peroxidase (GPx), glutathione reductase (GRd), catalase, and GSH S-transferase (GST) antioxidant enzymes were assayed according to previously reported methods.18,22 The GSH and the GSH/GSSG ratio were measured using a commercial kit (Cayman Chemical Co., Ann Arbor, MI, USA). SOD activity was assayed using a SOD assay kitWST (Dojindo Molecular Technologies Inc., Gaithersburg, MD, USA) as specified by the manufacturer. 2.7. Measurement of Nitric Oxide (NO). The levels of NO were assayed according to a previously reported method.23 Briefly, a gastrointestinal mucosal homogenate solution was mixed with the same volume of Griess reagent (5% phosphoric acid, 1% sulfanilamide, and 0.1% naphthyl ethylenediamine dihydrochloride) and incubated at room temperature for 10 min. The concentration of nitrite was measured by determining the optical density (OD) at 550 nm and calculated with a standard curve generated by Sodium nitrite. 2.8. Enzyme-Linked Immunosorbent Assay (ELISA). Int-407 cells were grown to 90% confluence in 6-well tissue culture plates and then added to the complete medium with camellia oil (prepared in DMSO) at 25, 50, and 75 μg/mL (final concentrations) for 24 h. The supernatants of Int-407 cells grown in conditioned medium and rat gastrointestinal mucosa protein samples was then harvested to be assayed for PGE2 (Cayman Chemical Company, Ann Arbor, MI, USA), VEGF (Invitrogen, Carlsbad, CA, USA), and IL-6 (eBioscience, San Diego, CA, USA) secretion using a specific ELISA kit as indicated by the manufacturer’s instructions. 2.9. RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The mRNA expression levels of GPx, HO1, and SOD were determined by RT-PCR. Total RNA was prepared using the TRIzol method (Life Technologies, Rockville, MD) following the manufacturer’s instructions. The following sense and antisense primer sequences used for RT-PCR analysis were: GPx, 5′-GCA GAG CCG GGG ACA AGA GAA-3′ (forward) and 5′-CTG CTC TTT CTC TCC ATT GAC′-3 (reverse); HO-1, 5′-AAG ATT GCC CAG AAA GCC CTG GAC-3′ (forward) and 5′-AAC TGT CGC CAC CAG AAA GCT GAG-3′ (reverse); SOD, 5′-TAC ACC CAG ATG AAC GAG C3′ (forward) and 5′-AGG AAC TTC TCA AAG TTC CAG GA-3′ (reverse); 18S, 5′-TTG GAG GGC AAG TCT GGT G-3′ (forward) and 5′-CCG TCC CAA GAT CCA ACT A-3′ (reverse). The amplification products were determined by electrophoresis on a 1.8% agarose gel containing 0.06 mg/L ethidium bromide. The gel was then photographed under UV transillumination. The results are expressed as ratios after normalization of the gene signal to the corresponding 18S or β-actin signal in each sample. 2.10. Pathological Histology Studies. The gastrointestinal tissues were fixed in formaldehyde (10%) and processed for histopathological studies according to the standard method and stained by hematoxylin/ eosin (H&E) solutions. The pathological features of the gastrointestinal inner surface were scored as described by de la Lastra et al.24 as follows: score 0 = no lesions; score 1 = vasodilatation or erythema; score 2 = 1− 10 scattered small ulcers (10 mm).

was due to stimulation of cyclooxygenase-2 (COX-2) enzyme production and nitric oxide (NO) synthase in gastric mucosal lesions. Liu et al.2 indicated that pro-inflammatory mediators, such as NO and interleukin-6 (IL-6), were generated by upregulation of COX-2 expression following ketoprofen treatment in Sprague−Dawley (SD) rats. Therefore, reduction of excess NO and IL-6 production as well as inhibition of COX-2 enzyme expression may be associated with the prevention and treatment of ketoprofen-induced inflammatory disease in the gastrointestinal mucosa. Camellia oil (obtained from tea seed, Camellia oleifera Abel.) is generally used as cooking oil in Taiwan and China and usually consists of an abundance of unsaturated fatty acids, vitamins, and various antioxidant factors. The naturally occurring active compounds in tea seeds are different from those in other types of oil seeds.15,16 Camellia oil is also traditionally used as a remedy for bowel, stomach, and burn injuries in China. Chen et al.17 indicated that the saponin in tea seed could lower the triglyceride, cholesterol, and low density-lipoprotein levels in the blood of rats. Our previous study indicated that administration of camellia oil strongly prevented the liver injuries induced by carbon tetrachloride (CCl4).18 However, the role of camellia oil on acute gastrointestinal ulcers induced by ketoprofen remains unknown. The objective of this study was to evaluate the effects of camellia oil on the activities of antioxidant enzymes and on ketoprofeninduced acute gastrointestinal oxidative damage.

2. MATERIALS AND METHODS 2.1. Materials. S(+)-Ketoprofen and 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Basal Medium Eagle (BME/ EBSS), bovine serum, trypsin-EDTA (T/E), L -glutamine, and penicillin/streptomycin (PS) were purchased from Gibco (Grand Island, NY, USA). The Trizol RNA isolation kit was purchased from Life Technologies (Rockville, MD, USA). Primers, dNTPs, reverse transcriptase, and Taq polymerase for RT-PCR were purchased from Gibco BRL (Cergy Pontoise, France). All chemicals and solvents used were of the highest purity available. The camellia oil was obtained from the HsinI Country Farmer’S Association (Nantou, Taiwan). 2.2. Cell Culture and Treatment. The human intestinal Int-407 cell line (BCRC 60022) was obtained from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in complete medium (90% BME/EBSS, 10% bovine serum, and 1% penicillin/streptomycin) at 37 °C, under humidified 5% CO2 incubator. The cells were detached with T/E (0.1% trypsin, 10 mM EDTA) solution and split weekly. The solution of camellia oil was prepared in DMSO, and according to the method described by Jang et al.,19 The control group received DMSO only and the concentration of DMSO used for the experiments was lower than in 0.1% (v/v). 2.3. Wound Scratch Assay. The human Int-407 cells were cultured to 90% confluence in 6-well tissue culture plates. A wound phenomenon was made by scratching the cells with a sterile 200 μL pipet tip and removing floating cells with PBS. Next, camellia oil (prepared in DMSO) was added to the complete medium at 25, 50, and 75 μg/mL (final concentrations) for 24 h. Cell migration was monitored and recorded under a phase contrast microscope. The cell migration ability was calculated using the following equation: migration area = [migration area (treated)/migration area (control)]. 2.4. Animal Treatment Procedures. Six-week-old (175−200 g) male SD rats were obtained from BioLASCO Co. (Taipei, Taiwan). Each rat was raised at 22 ± 2 °C, under 65−70% relative humidity on a 12/12 h light/dark cycles. The animals were fed a LabDiet 5001 Rodent Diet (24% protein, 5% fat, 5% fiber) (Nestle Purina, St. Louis, MO). All animal trial protocols were based on the National Institutes of Health (NIH) guidelines. These experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the National 643

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Figure 1. Effect of camellia oil on GPx, HO-1, and SOD mRNA expression in Int-407 cells. The cells were treated with camellia oil at 25, 50, and 75 μg/ mL for 12 h, and the levels of GPx, HO-1, and SOD mRNA expression were analyzed by RT-PCR. The results are presented as the mean ± SD for n = 3. Each group with different letter subscripts is significantly different (p < 0.05).

Figure 2. Effect of camellia oil on wound healing in Int-407 cells. (A) The cells were treated with camellia oil (25, 50, and 75 μg/mL) for 24 h, and a migration assay was performed to evaluate the wound-healing effects of camellia oil in Int-407 cells. (B) Graphical representation of cell migration ability. (C,D) Cells were treated with camellia oil at 25, 50, and 75 μg/mL for 24 h, and the production of VEGF and PGE2 was analyzed by ELISA. The results are presented as the mean ± SD for n = 3. Each group with different letter subscripts is significantly different (p < 0.05). 2.11. Immunohistochemical (IHC) Staining. For IHC staining of VEGF and HO-1 was determined following a previously reported method.25 Briefly, the tissue sections were blocked with StartingBlock blocking buffers prior to incubation with an anti-VEGF or anti-HO-1 antibody. Finally, the tissue sections were incubated with Envisionlabeled polymer reagent containing peroxidase-conjugated anti-IgG at room temperature, followed by detection with a 3,3-diaminobenzidine tetrahydrochloride solution and hematoxylin. 2.12. Statistical Analysis. The statistical analysis was carried out using analysis of variance (ANOVA) according to the SAS User’s Guide.

Duncan’s multiple range test was used to determine the significant differences (p < 0.05) between the means.

3. RESULTS 3.1. Effects of Camellia Oil on GPx, HO-1, and SOD mRNA Expression in Int-407 Cells. It has been suggested that upregulation of antioxidant enzyme activities may play an important role on cytoprotection against oxidative stress. In the present study, the induction effect of camellia oil on the activity 644

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Figure 3. Immunohistochemical analysis of VEGF and HO-1 in the gastrointestinal mucosa induced by oral administration of camellia oil. SD rats were orally administered camellia oil (1, 2, and 4 mL/kg/day) for 21 days, and VEGF and HO-1 expression levels were examined by immunohistochemical analysis. VEGF and HO-1 protein expression in the gastrointestinal mucosa is represented by brown color.

3.2. Effect of Camellia Oil on Wound Healing in Vitro and in Vivo. In the wound-healing assay, Int-407 cells were treated with various concentrations (25, 50, and 75 μg/mL) of camellia oil for 24 h. As shown in Figure 2A,B, the 75 μg/mL concentration exhibited the greatest effect on cell motility after 24 h of incubation relative to the control group (p < 0.05). VEGF (vascular endothelial growth factor) and prostaglandins play key roles in maintaining gastrointestinal mucosal integrity.26,27 As shown in Figure 2C,D, treatment of the cells with camellia oil at 25, 50, and 75 μg/mL for 24 h led to a dose-dependent increase in VEGF and PGE2 production. Moreover, some reports have

of antioxidant enzymes was evaluated, including GPx, HO-1, and SOD. Int-407 cells treated with camellia oil at 25, 50, and 75 μg/ mL for 12 h showed a dose-dependent increase in the mRNA expression of the antioxidant enzymes GPx, HO-1, and SOD, which reached a maximum at 75 μg/mL. Moreover, in the cells treated with camellia oil at 75 μg/mL for 12 h, the mRNA expression levels of GPx, HO-1, and SOD were increased by 1.80, 1.53, and 1.49-fold, respectively, compared with the control group (Figure 1, p < 0.05). These results suggest that camellia oil may potentially prevent oxidative stress-induced gastrointestinal injury. 645

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Figure 4. Effect of camellia oil on ketoprofen-induced changes in the activity levels of (A) catalase, (B) GRd, (C) GPx, (D) GST, (E) SOD, and the ratio of (F) GSH/GSSG in the gastrointestinal mucosa of SD rats. The animals were orally administered camellia oil (1, 2, and 4 mL/kg/day) for 21 consecutive days, and ketoprofen was then orally administered to all animals for 1 day. The data are presented as the mean ± SD for n = 6. Each group with different letter subscripts is significantly different (p < 0.05). KET, 50 mg ketoprofen/kg BW; COL, 1 mL camellia oil/kg BW; COM, 2 mL camellia oil/kg BW; COH, 4 mL camellia oil/kg B.W.

3.3. Effect of Camellia Oil on Ketoprofen-Induced Changes in Catalase, GRd, GPx, GST, and SOD Activities As Well as the GSH/GSSG Ratio in Gastrointestinal Mucosa. As shown in Figure 4, the treatment of SD rats with ketoprofen (50 mg/kg) resulted in a significant (p < 0.05) reduction in the activity of catalase, GRd, GPx, GST, and SOD as well as a reduction in the GSH/GSSH ratio in the gastrointestinal mucosa. However, oral administration of camellia oil (1, 2, and 4 mL/kg/day) exhibited a significant dose-dependent reversal of the impairment of the antioxidant enzyme system in the gastrointestinal mucosa compared with the ketoprofen-induced group (p < 0.05). The results indicate that camellia oil could

suggested that VEGF might prevent gastric mucosal injury by upregulation of HO-1 expression.27,28 In vitro, Int-407 cells treated with camellia oil at 25, 50, and 75 μg/mL exhibited a dose-dependent increase in HO-1 mRNA expression as well as VEGF production (Figures 1C and 2C). More importantly, the administration of camellia oil to animals at 1, 2, and 4 mL/kg for 21 days had significant effects on VEGF and HO-1 protein expression in the stomach (Figure 3A) and intestinal (Figure 3B) mucosa, as determined by immunohistochemical analysis. These results suggest that the induction of VEGF and HO-1 by camellia oil might play a key role in the regulation of wound healing. 646

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Figure 5. Effect of camellia oil on ketoprofen-induced changes in the levels of COX-2, NO, and IL-6 expression in rat gastrointestinal mucosal tissue. The animals were orally administered camellia oil (1, 2, and 4 mL/kg) for 21 consecutive days, and ketoprofen was then orally administered to all animals for 1 day. The data are presented as the mean ± SD for n = 6. Each group with different letter subscripts is significantly different (p < 0.05). KET, 50 mg ketoprofen/kg BW; COL, 1 mL camellia oil/kg BW; COM, 2 mL camellia oil/kg BW; COH, 4 mL camellia oil/kg BW.

reverse the impairment of the antioxidant system induced by ketoprofen in the gastrointestinal mucosa of SD rats. 3.4. Effect of Camellia Oil on Ketoprofen-Induced Changes in COX-2, NO, and IL-6 Expression Levels in the Gastrointestinal Mucosa. As shown in Figure 5A, treatment of rats with ketoprofen showed a significant (p < 0.05) increase in the level of COX-2 protein expression that was partly reduced by pretreatment with camellia oil (1, 2, and 4 mL/kg/day). NO and IL-6 are potent mediators of gastrointestinal mucosal inflammation caused by a wide variety of inflammatory stimuli such as toxic chemicals, viruses, and drugs. As shown in Figure 5B, NO production was abrogated by camellia oil in ketoprofen-treated SD rats (p < 0.05). As shown in Figure 5C, IL-6 production in animals treated with camellia oil (1, 2, and 4 mL/kg/day) was significantly inhibitory (p < 0.05), compared with the ketoprofen-induced group. However, no significant (p < 0.05) difference was found between any of dose-dependent manner. These results indicate that camellia oil might have the potential to protect patients with inflammation-associated gastrointestinal ulcers. 3.5. Pathological Histology of the Gastrointestinal Tract. The protective effects of camellia oil against oxidative injury to the gastrointestinal mucosa were investigated by hematoxylin/eosin staining. As shown in Figure 6, treatment of animals with ketoprofen at 50 mg/kg/day caused acute gastrointestinal injury as well as concavity of the gastrointestinal surface. The gastrointestinal tissues showed cloudy, swelled areas of variable size as well as necrosis and lymphocyte infiltration in the central zone and midzone. However, animals pretreated with camellia oil at 1, 2, and 4 mL/kg/day for 3 weeks prior to the administration of ketoprofen (50 mg/kg/day) showed decreased incidence of gastrointestinal mucosal injury.

Figure 6. Effect of camellia oil on ketoprofen-induced gastrointestinal mucosal damage in SD rats. (A) Histological injury score of the gastrointestinal mucosa after different doses of camellia oil in rats treated with ketoprofen. (B) The morphology of ketoprofen-induced gastrointestinal ulcers in SD rats pretreated with or without camellia oil for 21 d. Gastrointestinal damage is indicated with an arrow. The data are presented as the mean ± SD for n = 6. Each group with different letter subscripts is significantly different (p < 0.05). KET, 50 mg ketoprofen/ kg BW; COL, 1 mL camellia oil/kg BW; COM, 2 mL camellia oil/kg BW; COH, 4 mL camellia oil/kg BW. Hematoxylin/eosin staining, 100×.

4. DISCUSSION Camellia oil has been reported to have positive health effects and is used as a traditional remedy for stomachache. Several studies have suggested that vegetable oils (corn, olive, and sunflower oils) strongly inhibit gastrointestinal injuries induced by NSAID.20 Similar to olive oil, camellia oil is rich in oleic acid, which plays a key role in protection against ulcerogenesis.18 In the preliminary test (data not shown), our data found that methanol extract of camellia oil revealed the highest antioxidant activity (127.48 μM trolox/g extract) as determined by trolox equivalent antioxidant capacity (TEAC). The similar antioxidant activity was also obtained by the oxygen radical absorbance

capacity (ORAC) assay; it was the equal of 1.27 mM trolox/g extract. Moreover, methanol extract of camellia oil had the highest α-tocopherol (303.09 μg/g oil) and total phenolics (12.61 mg gallic acid/g extract) content as compare to soybean oil at concentrations of 24.27 (μg/g oil) and 1.72 (mg gallic acid/g extract), respectively. Chen et al.17 have demonstrated that administration of camellia oil could lower triglyceride, cholesterol, and low density-lipoprotein levels in the blood of rats. Moreover, we reported that two major active compounds in camellia oil, sesamin, and novel compound B (2,5-bis-benzo[1,3] dioxol-5-yl-tetrahydro-furo[3,4-d][1,3]dioxine) could effectively prevent carbon tetrachloride (CCl4)-induced liver oxidative 647

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damage in rats.18 However, the protective effect of camellia oil against injury of the gastrointestinal mucosa induced by ketoprofen remains unknown. Thus, the objective of this study was designed to study the effects of camellia oil on the activities of antioxidant enzymes against ketoprofen-induced acute gastrointestinal damage. NSAIDs such as ketoprofen are commonly used to treat inflammatory pain and as pyretic agents in clinical medicine. Despite their benefits, these drugs may cause peptic ulcer.2 Our previous study indicated that ketoprofen causes gastrointestinal ulcers through a variety of mechanisms, including increased mucosal lipid peroxidation and oxidative damage as well as decreased gastric mucus secretion in rats.13 Therefore, reducing oxidative stress and/or upregulating antioxidant enzymes may be effective approaches for preventing and treating gastrointestinal ulcers induced by ketoprofen. GPx, SOD, and, HO-1 are important antioxidant enzymes in the human body that have been shown to act as metal ion chelators and effective scavengers for ROS and reactive carbonyl species (RCS).11,12,29 As shown in Figure 1, camellia oil (75 μg/mL) strongly increased GPx, SOD, and HO-1 mRNA expression in Int-407 cells. Some reports have indicated that gastrointestinal isoenzyme-GPx is the mainly important gastric enzyme because it serves as a barrier against hydroperoxide formed during xenobiotic metabolism.30 Kirkby and Adin11 indicated that HO-1 breaks down heme to biliverdin and carbon monoxide, which can protect gastrointestinal cell against oxidative damage. Guo et al.12 reported that the upregulation of of HO-1 expression may reduce inflammation and tissue injury caused by ROS in the stomach and intestine. Our previous study showed that catechin effectively prevented injury of Int-407 cells induced by ketoprofen with upregulating HO-1 expression through the Nrf2 pathway.13 These results suggest that camellia oil inhibits oxidative injury of the gastrointestinal mucosa induced by ketoprofen through upregulating antioxidant enzymes, especially HO-1. Another approach for protection against ketoprofen-induced gastrointestinal mucosal damage may involve the upregulation of neovascularization in the gastrointestinal mucosal tissue. Neovascularization, which is the generation of new capillaries by sprouting of pre-existing microvessels, is under stringent control and normally occurs during embryonic and postembryonic development, the reproductive cycle, and wound repair.27,28 Taupin and Podolsky31 also indicated that enhanced stomach epithelial cell migration may be an effective approach for the treatment of stomach ulcers. VEGF is one of the most important factors for gastrointestinal mucosal reconstruction, mucosal defense, and ulcer healing.27 Sofalcone is used to treat gastrointestinal ulcers and protects the gastric mucosa. Shibuya et al.28 indicated that sofalcone might prevent gastric mucosal injury through the upregulation of VEGF production mediated via the Nrf2/HO-1 pathways. Similar to VEGF, prostaglandins play an important role in maintaining gastrointestinal mucosal integrity, which controls most gastric mucosa defense, and resolving inflammation.26,27 Megias et al.32 indicated that HO-1 modulated PGE2 production in osteoarthritic chondrocytes. Similarly, our results showed that treatment with camellia oil (25−75 μg/mL) not only increased the levels of HO-1, VEGF, and PGE2 expression but also enhanced the migration of human Int-407 cells (Figures 1C and 2). Furthermore, the VEGF and HO-1 protein levels in the stomach and intestinal mucosa of animals pretreated with camellia oil at 1, 2, and 4 mL/kg for 21 days were upregulated as evidenced by immunohistochemical analysis (Figure 3). These results indicate that camellia oil may

serve as barrier against ketoprofen-induced gastrointestinal mucosal injury, which may aid mucosal repair. Proton pump inhibitors (PPIs), such as lansoprazole, are usually used to treat gastric and duodenal ulcers.6,7 Many studies have suggested that administration of lansoprazole can reverse the effects of diclofena, ketoprofen, indomethacin, and piroxicam on the mucosal content of GSH, MDA, and myeloperoxidase (MPO).6−8 Similarly, our results indicated that camellia oil caused a significant (p < 0.05) dose-dependent reversal of the impairment of the antioxidant system during ketoprofen-induced gastrointestinal mucosal injury (Figure 4). To further investigate the protective effects of camellia oil against gastrointestinal injury in vivo induced by ketoproten in relation to antioxidant systems, SD rats were orally administered lansoprazole followed by gavage with ketoprofen. Our results showed that administration of lansoprazole (30 mg/kg) inhibited lipid peroxidation and reversed the reduction in the activities of GRd, GPx, GSH, and the GSH/GSSG ratio induced by ketoprofen (data not shown). These results suggest that camellia oil could reverse the impairment of the antioxidant enzymes activity in the gastrointestinal mucosa of SD rats induced by ketoprofen. On the other hand, numerous studies have indicated that the gastrointestinal toxicity of S(+)-ketoprofen in rats is due to stimulation of the production of COX-2 and NO synthase in gastric mucosal lesions.2,33,34 Pawlik et al.35 suggested that proinflammatory mediators, such as IL-6 and NO, are produced by COX-2. Erkan et al.36 indicated that COX-2 plays a key role in the stepwise process that ultimately causes gastric cancer. Blandizzi et al.8 reported that administration of lansoprazole prevented gastric mucosal injury caused by indomethacin, piroxicam, or ketoprofen by reducing COX-2 mRNA expression in SD rats. As shown in Figure 5, the administration of camellia oil to animals at 2 mL/kg/day reduced COX-2 protein expression as well as the production of NO and IL-6 in SD rats treated with ketoprofen. Moreover, the camellia oil group was no difference (p < 0.05) in food, water intake, and body weight compared with the levels in the control group and the ketoprofen-treated group (data not shown). More importantly, we found that administration of camellia oil (2 mL/kg/day) strongly prevented the ketoprofen-induced gastrointestinal mucosal damage, as demonstrated by the histopathological (H&E) staining of gastrointestinal tissues (Figure 6). Odabasoglu et al.20 indicated that animals treated with vegetable oils (corn, olive, and sunflower oils) at 2 mL/kg strongly inhibited indomethacin-induced gastric injuries in vivo. Furthermore, the animal dose of camellia oil was further extrapolated to a human equivalent dose following the calculated of Freireich et al.37 The result suggests that oral administration of camellia oil at 2 mL/kg is approximately equivalent in human dose to 0.3 mL/kg. Overall, these data indicate that camellia oil may offer a potential therapeutic approach for inflammation-associated disorders such as gastrointestinal ulcers. In conclusion, we demonstrated that intestinal epithelial cells treated with camellia oil (50−75 μg/mL) not only exhibited increased levels of GPx, HO-1, and SOD mRNA expression but also exhibited increased VEGF and PGE2 protein expression, which might serve as barrier against gastrointestinal mucosal oxidative injury. Moreover, the administration of camellia oil at 2 mL/kg/day strongly inhibited the ketoprofen-induced gastrointestinal mucosal damage in SD rats. These results confirm the health-promoting effects of camellia oil, which exhibits potent antiulcer effects against oxidative damage in the stomach and intestine induced by ketoprofen. 648

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AUTHOR INFORMATION

Corresponding Author

*Phone: 886-4-2287-9755. Fax: 886-4-2285-4378. E-mail: [email protected]. Funding

This research work was supported by the Council of Agriculture, Taiwan, under grant 100AS-3.1.4-FD-Z1(6). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. J. W. Liao of the Graduate Institute of Veterinary Pathobiology for his technical assistance on histological examination.

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ABBREVIATIONS USED CCl4, carbon tetrachloride; CAT, catalase; COX, cyclooxygenase; GSH, glutathione; GSSG, glutathione disulfide; GPx, glutathione peroxidase; GRd, glutathione reductase; HO-1, heme oxygenase-1; Int-407 cells, intestinal-407 cells; NSAIDs, nonsteroidal anti-inflammatory drugs; PGE2, prostaglandin E2; NO, nitric oxide; NQO1, NAD(P)H:(quinone-acceptor) oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SD rats, Sprague−Dawley rats; SOD, superoxide dismutase; VEGF, vascular endothelial growth factor

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