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Protective effects of camellia oil (Camellia brevistyla) against indomethacin-induced gastrointestinal mucosal damage in vitro and in vivo
Journal of Functional Foods 62 (2019) 103539
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Protective effects of camellia oil (Camellia brevistyla) against indomethacininduced gastrointestinal mucosal damage in vitro and in vivo Ruei-Yu Wanga,1, Yu-Tang Tungb,1, Sheng-Yi Chena,1, Ya-Lin Leec, Gow-Chin Yena,d,
T
⁎
a
Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan c Biotechnology Division, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, 189 Zhongzheng Road, Wufeng Dist., Taichung 41362, Taiwan d Graduate Institute of Food Safety, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Camellia oil Gastrointestinal mucosal damage Indomethacin Antioxidant enzyme Inflammation
Accumulating evidence reveals that nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, cause oxidative stress and inflammation, which consequently cause gastrointestinal (GI) mucosal damage. Camellia oil, a common edible oil used in Asia, has excellent antioxidative and anti-inflammatory characteristics. Herein, we examined the benefits and protective effects of camellia oil in indomethacin-induced human intestinal Int-407 cells and a mouse model of indomethacin-induced gastric mucosal damage. Camellia oil pretreatment significantly increased cell viability and wound healing and reduced reactive oxygen species production in indomethacin-induced Int-407 cells. In vivo experiments revealed that camellia oil preadministration prevented gastric wound generation by decreasing inflammatory mediators interleukin-6, tumor necrosis factorα, and cyclooxygenase-2 levels; increasing heme oxygenase-1 antioxidant protein level; and elevating transforming growth factor-β and vascular endothelial growth factor levels in indomethacin-induced BALB/c mice. Thus, camellia oil is a functional dietary oil that prevents oxidative damage and inflammation in NSAID-induced GI mucosal damage.
1. Introduction Peptic ulcer is a common disease of the digestive system that affects approximately 10% of the global population (Lanas & Chan, 2017). The gastrointestinal (GI) mucosa is the first line of defense against toxic substances, and long-term exposure to these toxic substances can cause gastric bleeding, ulceration, and perforation (Tseng et al., 2015). GI mucosal damage develops because of an imbalance between aggressive factors (drugs, excessive alcohol intake, smoking, stress, hyperacidity, and excessive secretion of gastric acid, pepsin, and bile acid) (Lanas & Chan, 2017; Soreide et al., 2015) and protective factors (mucin glycoproteins, bicarbonate secretion, and prostaglandin E synthase). Inflammation of the GI mucosa causes disruption of mucosal integrity (Yuan, Padol, & Hunt, 2006). Nonsteroidal anti-inflammatory drugs (NSAIDs) are aggressive factors causing GI mucosal damage; these include aspirin, ibuprofen, naproxen, sulindac, mefenamic acid, and indomethacin (Maddirevula,
Abanemai, & Alkuraya, 2016; Wolfe, Lichtenstein, & Singh, 1999). More than 30 million people take NSAIDs every day for their pain relieving and inflammation reducing effects (Bjarnason et al., 2018). Indomethacin, one of the most commonly prescribed NSAIDs, has been used for treating acute gout-like arthritis, preventing pancreatitis after endoscopic retrograde cholangiopancreatography, inducing apoptosis in small-cell lung cancer, promoting survival of new neurons, and treating hypokalaemia in Gitelman syndrome by regulating inflammatory response (Akhter, Pfau, & Gopal, 2016; Blanchard et al., 2015; de Groot et al., 2005; Gaffo & Saag, 2007; Hain et al., 2018). Nevertheless, numerous studies have demonstrated that indomethacin has adverse effects on the GI mucosa, such as gastric ulcers (Chiou et al., 2005; Ramadan, Bonin, Kennedy, Hambley, & Lay, 2005). In addition, indomethacin results in excessive gastric acid secretion and reactive oxygen species (ROS) production, and it interferes with mucosal cell regeneration (Maity et al., 2009; Shahin, Abdelkader, & Safar, 2018; Wallace, 2008). Therefore, at present, the priority is to effectively
Abbreviations: COX-2, cyclooxygenase-2; GI, gastrointestinal; HO-1, heme oxygenase-1; LPZ, lansoprazole; NSAIDs, nonsteroidal anti-inflammatory drugs; ROS, reactive oxygen species ⁎ Corresponding author at: Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan. E-mail address: [email protected] (G.-C. Yen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.103539 Received 2 April 2019; Received in revised form 23 August 2019; Accepted 26 August 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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containers at 4 °C in refrigerator until further use.
prevent or safely treat indomethacin-induced GI mucosal damage. In recent years, the search for traditional medicinal plants to treat ulcer has received increasing attention. The application of botanicals for treating GI disorder has generated a renewed interest (Anheyer et al., 2017; Simren & Tack, 2018). The seeds of Camellia brevistyla are rich in oil and suitable are for the extraction of high-quality camellia oils; this oil is generally used as edible oil in China, Japan, Korea, India, and Taiwan (Su, Shih, & Lin, 2014). Camellia oil has been traditionally used to treat burn wounds and GI cramps. Camellia oil contains abundant unsaturated fatty acids, including monounsaturated oleic acid (C18:1) and polyunsaturated linoleic acid (C18:2), phenolic compounds, catechins, α-tocopherol, and squalene (Wang, Zeng, Del Mar Contreras, & Wang, 2017; Yang, Liu, Chen, Lin, & Wang, 2016). Notably, our previous studies have demonstrated that camellia oil exhibits powerful antioxidant activity and health beneficial bioactive capacity for preventing diseases related to oxidative injury, protecting against CCl4-induced hepatic damage, and ameliorating GI mucosal injury caused by ketoprofen and ethanol treatment (Cheng, Lu, & Yen, 2015; Cheng et al., 2014; Lee, Shih, Hsu, & Yen, 2007; Lee & Yen, 2006; Lee, Tung, Wu, Tu, & Yen, 2018; Tu, Tung, Lee, & Yen, 2017). Therefore, camellia oil may be a potent candidate for further develop as functional food for treating GI disorders. However, the protective effect of camellia oils on indomethacin-induced GI mucosal damage remains unknown. In this study, we investigated its potential protective action against GI disorders by using human intestinal Int-407 cell lines and acute indomethacin-induced gastric mucosal damage mice models.
2.3. Cell culture The Int-407 human intestinal cell lines used in the study were purchased from the Bioresource Collection and Research Center (BCRC 60022, Hsinchu, Taiwan). The cells were grown in 90% BME with EBSS, 10% FBS, 0.37% sodium bicarbonate, and 1% PS and placed in a cell culture incubator (37 °C, 5% CO2). 2.4. MTT assay The cells were pretreated with 75, 100, or 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Lansoprazole, a well-known treatment for peptic ulcer disease, was used as a positive control. The dose of lansoprazole was based on that used in a previous study (Rybniker et al., 2015). After 6 h of incubation with test samples, cells were treated with 800 μM indomethacin for 24 h. After treatment, MTT assay was conducted according to the method reported in our previous study (Tu et al., 2017). 2.5. Intracellular ROS assessment Cells were pretreated with 75, 100, or 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Lansoprazole was used as a positive control. Following 6 h of incubation with test samples, cells were treated with 400 μM indomethacin for 1 h. Following 1 h of incubation, the 106 cells were collected and resuspended in 1 mL of phosphatebuffered saline (PBS). Cell suspension (195 μL) incubated with 5 μL of DCFH-DA (400 μM) was loaded into a 96-well plate for 1 h. The cells were then permeabilized with 0.05% Triton X-100 at room temperature for 15 min. The dichlorofluorescein fluorescence intensity was detected using a FLUOstar Galaxy reader at 485 nm (excitation) and an emission wavelength of 520 nm.
2. Materials and methods 2.1. Chemical reagents Indomethacin, dimethyl sulfoxide (DMSO), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Penicillin–streptomycin solution (PS), Basal Medium Eagle (BME), and Earle’s balanced salt solution (EBSS) were bought from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Cromwell, CT, USA). TRIzol and SYBR safe DNA gel stain were purchased from Invitrogen (Carlsbad, CA, USA). Ibidi Culture-Insert was purchased from ibidi GmbH (Martinsried, Germany). Disodium hydrogen phosphate (Na2HPO4) and dipotassium hydrogen phosphate (K2HPO4) were obtained from J.T. Baker (Chicago, IL, USA). Mouse interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were purchased from eBioscience (San Diego, CA, USA). Antibodies against β-actin, vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGF-β) were purchased from Novus Biologicals (Littleton, CO, USA). Heme oxygenase-1 (HO-1) antibody was obtained from Abcam (Cambridge, UK). Cyclooxygenase2 (COX-2) antibody was purchased from Cayman Chemical (Ann Arbor, MI, USA). A WesternBright ECL kit was purchased from Advansta (San Jose, CA, USA). iScript cDNA synthesis kit, Bio-Rad protein assay dye reagent concentrate, Bio-Rad protein ladder, and acrylamide were purchased from Bio-Rad (Hercules, CA, USA). A BCA protein assay kit was purchased from Pierce (Waltham, MA, USA).
2.6. Wound healing assay First, 105 cells were loaded into the ibidi Culture-Insert. The ibidi Culture-Insert was then removed when cell growth reached 90% confluence in 24-well tissue culture plates. Subsequently, the wound assay was performed and unattached cells were removed using PBS. After that, the cells were pretreated with 75, 100, or 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Lansoprazole was used as a positive control. Following 6 h of incubation with camellia oils or lansoprazole, the cells were treated with 100 μM indomethacin for 24 h. The cell migration distance was captured and recorded using an OLYMPUS IX71 inverted microscope (Osaka, Japan). 2.7. Protein extraction from human Int-407 cells The cells were pretreated with 75, 100, or 150 μg/mL of TMS camellia oil and 10 μM lansoprazole for 6 h before being incubated with 400 μM indomethacin for 3 h. Lansoprazole was used as a positive control. After stimulations, total protein was extracted using the Total Protein Extraction Kit (Millipore, Bedford, MA, USA) following the manufacturer’s instructions. Total quantification of the total protein in the human Int-407 cells was performed using a Pierce BCA protein assay kit (Waltham, MA, USA).
2.2. Preparation of camellia oils The camellia oils from Maokong and Shiding District (New Taipei City, Taiwan) were provided by Dr. Ya-Lin Lee (Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan). Camellia oils were prepared from camellia seeds (Camellia brevistyla CohenStuart) with roasting at 100 °C for 10 min prior to press the oil which was named as Taipei Maokong Shiding (TMS). Besides, Taipei Maokong Shiding extracted by low temperature and without roasting was named TML. Commercially refined soybean oil was purchased from Sigma–Aldrich (St. Louis, MO, USA). All oils were stored in airtight
2.8. Animals Four-week-old male BALB/c mice weighing 20–25 g were purchased from BioLASCO (Taipei, Taiwan). The animals were maintained under controlled temperature (23 ± 1 °C) and humidity (65–70%) and under a 12:12 light–dark cycle. They were fed the Laboratory Rodent Diet 5001 for 2 weeks to adapt to the environment. The experimental 2
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Fig. 1. Effects of camellia oil (TMS and TML) on cell viability (A) or ROS production (B) in indomethacin-induced human Int-407 cells. The cells were pretreated with 75, 100, and 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Following 6 h of incubation with camellia oils, the cells were treated with indomethacin. Cell viability was detected through MTT assay. Results are presented as the means ± SD. n = 3. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group.
2.11. ELISA
procedures and animal care were in accordance with the guidelines provided by the Institutional Animal Care and Use Committee (IACUC approval No. 105-004) of National Chung Hsing University (Taichung, Taiwan). In this study, indomethacin was used to induce GI mucosal damage according to the method by Morise, Granger, Fuseler, Anderson, and Grisham (1999) with slight modifications. Six-week-old BALB/c mice were randomly distributed into the following five groups (n = 8 per group): (1) control group; (2) indomethacin group; (3) lansoprazole + indomethacin group (LPZ + Indomethacin); (4) Camellia oil low dose (COL, 1 mL/kg b.w./day of camellia oil) + indomethacin group (COL + Indomethacin); and (5) Camellia oil high dose (COH, 2 mL/kg b.w./day of camellia oil) + indomethacin group (COH + Indomethacin). The COL and COH groups received oral gavage of TMS camellia oil (1 and 2 mL/kg b.w./day, respectively) for 3 weeks. The control and indomethacin groups were both administered soybean oil (2 mL/kg b.w./day) for 3 weeks, and the positive control group received lansoprazole (30 mg/kg b.w./day) for 7 days. On the 21st day, all the mice except for those in the control group received oral gavage of indomethacin (40 mg/kg b.w.) for 24 h. After 24 h, all mice were sacrificed using isoflurane.
The proteins in the mice gastric mucosa were assayed for the IL-6 and TNF-α productions using a specific ELISA kit in accordance with the manufacturer’s instructions.
2.12. Western blot analysis Western blotting for the detection of HO-1, VEGF, TGF-β, COX-2, and β-actin was performed according to our previous report (Cheng et al., 2014).
2.13. Statistical analysis All data are expressed as the mean ± standard deviation [SD] (n = 3 for the cell assay and n = 8 for the animal assay). Statistical data analysis was conducted using the Student's t-test for independent samples between independent groups. A P value < 0.05 was considered statistically significant.
2.9. Histopathologic studies 3. Results The stomach tissues collected from the BALB/c mice were fixed in 10% neutral formaldehyde and processed for histological examination. Hematoxylin–eosin (H&E) staining was performed and the histological injury scores of the gastric mucosa were assigned as described in our previous study (Tung, Huang, Lin, & Yen, 2018). The degrees of lesion were classified according to their severity as follows: 1 = minimal (degree of lesion < 1%); 2 = slight (degree of lesion approximately 1–25%); 3 = moderate (degree of lesion approximately 26–50%); 4 = moderate or severe (degree of lesion approximately 51–75%); and 5 = severe or high (degree of lesion approximately 76–100%).
3.1. Effects of camellia oils on the cell viability and ROS production in indomethacin- induced human Int-407 cells A previous study revealed that drug-induced GI tract damage can cause oxidative GI mucosal damage and cell death (Bjarnason et al., 2018). The effects of TMS and TML camellia oils and lansoprazole on the cell viability and ROS production in indomethacin- induced human Int-407 cells are presented in Fig. 1. The results indicated that pretreatment with 75–150 μg/mL of TMS and TML camellia oils and 10 μM lansoprazole significantly increased the cell viability in indomethacininduced human Int-407 cells (P < 0.05) (Fig. 1A). NSAIDs reduce the activities of antioxidant enzymes in the body, which results in excessive ROS accumulation and directly causes intestinal mucosal oxidative damage (Bhattacharyya, Chattopadhyay, Mitra, & Crowe, 2014). As illustrated in Fig. 1B, TMS and TML camellia oils had excellent preventive effects on ROS production. At the dose of 150 μg/mL, TMS and TML camellia oils both significantly inhibited ROS production in indomethacin-induced human Int-407 cells (P < 0.05).
2.10. Gastric mucosal homogenate preparation Gastric mucosal homogenate was executed on the basis of the method reported in our previous study (Cheng et al., 2014). The total protein levels of the gastric mucosa were quantified using a Pierce BCA protein assay kit (Waltham, MA, USA).
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Fig. 2. Effects of camellia oils (TMS and TML) on the cell migration ability with wound healing in indomethacin-induced human Int-407 cells (A and B). Representative phase-contrast images of cells into the wounded area in the wound healing assay. Cells were pretreated with 75, 100, and 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Following 6 h of incubation with camellia oils, cells were treated with 100 μM indomethacin for 24 h. Graphical presentation of the cell migration ability observed. Results are presented as the means ± SD. n = 3. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group.
In the wound healing assay, the migration area of indomethacininduced human Int-407 cells was measured. As illustrated in Fig. 2A and 2B, treatment with 75, 100, or 150 μg/mL of TMS or TML camellia oils enhanced wound healing in indomethacin-induced human Int-407 cells (P < 0.05). In addition, the results revealed that TMS camellia oil had a stronger effect on ROS production inhibition and wound healing capacity than TML camellia oil in indomethacin-induced human Int407 cells (Figs. 1B and 2). Therefore, the following in vitro and in vivo experiments were performed to identify the underlying molecular mechanisms of GI mucosal injury prevention were focused on TMS camellia oil intervention only.
integrity, and improves vascular permeability (Ohno et al., 2008). Therefore, VEGF plays a major role in ulcer healing. The results in the present study indicated that indomethacin significantly reduced VEGF protein expression (P < 0.05) and that pretreatment with 75, 100, and 150 μg/mL of TMS camellia oil considerably enhanced VEGF protein expression compared with pretreatment with the positive control (Fig. 3B). Recent studies have reported that several growth factors are involved in the healing process of gastric ulcers, such as VEGF and TGFβ (Chai, Norng, Tarnawski, & Chow, 2007; Ohno et al., 2008). Consistently, our results also revealed that indomethacin significantly reduced the expression of TGF-β protein and that pretreatment with 75, 100, and 150 μg/mL of TMS camellia oil was more effective than treatment with 10 μM lansoprazole in enhancing TGF-β protein expression (Fig. 3C).
3.3. Effect of TMS camellia oil on the HO-1, VEGF, and TGF-β protein expressions in indomethacin-induced human Int-407 cells
3.4. Effect of TMS camellia oil on gastric pathology improvement in indomethacin-induced GI mucosal damage BALB/c mice
As illustrated in Fig. 3, the protein expressions of HO-1, VEGF, and TGF-β in the indomethacin-induced human Int-407 cells decreased markedly after treatment with indomethacin (P < 0.05). HO-1 plays a crucial role in the preservation of intestinal function integrity (Onyiah et al., 2013). The results demonstrated that treatment with various concentrations (75, 100, and 150 μg/mL) of TMS camellia oil and 10 μM lansoprazole significantly increased the expression of HO-1 protein in indomethacin-induced human Int-407 cells (P < 0.05) (Fig. 3A). The present study revealed that TMS camellia oil had a stronger effect on HO-1 protein expression than did 10 μM lansoprazole (positive control). These results implied that pretreatment of Int-407 cells with TMS camellia oil for 6 h could maintain intestinal mucosal integrity. VEGF promotes angiogenesis, maintains normal blood vessel
NSAIDs are common antipyretic and analgesic drugs, but they induce adverse side effects on gastric lesions (Chiou et al., 2005). The morphology, H&E stain, and histological injury score of the gastric mucosa are presented in Fig. 4. Although the indomethacin group exhibited gastric mucosal bleeding, pretreatment with TMS camellia oil (1 and 2 mL/kg b.w./day) for 21 days or clinical antiulcer drug lansoprazole (30 mg/kg b.w.) for 7 days effectively reduced the gastric mucosal bleeding caused by indomethacin (Fig. 4A), suggesting that TMS camellia oil can protect the integrity of the gastric mucosa. The gastric mucosal tissue in the indomethacin group presented severe degeneration and necrosis with hemorrhage in the gastric mucosal layer (Fig. 4B and C). Pretreatment with TMS camellia oil (1 and 2 mL/kg b.w./day) for 21 days or lansoprazole (30 mg/kg b.w.) for 7 days reduced the
3.2. Effect of camellia oils on the wound healing in indomethacin-induced human Int-407 cells
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Fig. 3. Effect of TMS camellia oil on the protein expression of HO-1 (A), VEGF (B), and TGF-β (C) in indomethacin-induced human Int-407 cells. The cells were pretreated with 75, 100, and 150 μg/mL of TMS camellia oil and 10 μM lansoprazole. Following 6 h of incubation with camellia oils, the cells were treated with 400 μM indomethacin for 3 h. Results are presented as the means ± SD. n = 3. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group.
significantly reduced injury severity. The aforementioned results indicated that TMS camellia oil maintains the integrity of the gastric mucosa and has a satisfactory protective effect on the pathology in gastric mucosal injury models.
hemorrhage in the stomach mucosal layer. As illustrated in Fig. 4D and Supplementary Table 1, damage to the gastric mucosal tissue was significantly higher in the indomethacin-induced groups than in the control group. Oral administration of a low dose (1 mL/kg b.w./day) and high dose (2 mL/kg b.w./day) of TMS camellia oil for 21 days
Fig. 4. Effect of TMS camellia oil on the morphology (A), H&E stain 40× (B) and 400× (C), and histological injury score (D) of stomach in BALB/c mice induced by indomethacin. The animals were administered camellia oil (1 and 2 mL/kg b.w./day) orally for 21 consecutive days or lansoprazole (30 mg/kg b.w.) for 7 days. Indomethacin (40 mg/kg b.w.) was then orally administered to all animals for 24 h. The data represent the means ± SD of eight mice. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group. LPZ: 30 mg/kg b.w. of lansoprazole, COL: 1 mL/kg b.w./day of camellia oil, COH: 2 mL/kg b.w./day of camellia oil. 5
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Fig. 5. Effect of TMS camellia oil on IL-6 production (A), TNF-α production (B), and COX-2 protein expression (C) in the gastric mucosa of BALB/c mice induced by indomethacin. The animals were administered camellia oil (1 and 2 mL/kg b.w./day) orally for 21 consecutive days or lansoprazole (30 mg/kg b.w.) for 7 days. Indomethacin (40 mg/kg b.w.) was then orally administered to all animals for 24 h. The data represent the means ± SD of eight mice. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group. LPZ: 30 mg/kg b.w. of lansoprazole, COL: 1 mL/kg b.w./day of camellia oil, COH: 2 mL/kg b.w./day of camellia oil.
4. Discussion
3.5. Effect of TMS camellia oil on gastric mucosal IL-6, TNF-α, and COX-2 protein expressions in indomethacin-induced GI mucosal damage BALB/c mice
Accumulating evidence revealed that NSAIDs are extensively used for the prevention of cancer, pain, osteoarthritis, fever (antipyretic), hypertension, preeclampsia, Alzheimer’s disease, and inflammation (Felson, 2016; Hedberg et al., 2019; Taler, 2018; Varvel et al., 2009). The main risk arising from NSAID use is GI toxicity (Bjarnason et al., 2018), which induces gastric ulcers and inhibits ulcer healing (Lanas & Chan, 2017). Previous studies have demonstrated that GI mucosal injury is related to the accumulation of ROS and lipid peroxidation products in the mucosal tissue, and long-term oxidative stress in mucosal tissues leads to GI bleeding, ulceration, and perforation (Bhattacharyya et al., 2014; Bjarnason et al., 2018). Cumulative evidence suggests that diet plays a central role in GI health maintenance and protection against peptic ulcer (Cheng, Lu, & Yen, 2017; Farzaei, Abdollahi, & Rahimi, 2015; Veldhoen & Brucklacher-Waldert, 2012; Yen, Tsai, Lu, & Weng, 2018). Camellia oil contains a variety of antioxidant components such as oleic acid, phenolic compounds, catechin, α-tocopherol, and squalene (Cheng et al., 2014; Tu et al., 2017). The results of our study demonstrated that the phenolic content of TMS and TML camellia oils was 0.112 ± 0.007 and 0.286 ± 0.032 mg gallic acid/mL, respectively, and the α-tocopherol content was (378.32 ± 6.87 and 388.16 ± 12.57 ppm, respectively. In addition, the fatty acid compositions of TMS and TML camellia oils, respectively, were 6.0% and 7.1% palmitic acid (16:0), 0.8% and 3.3% stearic acid (18:0), 78.8% and 72.8% oleic acid (18:1), 12.2% and 15.6% linoleic acid (18:2), and 2.1% and 1.2% linolenic acid (18:3). Our previous studies have showed that camellia oil can ameliorate ketoprofen- and ethanol-induced GI mucosal damage (Cheng et al., 2014; Tu et al., 2017). Furthermore, oleic acid can prevent gastric ulcerogenesis (Cheng et al., 2015) and promote dermal reconstruction and wound closure through its antioxidant or anti-inflammatory effects (Donato-Trancoso, Monte-Alto-Costa, & Romana-Souza, 2016). Additionally, phenolic compounds possess a strong antiulcer ability that prevents gastric mucosal damage from NSAIDs (Cheng et al., 2017). Catechin ameliorates gastric mucosal injury induced by acetic acid, ethanol, and NSAIDs (Boligon et al., 2014; Cheng, Wu, Ho, & Yen, 2013; Qian et al., 2018). Furthermore, α-tocopherol prevents gastric lesions induced by Helicobacter pylori, NSAIDs, ethanol, stress, acid, and pyloric ligation (Kamisah, Qodriyah, Chua, & Nur Azlina, 2014). Moreover, squalene exhibits immune modulation, oxidative improvement, and prevention of dextran sulfate sodium (DSS)-induced acute colitis (Sanchez-Fidalgo, Villegas, Rosillo, Aparicio-Soto, & de la Lastra, 2015). All of the aforementioned facts indicate that camellia oil possesses considerable abilities for GI tract health management.
NSAID-induced gastric ulcers are associated with the expressions of IL-6 and TNF-α cytokines (Antonisamy et al., 2016; Higashimori et al., 2016). In addition, NSAIDs cause GI ulcer through the activation of COX, and COX can be divided into COX-1 and COX-2. COX-1 is mainly associated with kidney, platelet function, and gastric mucosal blood flow; COX-2 is primarily associated with inflammation (Battistella, Mamdami, Juurlink, Rabeneck, & Laupacis, 2005; Felson, 2016). However, the adverse effects caused by NSAIDs are presumed to be associated with the inhibition of COX-1 activity and the promotion of COX-2 activity. The effect of TMS camellia oil treatment on gastric mucosal IL-6, TNF-α, and COX-2 protein expressions in indomethacininduced GI mucosal damage BALB/c mice is presented in Fig. 5. IL-6 production, TNF-α secretion, and COX-2 protein expression were notably increased in the indomethacin-induced group compared with those in the control group (P < 0.05). Pretreatment with 2 mL/kg b.w./day of TMS camellia oil significantly reduced IL-6 production, TNF-α secretion, and COX-2 protein expression compared with indomethacin treatment, indicating that camellia oil can reduce inflammatory expressions in indomethacin-induced gastric mucosal damage.
3.6. Effect of TMS camellia oil on gastric mucosal HO-1, VEGF, and TGF-β protein expressions in indomethacin-induced GI mucosal damage BALB/c mice NSAIDs cause gastric ulcers, which are often accompanied by oxidative stress. Antioxidant enzyme HO-1 plays a vital role in the treatment of GI tract damage (Onyiah et al., 2013). In addition, NSAIDs inhibit the secretion and performance of the growth factors VEGF and TGF-β, leading to a delay in ulcer repair. The protein expressions of HO1, VEGF, and TGF-β in the gastric mucosa of BALB/c mice induced by indomethacin are presented in Fig. 6. Although the indomethacin group exhibited a marked decrease in the protein levels of HO-1, VEGF, and TGF-β compared with the control group (P < 0.05), the expressions of HO-1, VEGF, and TGF-β proteins were significantly increased in the lansoprazole + indomethacin group, COL + indomethacin group, and COH + indomethacin group (P < 0.05) (Fig. 6). This result evidenced that camellia oil might promote the healing of gastric mucosal wounds by enhancing the levels of antioxidant enzymes and growth factors.
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Fig. 6. Effect of TMS camellia oil on the protein expressions of HO-1 (A), VEGF (B), and TGF-β (C) in the gastric mucosa of BALB/c mice induced by indomethacin. The animals were administered camellia oil (1 and 2 mL/kg b.w./day) orally for 21 consecutive days or lansoprazole (30 mg/kg b.w.) for 7 days. Indomethacin (40 mg/kg b.w.) was then orally administered to all animals for 24 h. The data represent the means ± SD of eight mice. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group. LPZ: 30 mg/kg b.w. of lansoprazole, COL: 1 mL/kg b.w./day of camellia oil, COH: 2 mL/kg b.w./day of camellia oil.
enhancing growth factor TGF-β expression (Sharma, Ganguly, Paul, Maulik, & Swarnakar, 2012). In this study, the levels of VEGF and TGFβ proteins significantly reduced under gastric damage, and this reduction was reversed by camellia oil and lansoprazole. The effects of camellia oil and lansoprazole were similar (Fig. 6). Furthermore, the expression and regulation of HO-1 play crucial roles in the protection against ischemia-reperfusion injury, inflammatory bowel disease, necrotizing enterocolitis, radiation enteritis, acute pancreatitis, chronic pancreatitis, cytotoxic action of indomethacin, and DSS-induced colitis (Chang, Xue, Sharma, & Habtezion, 2015). Therefore, the upregulation of HO-1 in various organ system models may trigger an endogenous defensive mechanism against oxidative stress, inflammation, and tissue injury (Chang et al., 2015). As mentioned previously, the protein expressions of HO-1, VEGF, and TGF-β may play a fundamental role in accelerating GI wound healing following indomethacin-induced injury. In this study, the expressions of gastric mucosal HO-1, VEGF, and TGF-β proteins markedly decreased in indomethacin-treated BALB/c mice (P < 0.05). However, pretreatment with camellia oil markedly elevated the HO-1, VEGF, and TGF-β protein levels, thus indicating that camellia oil might promote wound healing by enhancing the levels of antioxidant enzymes and growth factors.
In the present study, pretreatment with TMS and TML camellia oils significantly increased the cell viability, reduced ROS production, and enhanced wound healing in indomethacin-damaged human Int-407 cells. These effects were similar to those of lansoprazole (Figs. 1 and 2). An increase in proinflammatory IL-6 and TNF-α cytokine secretion is highly associated with gastropathy caused by the constant use of indomethacin (Antonisamy et al., 2016; Higashimori et al., 2016). In addition, the adverse effects of NSAIDs are presumed to be associated with the suppression of COX-1 activity and promotion of COX-2 activity (Battistella et al., 2005; Felson, 2016). In the current study, an increase in inflammatory mediators IL-6, TNF-α, and COX-2 was observed in BALB/c mice with gastric mucosal damage induced by indomethacin (Fig. 5). Notably, camellia oil exhibited immunosuppressive effects by dramatically inhibiting these inflammatory mediators (IL-6, TNF-α, and COX-2) in indomethacin-induced gastric damage. In addition, the antiinflammatory effect (IL-6 and TNF-α) of lansoprazole was superior to that of camellia oil. The results of H&E staining were consistent with inflammatory cytokine expressions. It indicated indomethacin-induced severe degeneration and necrosis with hemorrhage of the stomach mucosal layer. Camellia oil had a beneficial protective effect against indomethacin-induced gastric injury and inflammation. Additionally, the protective effects of lansoprazole were stronger than those of camellia oil (Figs. 4 and 5). Previous studies have also reported the possible preventive capacity of camellia oil on ketoprofen- or ethanol-induced gastric mucosal damage through diminishing the injury and hemorrhage scores, inhibiting the production of inflammatory mediators, and reversing the antioxidant system (Cheng et al., 2014; Tu et al., 2017). NSAIDs restrict gastric cell proliferation and tissue repair and impede angiogenesis thus delaying the healing of gastroduodenal ulcers (Soreide, 2018; Soreide et al., 2015). Jones et al. (2001) reported that the healing of gastric ulcer wounds is associated with growth factor VEGF expression. VEGF can specifically act on tyrosine kinase receptors or kinase receptors in vascular endothelial cells, which promotes endothelial proliferation and migration, increases vascular permeability, and accelerates ulcer healing (Wallace, Dicay, McKnight, & Dudar, 2006). Indomethacin inhibits COX-1 expression and increases the endostatin/VEGF ratio, which could reduce VEGF release and result in angiogenesis suppression (Yadav et al., 2012). Therefore, VEGF plays a major role in promoting the restoration of connective tissues and angiogenesis during the repair of peptic ulcer, and it accelerates the healing of the ulcer area. A previous study revealed that curcumin, a natural phytochemical, promotes the healing of ulcer wounds by
5. Conclusions Based on the in vitro study conducted in this research, pretreatment with camellia oils significantly increased cell viability, reduced ROS production, and promoted wound healing in indomethacin-induced human intestine Int-407 cells. Similarly, the in vivo study provided compelling evidence that the preadministration of camellia oils dramatically prevents gastric wound generation in indomethacin-induced gastric damage BALB/c mice. This phenomenon may occur through an increase in the expressions of antioxidant enzyme HO-1, diminution of the levels of IL-6, TNF-α, and COX-2 inflammatory mediators, or the elevation of VEGF and TGF-β. The effects were similar to those observed for lansoprazole (positive control). In sum, these results evidence that camellia oils plays a key role in the prevention of adverse effects through its anti-inflammatory activity and antioxidant ability by upregulating growth factors in NSAID-induced GI damage. Ethics statement In this research work, the procedures for animal experiment were according to the rule of National Institutes of Health (NIH). The 7
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experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC No. 105-004) of National Chung Hsing University (Taichung, Taiwan).
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Acknowledgments This research was supported in part by Council of Agriculture, Taiwan under grant Nos. 1042008 and 105AS-15.3.1-FD-Z1(4). The authors thank Dr. J. W. Liao of the Graduate Institute of Veterinary Pathobiology, National Chung Hsing University for the assistance on the evaluation of pathological histology. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103539. References Akhter, A., Pfau, P. R., & Gopal, D. V. (2016). Rectal indomethacin to prevent post-ERCP pancreatitis. Gastroenterology, 151, 568–569. https://doi.org/10.1053/j.gastro.2016. 05.053. Anheyer, D., Frawley, J., Koch, A. K., Lauche, R., Langhorst, J., Dobos, G., & Cramer, H. (2017). Herbal medicines for gastrointestinal disorders in children and adolescents: A systematic review. Pediatrics, 139, pii: e20170062. https://doi.org/10.1542/peds. 2017-0062. Antonisamy, P., Arasu, M. V., Dhanasekaran, M., Choi, K. C., Aravinthan, A., Kim, N. S., ... Kim, J. H. (2016). Protective effects of trigonelline against indomethacin-induced gastric ulcer in rats and potential underlying mechanisms. Food & Function, 7, 398–408. https://doi.org/10.1039/c5fo00403a. Battistella, M., Mamdami, M. M., Juurlink, D. N., Rabeneck, L., & Laupacis, A. (2005). Risk of upper gastrointestinal hemorrhage in warfarin users treated with nonselective NSAIDs or COX-2 inhibitors. Archives of Internal Medicine, 165, 189–192. https://doi. org/10.1001/archinte.165.2.189. Bhattacharyya, A., Chattopadhyay, R., Mitra, S., & Crowe, S. E. (2014). Oxidative stress: An essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiological Reviews, 94, 329–354. https://doi.org/10.1152/physrev.00040.2012. Bjarnason, I., Scarpignato, C., Holmgren, E., Olszewski, M., Rainsford, K. D., & Lanas, A. (2018). Mechanisms of damage to the gastrointestinal tract from nonsteroidal antiInflammatory drugs. Gastroenterology, 154, 500–514. https://doi.org/10.1053/j. gastro.2017.10.049. Blanchard, A., Vargas-Poussou, R., Vallet, M., Caumont-Prim, A., Allard, J., Desport, E., ... Azizi, M. (2015). Indomethacin, amiloride, or eplerenone for treating hypokalemia in Gitelman syndrome. Journals of the American Society of Nephrology, 26, 468–475. https://doi.org/10.1681/ASN.2014030293. Boligon, A. A., de Freitas, R. B., de Brum, T. F., Waczuk, E. P., Klimaczewski, C. V., de Avila, D. S., ... de Freitas Bauermann, L. (2014). Antiulcerogenic activity of Scutia buxifolia on gastric ulcers induced by ethanol in rats. Acta Pharmaceutica Sinica B, 4, 358–367. https://doi.org/10.1016/j.apsb.2014.05.001. Chai, J., Norng, M., Tarnawski, A. S., & Chow, J. (2007). A critical role of serum response factor in myofibroblast differentiation during experimental oesophageal ulcer healing in rats. Gut, 56, 621–630. https://doi.org/10.1136/gut.2006.106674. Chang, M., Xue, J., Sharma, V., & Habtezion, A. (2015). Protective role of hemeoxygenase-1 in gastrointestinal diseases. Cellular and Molecular Life Sciences, 72, 1161–1173. https://doi.org/10.1007/s00018-014-1790-1. Cheng, Y. T., Lu, C. C., & Yen, G. C. (2015). Beneficial effects of Camellia oil (Camellia oleifera Abel.) on hepatoprotective and gastroprotective activities. Journal of Nutritional Science and Vitaminology (Tokyo), 61 Suppl, S100–S102. https://doi.org/ 10.3177/jnsv.61.S100. Cheng, Y. T., Lu, C. C., & Yen, G. C. (2017). Phytochemicals enhance antioxidant enzyme expression to protect against NSAID-induced oxidative damage of the gastrointestinal mucosa. Molecular Nutrition & Food Research, 61. https://doi.org/10.1002/mnfr. 201600659. Cheng, Y. T., Wu, C. H., Ho, C. Y., & Yen, G. C. (2013). Catechin protects against ketoprofen-induced oxidative damage of the gastric mucosa by up-regulating Nrf2 in vitro and in vivo. The Journal of Nutritional Biochemistry, 24, 475–483. https://doi.org/10. 1016/j.jnutbio.2012.01.010. Cheng, Y. T., Wu, S. L., Ho, C. Y., Huang, S. M., Cheng, C. L., & Yen, G. C. (2014). Beneficial effects of Camellia oil (Camellia oleifera Abel.) on ketoprofen-induced gastrointestinal mucosal damage through upregulation of HO-1 and VEGF. Journal of Agricultural and Food Chemistry, 62, 642–650. https://doi.org/10.1021/jf404614k. Chiou, S. K., Tanigawa, T., Akahoshi, T., Abdelkarim, B., Jones, M. K., & Tarnawski, A. S. (2005). Survivin: A novel target for indomethacin-induced gastric injury. Gastroenterology, 128, 63–73. https://doi.org/10.1053/j.gastro.2004.10.008. de Groot, D. J., Timmer, T., Spierings, D. C., Le, T. K., de Jong, S., & de Vries, E. G. (2005).
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Protective effects of camellia oil (Camellia brevistyla) against indomethacininduced gastrointestinal mucosal damage in vitro and in vivo Ruei-Yu Wanga,1, Yu-Tang Tungb,1, Sheng-Yi Chena,1, Ya-Lin Leec, Gow-Chin Yena,d,
T
⁎
a
Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan c Biotechnology Division, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan, 189 Zhongzheng Road, Wufeng Dist., Taichung 41362, Taiwan d Graduate Institute of Food Safety, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Camellia oil Gastrointestinal mucosal damage Indomethacin Antioxidant enzyme Inflammation
Accumulating evidence reveals that nonsteroidal anti-inflammatory drugs (NSAIDs), such as indomethacin, cause oxidative stress and inflammation, which consequently cause gastrointestinal (GI) mucosal damage. Camellia oil, a common edible oil used in Asia, has excellent antioxidative and anti-inflammatory characteristics. Herein, we examined the benefits and protective effects of camellia oil in indomethacin-induced human intestinal Int-407 cells and a mouse model of indomethacin-induced gastric mucosal damage. Camellia oil pretreatment significantly increased cell viability and wound healing and reduced reactive oxygen species production in indomethacin-induced Int-407 cells. In vivo experiments revealed that camellia oil preadministration prevented gastric wound generation by decreasing inflammatory mediators interleukin-6, tumor necrosis factorα, and cyclooxygenase-2 levels; increasing heme oxygenase-1 antioxidant protein level; and elevating transforming growth factor-β and vascular endothelial growth factor levels in indomethacin-induced BALB/c mice. Thus, camellia oil is a functional dietary oil that prevents oxidative damage and inflammation in NSAID-induced GI mucosal damage.
1. Introduction Peptic ulcer is a common disease of the digestive system that affects approximately 10% of the global population (Lanas & Chan, 2017). The gastrointestinal (GI) mucosa is the first line of defense against toxic substances, and long-term exposure to these toxic substances can cause gastric bleeding, ulceration, and perforation (Tseng et al., 2015). GI mucosal damage develops because of an imbalance between aggressive factors (drugs, excessive alcohol intake, smoking, stress, hyperacidity, and excessive secretion of gastric acid, pepsin, and bile acid) (Lanas & Chan, 2017; Soreide et al., 2015) and protective factors (mucin glycoproteins, bicarbonate secretion, and prostaglandin E synthase). Inflammation of the GI mucosa causes disruption of mucosal integrity (Yuan, Padol, & Hunt, 2006). Nonsteroidal anti-inflammatory drugs (NSAIDs) are aggressive factors causing GI mucosal damage; these include aspirin, ibuprofen, naproxen, sulindac, mefenamic acid, and indomethacin (Maddirevula,
Abanemai, & Alkuraya, 2016; Wolfe, Lichtenstein, & Singh, 1999). More than 30 million people take NSAIDs every day for their pain relieving and inflammation reducing effects (Bjarnason et al., 2018). Indomethacin, one of the most commonly prescribed NSAIDs, has been used for treating acute gout-like arthritis, preventing pancreatitis after endoscopic retrograde cholangiopancreatography, inducing apoptosis in small-cell lung cancer, promoting survival of new neurons, and treating hypokalaemia in Gitelman syndrome by regulating inflammatory response (Akhter, Pfau, & Gopal, 2016; Blanchard et al., 2015; de Groot et al., 2005; Gaffo & Saag, 2007; Hain et al., 2018). Nevertheless, numerous studies have demonstrated that indomethacin has adverse effects on the GI mucosa, such as gastric ulcers (Chiou et al., 2005; Ramadan, Bonin, Kennedy, Hambley, & Lay, 2005). In addition, indomethacin results in excessive gastric acid secretion and reactive oxygen species (ROS) production, and it interferes with mucosal cell regeneration (Maity et al., 2009; Shahin, Abdelkader, & Safar, 2018; Wallace, 2008). Therefore, at present, the priority is to effectively
Abbreviations: COX-2, cyclooxygenase-2; GI, gastrointestinal; HO-1, heme oxygenase-1; LPZ, lansoprazole; NSAIDs, nonsteroidal anti-inflammatory drugs; ROS, reactive oxygen species ⁎ Corresponding author at: Department of Food Science and Biotechnology, National Chung Hsing University, 145 Xingda Road, Taichung 40227, Taiwan. E-mail address: [email protected] (G.-C. Yen). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.103539 Received 2 April 2019; Received in revised form 23 August 2019; Accepted 26 August 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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containers at 4 °C in refrigerator until further use.
prevent or safely treat indomethacin-induced GI mucosal damage. In recent years, the search for traditional medicinal plants to treat ulcer has received increasing attention. The application of botanicals for treating GI disorder has generated a renewed interest (Anheyer et al., 2017; Simren & Tack, 2018). The seeds of Camellia brevistyla are rich in oil and suitable are for the extraction of high-quality camellia oils; this oil is generally used as edible oil in China, Japan, Korea, India, and Taiwan (Su, Shih, & Lin, 2014). Camellia oil has been traditionally used to treat burn wounds and GI cramps. Camellia oil contains abundant unsaturated fatty acids, including monounsaturated oleic acid (C18:1) and polyunsaturated linoleic acid (C18:2), phenolic compounds, catechins, α-tocopherol, and squalene (Wang, Zeng, Del Mar Contreras, & Wang, 2017; Yang, Liu, Chen, Lin, & Wang, 2016). Notably, our previous studies have demonstrated that camellia oil exhibits powerful antioxidant activity and health beneficial bioactive capacity for preventing diseases related to oxidative injury, protecting against CCl4-induced hepatic damage, and ameliorating GI mucosal injury caused by ketoprofen and ethanol treatment (Cheng, Lu, & Yen, 2015; Cheng et al., 2014; Lee, Shih, Hsu, & Yen, 2007; Lee & Yen, 2006; Lee, Tung, Wu, Tu, & Yen, 2018; Tu, Tung, Lee, & Yen, 2017). Therefore, camellia oil may be a potent candidate for further develop as functional food for treating GI disorders. However, the protective effect of camellia oils on indomethacin-induced GI mucosal damage remains unknown. In this study, we investigated its potential protective action against GI disorders by using human intestinal Int-407 cell lines and acute indomethacin-induced gastric mucosal damage mice models.
2.3. Cell culture The Int-407 human intestinal cell lines used in the study were purchased from the Bioresource Collection and Research Center (BCRC 60022, Hsinchu, Taiwan). The cells were grown in 90% BME with EBSS, 10% FBS, 0.37% sodium bicarbonate, and 1% PS and placed in a cell culture incubator (37 °C, 5% CO2). 2.4. MTT assay The cells were pretreated with 75, 100, or 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Lansoprazole, a well-known treatment for peptic ulcer disease, was used as a positive control. The dose of lansoprazole was based on that used in a previous study (Rybniker et al., 2015). After 6 h of incubation with test samples, cells were treated with 800 μM indomethacin for 24 h. After treatment, MTT assay was conducted according to the method reported in our previous study (Tu et al., 2017). 2.5. Intracellular ROS assessment Cells were pretreated with 75, 100, or 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Lansoprazole was used as a positive control. Following 6 h of incubation with test samples, cells were treated with 400 μM indomethacin for 1 h. Following 1 h of incubation, the 106 cells were collected and resuspended in 1 mL of phosphatebuffered saline (PBS). Cell suspension (195 μL) incubated with 5 μL of DCFH-DA (400 μM) was loaded into a 96-well plate for 1 h. The cells were then permeabilized with 0.05% Triton X-100 at room temperature for 15 min. The dichlorofluorescein fluorescence intensity was detected using a FLUOstar Galaxy reader at 485 nm (excitation) and an emission wavelength of 520 nm.
2. Materials and methods 2.1. Chemical reagents Indomethacin, dimethyl sulfoxide (DMSO), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Penicillin–streptomycin solution (PS), Basal Medium Eagle (BME), and Earle’s balanced salt solution (EBSS) were bought from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Cromwell, CT, USA). TRIzol and SYBR safe DNA gel stain were purchased from Invitrogen (Carlsbad, CA, USA). Ibidi Culture-Insert was purchased from ibidi GmbH (Martinsried, Germany). Disodium hydrogen phosphate (Na2HPO4) and dipotassium hydrogen phosphate (K2HPO4) were obtained from J.T. Baker (Chicago, IL, USA). Mouse interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were purchased from eBioscience (San Diego, CA, USA). Antibodies against β-actin, vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGF-β) were purchased from Novus Biologicals (Littleton, CO, USA). Heme oxygenase-1 (HO-1) antibody was obtained from Abcam (Cambridge, UK). Cyclooxygenase2 (COX-2) antibody was purchased from Cayman Chemical (Ann Arbor, MI, USA). A WesternBright ECL kit was purchased from Advansta (San Jose, CA, USA). iScript cDNA synthesis kit, Bio-Rad protein assay dye reagent concentrate, Bio-Rad protein ladder, and acrylamide were purchased from Bio-Rad (Hercules, CA, USA). A BCA protein assay kit was purchased from Pierce (Waltham, MA, USA).
2.6. Wound healing assay First, 105 cells were loaded into the ibidi Culture-Insert. The ibidi Culture-Insert was then removed when cell growth reached 90% confluence in 24-well tissue culture plates. Subsequently, the wound assay was performed and unattached cells were removed using PBS. After that, the cells were pretreated with 75, 100, or 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Lansoprazole was used as a positive control. Following 6 h of incubation with camellia oils or lansoprazole, the cells were treated with 100 μM indomethacin for 24 h. The cell migration distance was captured and recorded using an OLYMPUS IX71 inverted microscope (Osaka, Japan). 2.7. Protein extraction from human Int-407 cells The cells were pretreated with 75, 100, or 150 μg/mL of TMS camellia oil and 10 μM lansoprazole for 6 h before being incubated with 400 μM indomethacin for 3 h. Lansoprazole was used as a positive control. After stimulations, total protein was extracted using the Total Protein Extraction Kit (Millipore, Bedford, MA, USA) following the manufacturer’s instructions. Total quantification of the total protein in the human Int-407 cells was performed using a Pierce BCA protein assay kit (Waltham, MA, USA).
2.2. Preparation of camellia oils The camellia oils from Maokong and Shiding District (New Taipei City, Taiwan) were provided by Dr. Ya-Lin Lee (Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan). Camellia oils were prepared from camellia seeds (Camellia brevistyla CohenStuart) with roasting at 100 °C for 10 min prior to press the oil which was named as Taipei Maokong Shiding (TMS). Besides, Taipei Maokong Shiding extracted by low temperature and without roasting was named TML. Commercially refined soybean oil was purchased from Sigma–Aldrich (St. Louis, MO, USA). All oils were stored in airtight
2.8. Animals Four-week-old male BALB/c mice weighing 20–25 g were purchased from BioLASCO (Taipei, Taiwan). The animals were maintained under controlled temperature (23 ± 1 °C) and humidity (65–70%) and under a 12:12 light–dark cycle. They were fed the Laboratory Rodent Diet 5001 for 2 weeks to adapt to the environment. The experimental 2
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Fig. 1. Effects of camellia oil (TMS and TML) on cell viability (A) or ROS production (B) in indomethacin-induced human Int-407 cells. The cells were pretreated with 75, 100, and 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Following 6 h of incubation with camellia oils, the cells were treated with indomethacin. Cell viability was detected through MTT assay. Results are presented as the means ± SD. n = 3. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group.
2.11. ELISA
procedures and animal care were in accordance with the guidelines provided by the Institutional Animal Care and Use Committee (IACUC approval No. 105-004) of National Chung Hsing University (Taichung, Taiwan). In this study, indomethacin was used to induce GI mucosal damage according to the method by Morise, Granger, Fuseler, Anderson, and Grisham (1999) with slight modifications. Six-week-old BALB/c mice were randomly distributed into the following five groups (n = 8 per group): (1) control group; (2) indomethacin group; (3) lansoprazole + indomethacin group (LPZ + Indomethacin); (4) Camellia oil low dose (COL, 1 mL/kg b.w./day of camellia oil) + indomethacin group (COL + Indomethacin); and (5) Camellia oil high dose (COH, 2 mL/kg b.w./day of camellia oil) + indomethacin group (COH + Indomethacin). The COL and COH groups received oral gavage of TMS camellia oil (1 and 2 mL/kg b.w./day, respectively) for 3 weeks. The control and indomethacin groups were both administered soybean oil (2 mL/kg b.w./day) for 3 weeks, and the positive control group received lansoprazole (30 mg/kg b.w./day) for 7 days. On the 21st day, all the mice except for those in the control group received oral gavage of indomethacin (40 mg/kg b.w.) for 24 h. After 24 h, all mice were sacrificed using isoflurane.
The proteins in the mice gastric mucosa were assayed for the IL-6 and TNF-α productions using a specific ELISA kit in accordance with the manufacturer’s instructions.
2.12. Western blot analysis Western blotting for the detection of HO-1, VEGF, TGF-β, COX-2, and β-actin was performed according to our previous report (Cheng et al., 2014).
2.13. Statistical analysis All data are expressed as the mean ± standard deviation [SD] (n = 3 for the cell assay and n = 8 for the animal assay). Statistical data analysis was conducted using the Student's t-test for independent samples between independent groups. A P value < 0.05 was considered statistically significant.
2.9. Histopathologic studies 3. Results The stomach tissues collected from the BALB/c mice were fixed in 10% neutral formaldehyde and processed for histological examination. Hematoxylin–eosin (H&E) staining was performed and the histological injury scores of the gastric mucosa were assigned as described in our previous study (Tung, Huang, Lin, & Yen, 2018). The degrees of lesion were classified according to their severity as follows: 1 = minimal (degree of lesion < 1%); 2 = slight (degree of lesion approximately 1–25%); 3 = moderate (degree of lesion approximately 26–50%); 4 = moderate or severe (degree of lesion approximately 51–75%); and 5 = severe or high (degree of lesion approximately 76–100%).
3.1. Effects of camellia oils on the cell viability and ROS production in indomethacin- induced human Int-407 cells A previous study revealed that drug-induced GI tract damage can cause oxidative GI mucosal damage and cell death (Bjarnason et al., 2018). The effects of TMS and TML camellia oils and lansoprazole on the cell viability and ROS production in indomethacin- induced human Int-407 cells are presented in Fig. 1. The results indicated that pretreatment with 75–150 μg/mL of TMS and TML camellia oils and 10 μM lansoprazole significantly increased the cell viability in indomethacininduced human Int-407 cells (P < 0.05) (Fig. 1A). NSAIDs reduce the activities of antioxidant enzymes in the body, which results in excessive ROS accumulation and directly causes intestinal mucosal oxidative damage (Bhattacharyya, Chattopadhyay, Mitra, & Crowe, 2014). As illustrated in Fig. 1B, TMS and TML camellia oils had excellent preventive effects on ROS production. At the dose of 150 μg/mL, TMS and TML camellia oils both significantly inhibited ROS production in indomethacin-induced human Int-407 cells (P < 0.05).
2.10. Gastric mucosal homogenate preparation Gastric mucosal homogenate was executed on the basis of the method reported in our previous study (Cheng et al., 2014). The total protein levels of the gastric mucosa were quantified using a Pierce BCA protein assay kit (Waltham, MA, USA).
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Fig. 2. Effects of camellia oils (TMS and TML) on the cell migration ability with wound healing in indomethacin-induced human Int-407 cells (A and B). Representative phase-contrast images of cells into the wounded area in the wound healing assay. Cells were pretreated with 75, 100, and 150 μg/mL of TMS or TML camellia oils and 10 μM lansoprazole. Following 6 h of incubation with camellia oils, cells were treated with 100 μM indomethacin for 24 h. Graphical presentation of the cell migration ability observed. Results are presented as the means ± SD. n = 3. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group.
In the wound healing assay, the migration area of indomethacininduced human Int-407 cells was measured. As illustrated in Fig. 2A and 2B, treatment with 75, 100, or 150 μg/mL of TMS or TML camellia oils enhanced wound healing in indomethacin-induced human Int-407 cells (P < 0.05). In addition, the results revealed that TMS camellia oil had a stronger effect on ROS production inhibition and wound healing capacity than TML camellia oil in indomethacin-induced human Int407 cells (Figs. 1B and 2). Therefore, the following in vitro and in vivo experiments were performed to identify the underlying molecular mechanisms of GI mucosal injury prevention were focused on TMS camellia oil intervention only.
integrity, and improves vascular permeability (Ohno et al., 2008). Therefore, VEGF plays a major role in ulcer healing. The results in the present study indicated that indomethacin significantly reduced VEGF protein expression (P < 0.05) and that pretreatment with 75, 100, and 150 μg/mL of TMS camellia oil considerably enhanced VEGF protein expression compared with pretreatment with the positive control (Fig. 3B). Recent studies have reported that several growth factors are involved in the healing process of gastric ulcers, such as VEGF and TGFβ (Chai, Norng, Tarnawski, & Chow, 2007; Ohno et al., 2008). Consistently, our results also revealed that indomethacin significantly reduced the expression of TGF-β protein and that pretreatment with 75, 100, and 150 μg/mL of TMS camellia oil was more effective than treatment with 10 μM lansoprazole in enhancing TGF-β protein expression (Fig. 3C).
3.3. Effect of TMS camellia oil on the HO-1, VEGF, and TGF-β protein expressions in indomethacin-induced human Int-407 cells
3.4. Effect of TMS camellia oil on gastric pathology improvement in indomethacin-induced GI mucosal damage BALB/c mice
As illustrated in Fig. 3, the protein expressions of HO-1, VEGF, and TGF-β in the indomethacin-induced human Int-407 cells decreased markedly after treatment with indomethacin (P < 0.05). HO-1 plays a crucial role in the preservation of intestinal function integrity (Onyiah et al., 2013). The results demonstrated that treatment with various concentrations (75, 100, and 150 μg/mL) of TMS camellia oil and 10 μM lansoprazole significantly increased the expression of HO-1 protein in indomethacin-induced human Int-407 cells (P < 0.05) (Fig. 3A). The present study revealed that TMS camellia oil had a stronger effect on HO-1 protein expression than did 10 μM lansoprazole (positive control). These results implied that pretreatment of Int-407 cells with TMS camellia oil for 6 h could maintain intestinal mucosal integrity. VEGF promotes angiogenesis, maintains normal blood vessel
NSAIDs are common antipyretic and analgesic drugs, but they induce adverse side effects on gastric lesions (Chiou et al., 2005). The morphology, H&E stain, and histological injury score of the gastric mucosa are presented in Fig. 4. Although the indomethacin group exhibited gastric mucosal bleeding, pretreatment with TMS camellia oil (1 and 2 mL/kg b.w./day) for 21 days or clinical antiulcer drug lansoprazole (30 mg/kg b.w.) for 7 days effectively reduced the gastric mucosal bleeding caused by indomethacin (Fig. 4A), suggesting that TMS camellia oil can protect the integrity of the gastric mucosa. The gastric mucosal tissue in the indomethacin group presented severe degeneration and necrosis with hemorrhage in the gastric mucosal layer (Fig. 4B and C). Pretreatment with TMS camellia oil (1 and 2 mL/kg b.w./day) for 21 days or lansoprazole (30 mg/kg b.w.) for 7 days reduced the
3.2. Effect of camellia oils on the wound healing in indomethacin-induced human Int-407 cells
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Fig. 3. Effect of TMS camellia oil on the protein expression of HO-1 (A), VEGF (B), and TGF-β (C) in indomethacin-induced human Int-407 cells. The cells were pretreated with 75, 100, and 150 μg/mL of TMS camellia oil and 10 μM lansoprazole. Following 6 h of incubation with camellia oils, the cells were treated with 400 μM indomethacin for 3 h. Results are presented as the means ± SD. n = 3. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group.
significantly reduced injury severity. The aforementioned results indicated that TMS camellia oil maintains the integrity of the gastric mucosa and has a satisfactory protective effect on the pathology in gastric mucosal injury models.
hemorrhage in the stomach mucosal layer. As illustrated in Fig. 4D and Supplementary Table 1, damage to the gastric mucosal tissue was significantly higher in the indomethacin-induced groups than in the control group. Oral administration of a low dose (1 mL/kg b.w./day) and high dose (2 mL/kg b.w./day) of TMS camellia oil for 21 days
Fig. 4. Effect of TMS camellia oil on the morphology (A), H&E stain 40× (B) and 400× (C), and histological injury score (D) of stomach in BALB/c mice induced by indomethacin. The animals were administered camellia oil (1 and 2 mL/kg b.w./day) orally for 21 consecutive days or lansoprazole (30 mg/kg b.w.) for 7 days. Indomethacin (40 mg/kg b.w.) was then orally administered to all animals for 24 h. The data represent the means ± SD of eight mice. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group. LPZ: 30 mg/kg b.w. of lansoprazole, COL: 1 mL/kg b.w./day of camellia oil, COH: 2 mL/kg b.w./day of camellia oil. 5
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Fig. 5. Effect of TMS camellia oil on IL-6 production (A), TNF-α production (B), and COX-2 protein expression (C) in the gastric mucosa of BALB/c mice induced by indomethacin. The animals were administered camellia oil (1 and 2 mL/kg b.w./day) orally for 21 consecutive days or lansoprazole (30 mg/kg b.w.) for 7 days. Indomethacin (40 mg/kg b.w.) was then orally administered to all animals for 24 h. The data represent the means ± SD of eight mice. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group. LPZ: 30 mg/kg b.w. of lansoprazole, COL: 1 mL/kg b.w./day of camellia oil, COH: 2 mL/kg b.w./day of camellia oil.
4. Discussion
3.5. Effect of TMS camellia oil on gastric mucosal IL-6, TNF-α, and COX-2 protein expressions in indomethacin-induced GI mucosal damage BALB/c mice
Accumulating evidence revealed that NSAIDs are extensively used for the prevention of cancer, pain, osteoarthritis, fever (antipyretic), hypertension, preeclampsia, Alzheimer’s disease, and inflammation (Felson, 2016; Hedberg et al., 2019; Taler, 2018; Varvel et al., 2009). The main risk arising from NSAID use is GI toxicity (Bjarnason et al., 2018), which induces gastric ulcers and inhibits ulcer healing (Lanas & Chan, 2017). Previous studies have demonstrated that GI mucosal injury is related to the accumulation of ROS and lipid peroxidation products in the mucosal tissue, and long-term oxidative stress in mucosal tissues leads to GI bleeding, ulceration, and perforation (Bhattacharyya et al., 2014; Bjarnason et al., 2018). Cumulative evidence suggests that diet plays a central role in GI health maintenance and protection against peptic ulcer (Cheng, Lu, & Yen, 2017; Farzaei, Abdollahi, & Rahimi, 2015; Veldhoen & Brucklacher-Waldert, 2012; Yen, Tsai, Lu, & Weng, 2018). Camellia oil contains a variety of antioxidant components such as oleic acid, phenolic compounds, catechin, α-tocopherol, and squalene (Cheng et al., 2014; Tu et al., 2017). The results of our study demonstrated that the phenolic content of TMS and TML camellia oils was 0.112 ± 0.007 and 0.286 ± 0.032 mg gallic acid/mL, respectively, and the α-tocopherol content was (378.32 ± 6.87 and 388.16 ± 12.57 ppm, respectively. In addition, the fatty acid compositions of TMS and TML camellia oils, respectively, were 6.0% and 7.1% palmitic acid (16:0), 0.8% and 3.3% stearic acid (18:0), 78.8% and 72.8% oleic acid (18:1), 12.2% and 15.6% linoleic acid (18:2), and 2.1% and 1.2% linolenic acid (18:3). Our previous studies have showed that camellia oil can ameliorate ketoprofen- and ethanol-induced GI mucosal damage (Cheng et al., 2014; Tu et al., 2017). Furthermore, oleic acid can prevent gastric ulcerogenesis (Cheng et al., 2015) and promote dermal reconstruction and wound closure through its antioxidant or anti-inflammatory effects (Donato-Trancoso, Monte-Alto-Costa, & Romana-Souza, 2016). Additionally, phenolic compounds possess a strong antiulcer ability that prevents gastric mucosal damage from NSAIDs (Cheng et al., 2017). Catechin ameliorates gastric mucosal injury induced by acetic acid, ethanol, and NSAIDs (Boligon et al., 2014; Cheng, Wu, Ho, & Yen, 2013; Qian et al., 2018). Furthermore, α-tocopherol prevents gastric lesions induced by Helicobacter pylori, NSAIDs, ethanol, stress, acid, and pyloric ligation (Kamisah, Qodriyah, Chua, & Nur Azlina, 2014). Moreover, squalene exhibits immune modulation, oxidative improvement, and prevention of dextran sulfate sodium (DSS)-induced acute colitis (Sanchez-Fidalgo, Villegas, Rosillo, Aparicio-Soto, & de la Lastra, 2015). All of the aforementioned facts indicate that camellia oil possesses considerable abilities for GI tract health management.
NSAID-induced gastric ulcers are associated with the expressions of IL-6 and TNF-α cytokines (Antonisamy et al., 2016; Higashimori et al., 2016). In addition, NSAIDs cause GI ulcer through the activation of COX, and COX can be divided into COX-1 and COX-2. COX-1 is mainly associated with kidney, platelet function, and gastric mucosal blood flow; COX-2 is primarily associated with inflammation (Battistella, Mamdami, Juurlink, Rabeneck, & Laupacis, 2005; Felson, 2016). However, the adverse effects caused by NSAIDs are presumed to be associated with the inhibition of COX-1 activity and the promotion of COX-2 activity. The effect of TMS camellia oil treatment on gastric mucosal IL-6, TNF-α, and COX-2 protein expressions in indomethacininduced GI mucosal damage BALB/c mice is presented in Fig. 5. IL-6 production, TNF-α secretion, and COX-2 protein expression were notably increased in the indomethacin-induced group compared with those in the control group (P < 0.05). Pretreatment with 2 mL/kg b.w./day of TMS camellia oil significantly reduced IL-6 production, TNF-α secretion, and COX-2 protein expression compared with indomethacin treatment, indicating that camellia oil can reduce inflammatory expressions in indomethacin-induced gastric mucosal damage.
3.6. Effect of TMS camellia oil on gastric mucosal HO-1, VEGF, and TGF-β protein expressions in indomethacin-induced GI mucosal damage BALB/c mice NSAIDs cause gastric ulcers, which are often accompanied by oxidative stress. Antioxidant enzyme HO-1 plays a vital role in the treatment of GI tract damage (Onyiah et al., 2013). In addition, NSAIDs inhibit the secretion and performance of the growth factors VEGF and TGF-β, leading to a delay in ulcer repair. The protein expressions of HO1, VEGF, and TGF-β in the gastric mucosa of BALB/c mice induced by indomethacin are presented in Fig. 6. Although the indomethacin group exhibited a marked decrease in the protein levels of HO-1, VEGF, and TGF-β compared with the control group (P < 0.05), the expressions of HO-1, VEGF, and TGF-β proteins were significantly increased in the lansoprazole + indomethacin group, COL + indomethacin group, and COH + indomethacin group (P < 0.05) (Fig. 6). This result evidenced that camellia oil might promote the healing of gastric mucosal wounds by enhancing the levels of antioxidant enzymes and growth factors.
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Fig. 6. Effect of TMS camellia oil on the protein expressions of HO-1 (A), VEGF (B), and TGF-β (C) in the gastric mucosa of BALB/c mice induced by indomethacin. The animals were administered camellia oil (1 and 2 mL/kg b.w./day) orally for 21 consecutive days or lansoprazole (30 mg/kg b.w.) for 7 days. Indomethacin (40 mg/kg b.w.) was then orally administered to all animals for 24 h. The data represent the means ± SD of eight mice. # indicates P < 0.05 compared with the untreated control group; * indicates P < 0.05 compared with the indomethacin-only group. LPZ: 30 mg/kg b.w. of lansoprazole, COL: 1 mL/kg b.w./day of camellia oil, COH: 2 mL/kg b.w./day of camellia oil.
enhancing growth factor TGF-β expression (Sharma, Ganguly, Paul, Maulik, & Swarnakar, 2012). In this study, the levels of VEGF and TGFβ proteins significantly reduced under gastric damage, and this reduction was reversed by camellia oil and lansoprazole. The effects of camellia oil and lansoprazole were similar (Fig. 6). Furthermore, the expression and regulation of HO-1 play crucial roles in the protection against ischemia-reperfusion injury, inflammatory bowel disease, necrotizing enterocolitis, radiation enteritis, acute pancreatitis, chronic pancreatitis, cytotoxic action of indomethacin, and DSS-induced colitis (Chang, Xue, Sharma, & Habtezion, 2015). Therefore, the upregulation of HO-1 in various organ system models may trigger an endogenous defensive mechanism against oxidative stress, inflammation, and tissue injury (Chang et al., 2015). As mentioned previously, the protein expressions of HO-1, VEGF, and TGF-β may play a fundamental role in accelerating GI wound healing following indomethacin-induced injury. In this study, the expressions of gastric mucosal HO-1, VEGF, and TGF-β proteins markedly decreased in indomethacin-treated BALB/c mice (P < 0.05). However, pretreatment with camellia oil markedly elevated the HO-1, VEGF, and TGF-β protein levels, thus indicating that camellia oil might promote wound healing by enhancing the levels of antioxidant enzymes and growth factors.
In the present study, pretreatment with TMS and TML camellia oils significantly increased the cell viability, reduced ROS production, and enhanced wound healing in indomethacin-damaged human Int-407 cells. These effects were similar to those of lansoprazole (Figs. 1 and 2). An increase in proinflammatory IL-6 and TNF-α cytokine secretion is highly associated with gastropathy caused by the constant use of indomethacin (Antonisamy et al., 2016; Higashimori et al., 2016). In addition, the adverse effects of NSAIDs are presumed to be associated with the suppression of COX-1 activity and promotion of COX-2 activity (Battistella et al., 2005; Felson, 2016). In the current study, an increase in inflammatory mediators IL-6, TNF-α, and COX-2 was observed in BALB/c mice with gastric mucosal damage induced by indomethacin (Fig. 5). Notably, camellia oil exhibited immunosuppressive effects by dramatically inhibiting these inflammatory mediators (IL-6, TNF-α, and COX-2) in indomethacin-induced gastric damage. In addition, the antiinflammatory effect (IL-6 and TNF-α) of lansoprazole was superior to that of camellia oil. The results of H&E staining were consistent with inflammatory cytokine expressions. It indicated indomethacin-induced severe degeneration and necrosis with hemorrhage of the stomach mucosal layer. Camellia oil had a beneficial protective effect against indomethacin-induced gastric injury and inflammation. Additionally, the protective effects of lansoprazole were stronger than those of camellia oil (Figs. 4 and 5). Previous studies have also reported the possible preventive capacity of camellia oil on ketoprofen- or ethanol-induced gastric mucosal damage through diminishing the injury and hemorrhage scores, inhibiting the production of inflammatory mediators, and reversing the antioxidant system (Cheng et al., 2014; Tu et al., 2017). NSAIDs restrict gastric cell proliferation and tissue repair and impede angiogenesis thus delaying the healing of gastroduodenal ulcers (Soreide, 2018; Soreide et al., 2015). Jones et al. (2001) reported that the healing of gastric ulcer wounds is associated with growth factor VEGF expression. VEGF can specifically act on tyrosine kinase receptors or kinase receptors in vascular endothelial cells, which promotes endothelial proliferation and migration, increases vascular permeability, and accelerates ulcer healing (Wallace, Dicay, McKnight, & Dudar, 2006). Indomethacin inhibits COX-1 expression and increases the endostatin/VEGF ratio, which could reduce VEGF release and result in angiogenesis suppression (Yadav et al., 2012). Therefore, VEGF plays a major role in promoting the restoration of connective tissues and angiogenesis during the repair of peptic ulcer, and it accelerates the healing of the ulcer area. A previous study revealed that curcumin, a natural phytochemical, promotes the healing of ulcer wounds by
5. Conclusions Based on the in vitro study conducted in this research, pretreatment with camellia oils significantly increased cell viability, reduced ROS production, and promoted wound healing in indomethacin-induced human intestine Int-407 cells. Similarly, the in vivo study provided compelling evidence that the preadministration of camellia oils dramatically prevents gastric wound generation in indomethacin-induced gastric damage BALB/c mice. This phenomenon may occur through an increase in the expressions of antioxidant enzyme HO-1, diminution of the levels of IL-6, TNF-α, and COX-2 inflammatory mediators, or the elevation of VEGF and TGF-β. The effects were similar to those observed for lansoprazole (positive control). In sum, these results evidence that camellia oils plays a key role in the prevention of adverse effects through its anti-inflammatory activity and antioxidant ability by upregulating growth factors in NSAID-induced GI damage. Ethics statement In this research work, the procedures for animal experiment were according to the rule of National Institutes of Health (NIH). The 7
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experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC No. 105-004) of National Chung Hsing University (Taichung, Taiwan).
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