European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastrointestinal and urogenital pharmacology

Quercetin protects gastric epithelial cell from oxidative damage in vitro and in vivo Xin-Ting Hu a, Chen Ding a, Nan Zhou b, Chen Xu a,n a b

State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, PR China School of Foreign Languages and Literature, Chongqing Normal University, Chongqing 401331, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 16 September 2014 Received in revised form 3 February 2015 Accepted 10 February 2015

Epithelial injury caused by reactive oxygen species (ROS) including H2O2 plays a critical role in the pathogenesis of gastric disorders. Therefore, pharmacological intervention targeting reactive oxygen species elimination has highly clinical values in therapy of gastric diseases. Although quercetin has been found to possess gastroprotective activity, whether it has a protective activity againress related injury to gastric epithelial cells remains unknown. The aim of the study is herein to investigate a possible protective effect of quercetin against oxidative stress in vitro and vivo. Human gastric epithelial GES-1 cells were pretreated with quercetin and then challenged with H2O2. In vivo reactive oxygen species production in acute gastric mocosa injury was assessed using a chemiluminescent probe L-012 (8amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4-(2H,3H)dione) after quercetin was administered to mice. In GES-1 cells, pretreatment of quercetin can significantly diminish H2O2-induced cell viability loss; decrease intracellular reactive oxygen species and Ca2 þ influx; restore H2O2-induced ΔΨm dissipation. It also upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC-1α) expression under the state of oxidative stress, and the downstream cell apoptosis significantly decreased. In vivo, chemiluminescence imaging shows that quercetin attenuates reactive oxygen species production and gastric damages in acute gastric mucosal injury. We first reported the evidence that quercetin can protect gastric epithelial GES-1 cells from oxidative damage and ameliorate reactive oxygen species production during acute gastric mucosal injury in mice. This might be ascribed to its inhibition of oxidative stress, regulation of mitochondrial dysfunction, initiation of antioxidant defense and inhibition of apoptosis. & 2015 Published by Elsevier B.V.

Chemical compounds studied in this article: Quercetin (PubChem CID: 5280343) H2O2 (PubChem CID: 784) L-012 (8-amino-5-chloro-7-phenylpyrido [3,4-d]pyridazine-1,4-(2H,3H)dione) (PubChem CID: 126741) MTT (Methyl-thiazol Tetrazolium) (PubChem CID: 64965) DMSO (dimethyl sulphoxide) (PubChem CID: 679) H2DCF-DA (20 ,70 dichlorodihydrofluorescein diacetate) (PubChem CID: 77718) Hoechst 33258 (PubChem CID: 5326397) Ethanol (PubChem CID: 702) Keywords: Gastric epithelial cell Oxidative stress Mitochondrial dysfunction In vivo chemiluminescence imaging

1. Introduction The impact of gastric diseases on human health, such as chronic gastritis, duodenal and gastric ulceration, adenocarcinoma and gastric MALT lymphoma, remains a big issue deserving worldwide attention, due to the growing consumption of nonsteroidal antiinflammatory drugs (NSAIDs), prevailing Helicobacter pylori, psychological stresses, alcohol consumption and cigarette smoking in modern society (Boland et al., 2005). In all cases, chronic increased oxidative stress is the major gastric pathologic feature and plays an essential role in the multiple progressions of gastric diseases (Bhattacharyya et al., 2014). Under

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Corresponding author. Tel./fax: þ 86 25 83585450. E-mail address: [email protected] (C. Xu).

physiological conditions, gastric epithelium is exposed to higher reactive oxygen species level which is derived from physical, chemical or microbiological agents from the gastric lumen, far higher than other tissues or biological fluids (Halliwell et al., 2000; Hiraishi et al., 1994). Intracellular accumulation of reactive oxygen species is generated as a consequence of incomplete reduction of oxygen in the normal metabolic processes and also directly produced by a range of oxidase enzymes such as NADPH oxidases (Valko et al., 2007). The imbalance of reactive oxygen species generation and the endogenous antioxidant defense system leads to oxidative stress (Valko et al., 2007). Among various reactive oxygen species, H2O2 is stable, small and uncharged molecule, facilitating it to freely diffuse across cell membranes and acts as a physiological second messenger. However, excessive level of H2O2 causes detrimental consequences such as apoptosis, necrosis and other oxidative damage (Veal et al., 2007). Therefore, interventions to ameliorate the processes of oxidative

http://dx.doi.org/10.1016/j.ejphar.2015.02.007 0014-2999/& 2015 Published by Elsevier B.V.

Please cite this article as: Hu, X.-T., et al., Quercetin protects gastric epithelial cell from oxidative damage in vitro and in vivo. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.007i

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stress and its related pathway are applicable to many different gastric indications. Increased dietary intake and botanicals consumption may reduce the risk for gastric mucosal oxidative injury and accordingly draw much attention (Mahady et al., 2005; Patra et al., 2014; Riceevans et al., 1995). More recently, extracts from apple, strawberry and black rice have been reported to possess extraordinary gastric mucosa protective effect, mainly owing to the abundance in polyphenols and their antioxidant activities (Alvarez-Suarez et al., 2011; Graziani et al., 2005; Kim et al., 2011). Dietary antioxidants, being able to scavenge reactive oxygen species, chelate metal ion and break lipid chain peroxidation reactions, play a crucial role in maintaining gastric homeostasis and polyphenolic compounds constitute the major class (Handique and Baruah, 2002). One of the most extensively studied groups of such substances is the flavonoids which are abundant in fruits, vegetables and red wine of plant origin. Of the flavonoids, quercetin is a representative compound. Dietary intake of quercetin and other flavonoids can cause much higher plasma concentrations, while under normal culture conditions, they can also be uptaken intracellular in vitro (Manach et al., 2004). Furthermore, extensive bioactive studies found that quercetin exhibits many beneficial effects on human health, including neuroprotective, anticarcinogenic, antiinflammatory, antiallergic, and antiviral activities, in particular antioxidant and free radical-scavenging activities (Boots et al., 2008). Although quercetin has been found to protect gastric mucosa against ischemia/reperfusion, ethanol, indomethacin, cold restraint stress and H. pylori in different animal models (Arsic et al., 2010; GonzalezSegovia et al., 2008; Kahraman et al., 2003; Mojzis et al., 2001; Yan et al., 2011), whether quercetin has a protective activity against oxidative stress related injury to gastric epithelial cells remains unknown. Herein, in the present study, we investigate whether quercetin protects reactive oxygen species induced damage on a human gastric epithelial cell line, GES-1, challenged with oxidative stress mediated by H2O2, and explore the possible mechanisms underlining such action. More importantly, we apply a chemiluminescent probe L-012 to assess the effect of quercetin on reactive oxygen species production in vivo, trying to offer a direct evidence for its effect against oxidative damage to gastric epithelial cells.

2. Materials and methods 2.1. Cell lines, cell culture, and chemicals Human gastric epithelial cell, GES-1 cell line, was obtained from the Xiangya School of Medicine, Central South University (Changsha, China). Dulbecco's Modified Eagle's Medium (DMEM/ HIGH GLUCOSE) was purchased from HyClone (Peking, China) and fetal bovine serum (FBS) was purchased from HyClone (South American). GES-1 was cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 4 mM L-glutamine, and 4500 mg/l glucose and grown in suspension at 37 1C in a 5% CO2 humidified incubator. Quercetin was purchased from Sigma. 2.2. Cell viability assay Cells were seeded in 96-well-plate at a density of 1  105 cells/ ml in growth medium for approximately 16 h before drug was added. After added quercetin with 2% fetal bovine serum for 24 h incubation, cells were exposed to treatments of 400 mM H2O2 or phosphate balanced solution (PBS) for 1 h, then the old medium was discarded and new medium with 2% fetal bovine serum was added to plates. Methyl-thiazol Tetrazolium (MTT) was dissolved in PBS at a concentration of 5 mg/ml, 20 ml of the MTT solution was added to each well of 96-well-plate and incubated for 4 h at 37 1C in a humidified atmosphere of 5% CO2. Finally the MTT-containing

medium was removed and 150 ml of dimethyl sulphoxide (DMSO) was added to each well. The absorbance was read at 490 nm using a microplate reader (Tecan Safire, Crailsheim, Germany) and for each treatment eight replicate wells were examined. The A490 was taken as an index of cell viability. 2.3. Measurement of intracellular reactive oxygen species Intracellular reactive oxygen species level was monitored by using the fluorescent probe 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma), which crosses cell membranes and is hydrolyzed by intracellular nonspecific esterases to nonfluorescent DCFH. DCFH then is oxidized by reactive oxygen species to highly fluorescent compound DCF (Lebel et al., 1992). GES-1 cells were seeded into 96-well plates at a density of 5  104/ml. After overnight growth, cells were then treated with different concentrations of quercetin at 37 1C for 24 h. Cells were rinsed with PBS and then incubated with 100 ml of 5 μM DCFH-DA at 37 1C for 30 min. The fluorescence intensity of DCF was measured in a microplatereader (Tecan Safire, Crailsheim, Germany) at excitation wavelength 485 nm and emission wavelength 530 nm. Then the cells were exposed to 300 mM H2O2 for 30 and 60 min at 37 1C and the fluorescence intensity of DCF was measured in a microplate-reader (Tecan Safire, Crailsheim, Germany) at excitation wavelength 485 nm and emission wavelength 538 nm. Data are presented as the fluorescence intensity ratios (after H2O2 added)/(before H2O2 added). 2.4. Intracellular free Ca2 þ ([Ca2 þ ]i) measurements [Ca2 þ ]i was determined with the Ca2 þ -sensitive fluorescent dye Fura-2/AM (Sigma, St. Louis, MO) according to previous procedure with modifications (Sareen et al., 2007). GES-1 cells were seeded in 96-well plates at the density of 5  104/ml. After overnight growth, cells were treated with quercetin with 2% fetal bovine serum for 24 h. Then cells were rinsed with HBSS (or Ca2 þ free HBSS) and loaded with 2 μM Fura-2/AM in HBSS (or Ca2 þ -free HBSS) containing 4% BSA at 37 1C for 30 min. Thereafter, cells were washed with HBSS or Ca2 þ -free HBSS in the presence of 4% BSA once and exposed to 300 mM H2O2 or HBSS (or Ca2 þ -free HBSS) for different incubation times. The fluorescence signal of [Ca2 þ ]i was determined in a microplate-reader (Tecan Safire, Crailsheim, Germany) at excitation wavelength of 340 nm or 380 nm and emission wavelength of 510 nm. Data are presented as the ratio of the fluorescence at 340 nm excitation to that at 380 nm excitation. 2.5. Measurement of mitochondrial inner membrane potential (ΔΨm) ΔΨm was monitored by applying the fluorescence dye JC-1 (Molecular Probes). For this purpose, GES-1 cells were seeded in 96-well plates at the density of 5  104/ml. After overnight growth, cells were incubated with quercetin at the concentrations of 25, 50 and 100 mM for 24 h. Cells were exposed to 400 mM H2O2 or PBS for 3 h, then the old medium was removed. Cells were then subjected to 100 ml of 10 μM JC-1 at 37 1C for 30 min. The medium was then discarded, and the cells were washed three times with PBS. The fluorescence intensity of JC-1 was measured in a microplate-reader (Tecan Safire, Crailsheim, Germany) at excitation wavelength of 485 nm or 550 nm and emission wavelength of 535 nm or 600 nm. 2.6. Real time RT-PCR Cells were seeded in 6-well-plate at a density of 5  105 cells/ well for approximately 16 h before drug was added. After added

Please cite this article as: Hu, X.-T., et al., Quercetin protects gastric epithelial cell from oxidative damage in vitro and in vivo. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.007i

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quercetin with 2% fetal bovine serum medium for 24 h incubation, cell lines were exposed to treatments of 400 mM H2O2 or PBS. Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturers' instructions. RNA was resuspended in RNasefree water (Takara, Dalian, China) and 1 ml aliquots in duplicate were quantified using BioPhotometer plus (Eppendorf, Germany). cDNA was generated from 2 mg of RNA by reverse transcription performed at 42 1C for 60 min followed by 70 1C for 10 min. Primers for human PGC-1α and GAPDH were designed by Primer Premier. Relative gene expression was determined by quantitative real-time reverse transcription-PCR relative to GAPDH. PGC-1α is a regulator of mitochondrial biogenesis and function. PCR reactions were done in triplicate using the following protocol: 95 1C for 5 min followed by 40 cycles of 95 1C for 30 s, 60 1C for 30 s, and 72 1C for 30 s. The PCR reaction mix consisted of 1 ml template cDNA in 20 ml reaction, 10  PCR Buffer, 20  Eva Green (fluorescein dye), and 0.2 mM each of a gene-specific oligonucleotide pair. An extension step was used to measure the increase in fluorescence and melting curves obtained immediately after amplification by increasing temperature in 0.5 1C increments from 65 1C to 95 1C of 5 s each analyzed (CFX Manager software; Bio-Rad). The fold difference in PGC-1α expression between treated and control was normalized to GAPDH and calculated by the comparative threshold (CT) method. Primers for Q-PCR were PGC1α: forward 50 -GGAGACGTGACCACTGACAATGA-30 , backward 50 TGTTGGCTGGTGCCAGTAAGAG-30 ; GAPDH: forward 50 -GCACCGTCAAGGCTGAGAAC-30 , backward 50 -TGGTGAAGACGCCAGTGGA-30 . 2.7. Apoptosis assay The apoptotic cell fraction in treated GES-1 cells was assessed by two independent methods. Morphological observation of apoptotic GES-1 cells was performed with Hoechst 33258 staining. Nuclei can be stained with Hoechst 33258 to detect chromatin condensation or nuclear fragmentation characteristic of apoptosis. Briefly, cells were exposed to 400 mM H2O2 or PBS in 2% fetal bovine serum medium for different times. Cells were fixed with ice-cold PBS containing 4% paraformaldehyde (PFA) for 15 min at room temperature. Cells were washed twice with ice-cold PBS and stained with Hoechst 33258 (Sigma, St. Louis, MO) for 5 min at room temperature, then washed to remove unbound dye. Cell morphology was observed under a Nikon fluorescence microscope. Quantification of apoptotic GES-1 cells was performed with an Annexin V-FITC and propidium iodide (PI) apoptosis detection kit (KEYGEN BIOTECH, Nanjing, China) according to the manufacturer's instructions. In brief, cells were seeded in 24-well-plate at a density of 2  105 cells/well. After overnight growth, cells were treated with quercetin in 2% fetal bovine serum medium for 24 h, and then exposed to 400 mM H2O2 or PBS for different times. Thereafter, cells were collected by trypsin, washed with PBS, resuspended in 500 ml of binding buffer. Finally, 5 ml Annexin V-FITC and 5 ml PI were added to the cells and incubated at room temperature in dark for 5 min, and the 10,000 ungated cells were immediately acquired by flow cytometry (BD FACScalibur) and analyzed by Flowjo software. For each treatment four replicate wells were examined. 2.8. In vivo reactive oxygen species detection and induction of gastric mucosal damage

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Mice were randomly divided into six groups (5 mice in each group). Control group administered saline containing 1% DMSO without ethanol-induced lesions; ethanol group received saline containing 1% DMSO for 7 days and then 5 ml/kg ethanol; omeprazole group received omeprazole dissolved in saline with 1% DMSO (20 mg/kg of body weight) for 7 days; omeprazole þethanol group received omeprazole dissolved in saline with 1% DMSO (20 mg/kg of body weight) for 7 days and then 5 ml/kg ethanol; quercetin group received quercetin dissolved in saline with 1% DMSO (25 mg/kg of body weight) for 7 days; quercetinþethanol group received quercetin dissolved in saline with 1% DMSO (25 mg/kg of body weight) for 7 days and then 5 ml/kg ethanol. Before the day of induction of gastric lesions, mice were fasted with free access to water for 18 h. The following day, 60 min after the last administration, absolute ethanol (5 ml/kg, b.wt.) was given via gavage to induce gastric lesions. Thirty minutes after i.g. administration of ethanol, 25 mg/kg L012 (Wako Chemicals, Japan) in ultrapure H2O was given to mice subcutaneously. Afterwards, mice were anesthetized with 350 mg/ kg intraperitoneal injection of 10% chloral hydrate. Then chemiluminescence images were captured in a period of 30 min by an IVIS Lumina XR imaging system (Caliper Instruments, Alameda, CA, USA), which consists of a light tight chamber equipped with a cooled CCD camera. Then, mice were sacrificed and dissected. The stomach was removed, opened and rinsed gently in PBS, followed by the macroscopic examination. The degree of gastric mucosal lesions was assessed and rated according to the ulcer score scales as previously described (Salga et al., 2012). Subsequently, the gastric mucosa were taken and fixed in 10% formalin for histological H&E analyses, and the histological damage was quantified according to the scoring system by Chiu scale (Chiu et al., 1970). 2.9. Statistical analysis All the data were presented as mean value 7standard deviation. Differences were evaluated with Student's t-test and Pvalue o0.05 was considered as statistically significant.

3. Results 3.1. Quercetin protects GES-1 cells from oxidative stress-mediated cell viability loss As shown in Fig. 1A, quercetin alone has no apparent effect on cell viability at concentrations of 6.25, 50 and 100 mM, while a significant cell growth is observed when quercetin concentration is 12.5 and 25 mM (Po0.01 and Po0.05, respectively). One-hour exposure to H2O2 (400 mM) significantly decreases the viability of GES-1 cells by 53% of untreated control (Po0.01). Pretreatment of quercetin at 25 and 50 mM remarkably diminishes this decrease by 75% and 79% of the control, respectively (Po0.01 and Po0.001). These results indicate that quercetin protects GES-1 cells from oxidative stress-induced cytotoxicity in a concentration-dependent manner. The concentration of 50 mM is also chosen for the following investigation of quercetin. 3.2. Quercetin reduces intracellular reactive oxygen species

Balb/c mice (male, 25–30 g body weight) were purchased from the Laboratory Animal Center of Academy of PLA Military Medical Sciences (AMMS, Beijing, China) and used in all in vivo studies. The mice were maintained in standard cages, at a controlled temperature (23 71 1C), with a 12 h dark/12 h light cycle, and food and water ad libitum. Animal protocol was in accordance with the national guidelines, and approved by the Medicine Animal Care Committee of Nanjing University (Nanjing, China).

To determine the effect of quercetin on intracellular reactive oxygen species level induced by H2O2 in GES-1 cells, reactive oxygen species production is detected using a DCF-DA fluorescent probe. The intensity of DCF fluorescence reflects the reactive oxygen species level in cells. As shown in Fig. 1B, 30 min-exposure to H2O2 causes a rapid and significant increase in DCF fluorescence by 2.4-fold compared

Please cite this article as: Hu, X.-T., et al., Quercetin protects gastric epithelial cell from oxidative damage in vitro and in vivo. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.007i

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Fig. 1. Effect of quercetin (Qt) on H2O2-induced cell viability loss, reactive oxygen species increase, [Ca2 þ ]i elevation and ΔΨm dissipation in GES-1 cells. (A) Cell viability was determined by a MTT assay. The cells were pretreated with quercetin for 24 h, followed by exposure to 400 mM H2O2 for 1 h. (B) Reactive oxygen species were monitored by using the fluorescent probe DCFH-DA. The cells were pretreated with quercetin for 24 h and then loaded with 100 ml of 5 μM DCFH-DA at 37 1C for 30 min. After exposed to 300 mM H2O2 for 30 and 60 min, fluorescence intensity was measured. (C) [Ca2 þ ]i level was detected by Fura-2-AM staining. Cells were pretreated with quercetin for 24 h and then loaded with 2 μM Fura-2-AM. After exposure to H2O2 challenge (300 mM, 3 h), fluorescence intensity was measured. (D) ΔΨm were determined by using the fluorescent probe JC-1. Cells were pretreated with quercetin for 24 h, and then exposed to 400 mM H2O2 for 3 h. Afterwards, cells were stained with 100 ml of 10 μM JC-1 at 37 1C for 30 min. Values are mean7 standard deviation (m.v. 7 S.D.) of data. nPo 0.05 and nnPo 0.01 vs. control (without treatment); P o0.01 and Po 0.001 vs. H2O2 without quercetin.

with control (P o0.05 vs. control). However, 24 h pretreatment of quercetin significantly attenuates this increase in a dosedependent manner, with 66.9% and 61.3% of fluorescence of H2O2-treated group at 25 and 50 mM respectively (P o0.05, P o0.05 vs. H2O2-treated). When H2O2 exposure extends to a longer time of 60 min, a significant increase in reactive oxygen species level is also observed with 2.2-fold compared to control (P o0.05 vs. control). However, no significant differences are observed among groups when quercetin applies to cells exposed to H2O2 for 60 min. 3.3. Quercetin decreases intracellular free Ca2 þ ([Ca2 þ ]i) level In this study, [Ca2 þ ]i was measured using the Fura-2-AM calcium probe. As shown in Fig. 1C, incubation of 400 mM H2O2 for 3 h significantly increases [Ca2 þ ]i by 2.6-fold of control (P o0.05), and pre-treatment of 25 and 50 mM quercetin significantly inhibited the H2O2-induced [Ca2 þ ]i elevation by 51% and 74%, respectively (P o0.05), reversing [Ca2 þ ]i almost to the resting level. However, when Ca2 þ in the extracellular buffer is removed, the increase of [Ca2 þ ]i induced by H2O2 is prevented, and pretreatment of quercetin could not alter [Ca2 þ ]i level. These results suggest that the increase of intracellular calcium induced by H2O2 is ascribed to the Ca2 þ influx from the extracellular medium, and quercetin could decrease the intracellular calcium influx induced by H2O2. 3.4. Quercetin restores mitochondrial inner membrane potential (ΔΨm)

50% of control (Po0.05 vs. control). However, when pretreated with quercetin, ΔΨm remarkably increases in a dose-dependent way with the increase of 72% and 73% of control at quercetin concentrations of 50 and 100 mM respectively (Po0.05 compared to H2O2). The results display that in a medium containing 400 mM H2O2, treatment of cells with quercetin attenuates the mitochondrial membrane depolarization. 3.5. Quercetin increases PGC-1α gene expression We first examine transcript levels of PGC-1α in GES-1 cells under normal state. No significant difference is found between control and quercetin-treated cells (P¼ 0.15, Fig. 2I). We further measure PGC-1α expression in GES-1 cells at different periods of time under oxidative stress. There are no significant differences among groups when cells are treated with H2O2 at 400 mM for 1 h (P¼ 0.11 vs. control, P ¼0.071 vs. H2O2treated, Fig. 2II), although H2O2 alone decreases PGC-1α expression by 80% of control and quercetin promotes PGC-1α by 2.2-fold compared with H2O2 treated alone. However, as the time of oxidative stress challenge increases to 12 h, PGC-1α expression displays a different profile. H2O2 significantly lowers PGC-1α expression by 43% of control (Po0.05 vs. control, Fig. 2III), and pretreatment with 50 mM quercetin markedly promotes PGC-1α by 2.4-fold compared with H2O2 treated alone (Po0.05 vs. H2O2-treated, Fig. 2III). These data demonstrates that quercetin promotes PGC-1α expression in GES-1 cells in the state of oxidative stress. 3.6. Quercetin prevents oxidative stress-induced cell apoptosis

Mitochondrial membrane depolarization is indicated by decrease of JC-1 aggregates and increase of JC-1 monomers. As shown in Fig. 1D, H2O2 at 400 mM significantly decreases ΔΨm in GES-1 cells by about

Hoechst 33258 staining reveals that exposure of GES-1 cells to H2O2 for 16 h leads to time-dependent apoptosis, but the degree of

Please cite this article as: Hu, X.-T., et al., Quercetin protects gastric epithelial cell from oxidative damage in vitro and in vivo. Eur J Pharmacol (2015), http://dx.doi.org/10.1016/j.ejphar.2015.02.007i

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Fig. 2. Effect of quercetin on PGC-1α gene expression. Relative PGC-1α expression was measured after GES-1 cells were incubated with 50 mM quercetin, followed by exposure without H2O2 (I), with 400 mM H2O2 for 1 h (II) and 12 h (III). Values represent mean 7 S.D., and GAPDH serves as control. nPo 0.05 vs. control (without treatment). P o 0.05 vs. H2O2 without quercetin.

apoptosis is much lower in the quercetin-pretreated group in a concentration-dependent manner. As shown in Fig. 3A, cells have regular and smoother nuclei and did not show any morphological changes among groups when cells were treated with H2O2 for 1 h. However, when the H2O2 incubation time increases to 6 h, the H2O2-treated cells exhibit apparent nuclear condensation and fragmentation, while the presence of quercetin significantly reduces the apoptotic nuclei. As the H2O2 incubation time increases to 12 h, more nuclear condensation and fragmentation are observed in H2O2-treated cells, and quercetin still presents inhibitory effects as observed at 6 h. The results of flow cytometric analysis of cells double stained with Annexin V-FITC and PI are consistent with those of the Hoechst data. As shown in Fig. 3B, after exposure of the cells to 400 mM H2O2 for 1 h, the percentage of apoptotic cells (early and late-stage apoptosis) significantly increases (12.25 70.36%, P o0.001 vs. control of 5.29 70.89%), whereas pretreatment of quercetin at 25 and 50 mM remarkably decreases the number of apoptotic cells compared to H2O2-treated group (10.46 70.08%, P o0.01 vs. H2O2-treated, and 5.437 0.38%, P o0.001 vs. H2O2treated) respectively. Following the incubation time with H2O2 increases to 5 h, the percentage of apoptotic cells increases (28.337 4.61%, P o0.001 vs. control of 6.57 72.18%), while 50 mM quercetin decreases the percentage of apoptotic cells (9.68 70.94%, P o0.01 vs. H2O2-treated) and no significant differences are observed between 25 mM quercetin and H2O2-treated group. These results collectively display that quercetin protected GES-1 cells from oxidative stress-induced apoptosis in a concentration-dependant manner. 3.7. Quercetin alleviates gastric reactive oxygen species accumulation and mucosal damage in ethanol-induced gastric injury We then sought to investigate whether quercetin counteracts oxidative stress in vivo, thereby exerting the protective effect in gastric mucosa. Reactive oxygen species accumulation surrounding injured gastric mucosa induced by ethanol was assessed using a reactive oxygen species-sensitive L-012 probe. The in vivo images (Fig. 4A) present that a prominent chemiluminescence signal could be detected at the stomach area in ethanol-treated mice, whereas only minimal signal is detected when the ethanol mice are pretreated with either quercetin or omeprazole. Analysis of L012 chemiluminescence intensity (Fig. 4B) confirms that luminescent signal in ethanol-treated mouse is approximately 69 times higher than that of control sample. By contrast, in ethanol-induced mice, quercetin significantly reduces the amount of luminescence by approximately 82% (P o0.05 vs. ethanol), while omeprazole

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67 reduces the amount of luminescence by approximately 87% 68 (P o0.05 vs. ethanol). These results suggest that quercetin 69 attenuates reactive oxygen species accumulation in acute gastric 70 injury mice. 71 Furthermore, the macroscopic evaluation and histological ana72 lysis were subsequently conducted in support for the in vivo 73 reactive oxygen species assay. As shown in Fig. 5A, ethanol acutely 74 causes multiple visible gastric bleedings and hemorrhagic ero75 sions. Administration of quercetin prevented macroscopic injury to 76 the mice glandular stomach. A similar efficacy was observed in 77 omeprazole-treated mice. 78 The histological H&E staining (Fig. 5B) showed ethanol caused 79 inflammation infiltration, prominent presence of edema, moderate 80 hemorrhage and a great loss of epithelium cells, compared with 81 untreated control. By contrast, quercetin alleviated inflammation, 82 suppressed edema, protected epithelium and crypt damage. These histological appearances are similarly observed in those pretreated 83 with omeprazole, serving as the reference drug. 84 Quantitatively (Fig. 5C and D), pretreatment of quercetin 85 caused an approximate 75% decrease in the extent of macroscopic 86 injury (Po 0.01), as well as a 75% reduction in the degree of 87 histological injury (Po 0.001) compared with ethanol-treated 88 mice. Omeprazole had a comparable efficiency with a decrease Q3 89 90 of 79% in the extent of macroscopic injury and a decrease of 71% in 91 degree of histological injury (P o0.01 and P o0.01 vs. ethanol92 treated group). These results collectively indicate that quercetin 93 could counteract reactive oxygen species accumulation surrounding 94 injured gastric mucosa induced by ethanol and prevent this gastric 95 mucosal damage, as effective as omeprazole. 96 97 98 4. Discussion 99 100 It has been well established that the elevated reactive oxygen 101 species is an essential factor in the onset and development of 102 gastric epithelial injury and various gastric disorders. In this 103 regard, the approach of reducing reactive oxygen species produc104 tion and oxidative damage may provide a beneficial intervention 105 to combat gastric diseases (Oliveira et al., 2003). Quercetin is well106 known as a powerful free radical scavenger and has been reported 107 to counteract oxidative stress in different cell lines (Gonzalez108 Segovia et al., 2008; Kahraman et al., 2003). In this study, we first 109 provide evidence that quercetin can protect gastric epithelial 110 GES-1 cells from oxidative damage and ameliorate reactive oxygen 111 species production during acute gastric mucosal injury in mice. It is 112 further found that quercetin protects oxidative damages through 113 inhibition of oxidative stress, regulation of mitochondrial dysfunc114 tion, initiation of antioxidant defense and inhibition of apoptosis. 115 Different from endogenous H2O2, exogenously added H2O2 is 116 less effective at eliciting a signaling response (Choi et al., 2005), 117 whereas it can rapidly diffuse across membranes and immediately 118 increase intracellular reactive oxygen species level including H2O2, 119 O2 and NO, and then oxidize various biomolecules, resulting in 120 their function loss and causing cell damage that ultimately 121 compromises cellular function (Ferreira et al., 2010; Jarrett et al., 122 2010). Our results showed that intracellular reactive oxygen species 123 level elevates immediately after H2O2 exposure (Fig. 1B), and 124 GES-1 cells showed a dose-dependent viability loss (Fig. 1A), in 125 good agreement with other reports (Lu and Gong, 2009). Mean126 while, pretreated with different concentration of quercetin sig127 nificantly reduced the intracellular oxidants and decreased the 128 subsequent cell viability loss, suggesting that cell damage and 129 cytotoxicity induced by intracellular reactive oxygen species can be 130 prevented by quercetin. These results suggest that quercetin may 131 exert gastroprotective effect through an antioxidant pathway, 132 which is in turn agreement with the result of other reports that

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Fig. 3. Effect of quercetin on oxidative stress-induced apoptosis. (A) Hoechst 33258 staining. The cells were pre-treated with quercetin (25, 50 mM) then exposed to 400 mM H2O2 for 1, 6 and 12 h. Cells were visualized using Hoechst 33258 under a Nikon inverted-fluorescence microscope with magnification at 400  . Apoptotic cells showed a characteristic of nuclear condensation and fragmentation (marked in dashed circle). (B) Density plot of flow cytometric analysis of Annexin V-FITC and PI double-stained cells. Cells staining Annexin V þ /PI  and Annexin V þ /PI þ represented the early and late apoptotic events, respectively.

Fig. 4. Effect of quercetin on reactive oxygen species production in acute gastric mucosal injury mice. (A) In vivo imaging of mouse injected with L-012. Con is baseline. Ethanol, ethanol þQt and ethanol þ Ome are protocol of pre-administration with saline, quercetin or omeprazole respectively, then followed by ethanol treatment. (B) Average luminescence intensity. Error bars are S.D. nnPo 0.01 and nnn P o0.001 vs. ethanol sample.

have indicated that antioxidant strategies may protect cells from H2O2-induced toxicity. Excessive reactive oxygen species can affect Ca2 þ homeostasis due to the deterioration of membranes. The combination of reactive oxygen species and Ca2 þ dyshomeostasis will lead to loss of mitochondrial membrane potential, opening of mitochondrial

permeability transition pore (MPTP) and damage to cell (Brookes et al., 2004). The present study showed that H2O2 induces increase of [Ca2 þ ]i, which highly depends on the calcium ion in extracellular buffer (Fig. 1C). This result suggests that the elevation of Ca2 þ is likely dependent on Ca2 þ influx from the extracellular calcium, rather than the intracellular Ca2 þ stores. Another indication is that an increase on [Ca2 þ ]i can be detected until 3 h after H2O2 treatment, and there is however no obvious change at 30 min and 1 h (data not shown). This is inconsistent to some literatures in which an increase of [Ca2 þ ]i can be detected as early as several minutes or even 1 h after H2O2 treatment (Hung et al., 2003). This can be explained that the reported experimental setup both on H2O2 concentration and the sensitivity of the assay is much higher than that of ours. Besides, it may be a secondary event of reactive oxygen species action on proteins of calcium pathway in GES-1 cell because the H2O2-induced increase in reactive oxygen species is ahead of the increase in [Ca2 þ ]i (0.5 vs. 3 h). The high level of cytosolic calcium will further induce the uptake of calcium into mitochondria through the calcium uniporter, and the sustained high Ca2 þ level will further lead to chronic, damaging calcium accumulation within mitochondria (Rasola and Bernardi, 2011). Thus, the increase in cytosolic Ca2 þ , oxidative damage, and decreased ATP synthesis constitutes a vicious cycle in mitochondria, leading to a more detrimental Ca2 þ overload and even higher oxidative stress that ultimately compromises cellular function (Giorgi et al., 2012). Quercetin can significantly suppress H2O2-evoked [Ca2 þ ]i elevation, consequently disrupt this vicious cycle and greatly benefit for mitochondrial function. These results indicate that quercetin might prevent gastric epithelial from oxidative stress by restoring calcium dysregulation and inhibiting Ca2 þ -activated pathways through suppressing the elevation of intracellular Ca2 þ . Similar results are also observed in PC12 cells and lung epithelial cells

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Fig. 5. Effect of quercetin on ethanol-induced acute gastric mucosal injury in mice. Mice were pretreated with saline, or 25 mg/kg quercetin or 20 mg/kg omeprazole for 7 days, then ingested with a dose of ethanol. (A) Representative macroscopic images of ethanol-induced acute gastric mucosal injury. (B) Histological HE staining (200  ) of ethanol-induced ulcerated gastric mucosa. (C) Macroscopic damage was quantified by measuring the extent of the gastric mucosal lesions. (D) Quantification of histological injury in gastric mucosa of each group of mice. Values are mean 7standard deviation (m.v. 7 S.D.) of data. *Po 0.05 and **Po 0.01 vs. control (without treatment); P o0.05, Po 0.01 and Po 0.001 vs. ethanol-treated group.

(Boots et al., 2007; Wang and Joseph, 1999). However, the mechanism underlining this effect is still undiscovered. It is perhaps associated with its strong antioxidant property (Shi et al., 2010), or might be related to directly act on the proteins in Ca2 þ -activated pathways. Sustained oxidative stress and Ca2 þ dyshomeostasis endanger mitochondrial function and integrity, resulting in mitochondrial dysfunction including mitochondrial oxidative damage, ATP depletion and calcium overload, subsequently inducing MPTP opening and mitochondrial membrane potential disruption (Smith et al., 2012). Mitochondrial dysfunction has long been implicated in the early process of exogenously added H2O2-induced apoptosis (Polster and Fiskum, 2004). ΔΨm loss, as a typical biochemical characteristic of mitochondrial dysfunction, was measured using fluorescent probe JC-1 in the present study. As expected, exposure to H2O2 for 3 h causes mitochondrial depolarization (Fig. 1D), which may release apoptosis-involved factors such as cytochrome c which activate caspase cascade, cause nuclear condensation, and generate secondary reactive oxygen species. Quercetin dosedependently diminishes this ΔΨm loss, demonstrating that quercetin may restore mitochondrial dysfunction under oxidative challenges. It is noted here that quercetin at the highest dose of 100 mM did not have a remarkable effect on either of the 4 parameters in Fig. 1, which is coincided with other reporting that quercetin at 100 mM decreases antioxidant capacity and thiol content, ultimately causing cytotoxicity (Boots et al., 2007). It is reported that quercetin shows both antioxidant and prooxidant properties (Gibellini et al., 2011). Under oxidative state, quercetin protects cells against oxidative damages to cells, due to its potent antioxidant activity as a reactive oxygen species scavenger and metal chelator. On the other hand, quercetin chemically reacts with free radicals forming oxidized quercetin, a potential prooxidant, which further arylates

glutathione and protein thiol groups, causing glutathione depletion, increase in cytosolic calcium concentration and LDH leakage, eventually leading to cytotoxicity and cell death (Boots et al., 2007). The paradox of quercetin might relate to its dose and the redox state of the cell (Ranawat et al., 2013). Indeed, in different normal cell models low concentrations of quercetin (r50 mM) induce cell proliferation and increase the antioxidant capacity of the cells, whereas higher concentrations of quercetin (Z100 mM) induce cytotoxicity (Robaszkiewicz et al., 2007). Oxidative stress is mainly caused by disturbance of reactive oxygen species production and endogenous antioxidant defense system, including GSH and antioxidant enzymes of SOD, CAT etc. Cellular redox state tightly regulates antioxidant genes such as PGC-1α, HO-1 and Nrf-2, which subsequently regulate numerous reactive oxygen species-detoxifying enzymes (Austin and St-Pierre, 2012). Thus, PGC-1α plays a central role in cellular antioxidant defense system under oxidative stress. Here interestingly, one hour-exposure to H2O2 did not show obvious effect on PGC-1α, while 12 h-exposure decreased PGC-1α expression in GES-1 cells (Fig. 2). These results are not in line with other literatures. For example, it was found that PGC-1α expression was increased in neural cells treated with 1 mM H2O2 for 2 h (St-Pierre et al., 2006), and PGC-1α mRNA levels were elevated in primary human umbilical vein endothelial cells exposed to 300 mM H2O2 for 2 h (Qian et al., 2010). These seemingly opposite results might be explained by H2O2 exposure time among different experiments. As shown above, 12 h-exposure to H2O2 has led to mitochondrial dysfunction, thus it is plausible that PGC-1α expression decreased after H2O2 exposure in this case. We also found that in GES-1 cells quercetin could not affect PGC-1α expression under normal state. This is seemingly inconsistent to previous results conducted in HepG2 cells (Rayamajhi et al., 2013) and skeletal muscle and brain of mice (Davis et al.,

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2009), in which quercetin enhances PGC-1α level and stimulates mitochondrial biogenesis. More interestingly, in the state of oxidative stress, quercetin upregulates PGC-1α expression, suggesting that compensatory induction of antioxidant defense is initiated to counteract the heavier oxidative challenge. Thus, the endogenous oxidative stress will accordingly be ameliorated with the treatment of quercetin. Apart from the direct antioxidant effects acting as reactive oxygen species scavenger, the regulation of antioxidant gene such as PGC-1a under oxidative state by quercetin might also contribute to its anti-oxidant effect in cells. The downstream cellular event of exogenously added H2O2 in cells would be apoptosis and our results indeed showed the apoptotic cells were increased by H2O2 treatment. Quercetin markedly alleviates H2O2-induced cellular apoptosis (Fig. 3A and B). This reduction could be due to the consequence of regulating upstream mitochondrial dysfunction and antioxidation. It is noted that an apparent apoptosis occurred only when H2O2 exposure for 5–6 h lags behind calcium dyshomeostasis and ΔΨm loss, showing a sequential cellular event during oxidative stress. Considering the central role of mitochondria in relation between reactive oxygen species damage, calcium homeostasis and PGC-1α, we postulate that the protective effect of quercetin on mitochondria may be associated with its inhibition of the intracellular accumulation of reactive oxygen species and contributed to its inhibition of cell apoptosis. Gastric mucosal layers, as a dynamic barrier in counteracting the effects of noxious agents through a series of endogenous antioxidant defense systems, were attacked by high levels of various reactive oxygen species in both physiological and pathological conditions. Accordingly, it would be much more important if quercetin can combat oxidative damage in vivo. In this study, ethanol was employed to produce a typical gastric mucosal injury. Ethanol can diffuse into gastric mucosa and injure both epithelial and endothelial cells. Meanwhile, the accumulated inflammatory cells around the damaged mucosa, in particular neutrophils, release inflammatory mediators and generate more reactive oxygen species, thereby deteriorating inflammation and injuries (Rahim et al., 2014). Accordingly, the elevated reactive oxygen species is considered to play an important role in the onset and development of ethanol-induced mucosal injury (Hiraishi et al., 1999). Herein, a highly sensitive chemiluminescent probe L-012, an analog of luminal, was applied to noninvasively monitor the production of reactive oxygen species in mice, based on its property of reacting with various types of reactive oxygen species generated by activated inflammatory cells (Imada et al., 1999). As shown in Figs. 4 and 5, ethanol ingestion caused an increment of reactive oxygen species accumulation in gastric mucosa, accompanied with a severe gastric hemorrhagic lesion and pathological change, indicating the involvement of oxidative stress in this process, highly consistent with other reports (Alvarez-Suarez et al., 2011; Nordin et al., 2014). Administration of quercetin significantly attenuates reactive oxygen species accumulation in damaged gastric mucosa, and provides a potent protection for gastric mucosa at both macroscopic and microscopic levels. To date, quercetin and quercetinenriched extracts have been reported to possess gastro-protective activity in different animal models (Alarcon de la Lastra et al., 1994; Suzuki et al., 1998) and some efforts have focused on its possible mechanism. In this respect, Yan et al. (2011) found that quercetin derivative induces mucus secretion and down-regulates ICAM-1 expression in protecting gastric damage induced by indomethacin. Klipinska et al. (2006) reported that quercetin protects DNA damage induced by dietary irritants in normal and H. pylori-infected gastric mucosa cells. Alvarez-Suarez et al. (2011) showed that quercetin could activate antioxidant enzymes and attenuate MDA increase in ethanol-induced damage to rats gastric

mucosa. Here, we first display a direct evidence, as indicated by chemiluminescence intensity of L-012 probe, that quercetin indeed act on reactive oxygen species generation, which at least in part account for its excellent gastroprotective activity. Moreover, this in vivo result reinforces the above in vitro data, strongly supporting that quercetin has the beneficial effect against oxidative stress and damage in reactive oxygen species-related gastropathy, thereby facilitating to maintain the integrity of gastric mucosa. This could be due to quercetin's direct antioxidant property (Boots et al., 2007), and more possibly its regulation on the complex crosstalk between mitochondria, reactive oxygen species and PGC-1α presented in this work. Omeprazole was used as a positive control in this study and we found that omeprazole significantly reduces reactive oxygen species accumulation and prevents the gastric damage induced by ethanol. This result is highly in accordance with other reports (Nordin et al., 2014; Tohda et al., 2006). Although it is well known for its inhibitory action on gastric acid secretion as a proton pump inhibitor, omeprazole was found to possess antioxidant and antiinflammatory activities in recent years (Blandizzi et al., 2000; Lapenna et al., 1996) and increasing studies that focusing on gastroprotection have used omeprazole as a positive control (Boligon et al., 2014; Nordin et al., 2014). It was shown that omeprazole protects gastric oxidative damage through scavenging hydroxyl radical (Biswas et al., 2003). It was also reported that omeprazole is a powerful scavenger of hypochlorous acid (Simon et al., 2006). Additionally, it was demonstrated that omeprazole could increase endogenous antioxidant enzymes of SOD, GSH and CAT in animals (Nordin et al., 2014). Furthermore, omeprazole was found to protect against acute gastric mucosal lesions induced by compound 48/80 in rats by a mechanism related to its antiinflammatory, antioxidant, and/or gastric mucus secretionenhancing actions (Kobayashi et al., 2002). Based on our results and scientific literatures, we postulate that the action of omeprazole in reducing chemiluminescence of L-012 in vivo reactive oxygen species assay is probably due to its free radical scavenging activity and anti-inflammatory effect, which alleviate reactive oxygen species generation around activated inflammatory cells. These results further support that omeprazole protects gastric mucosa through a mechanism related to its antioxidant, antiinflammatory effect. At the same time, the comparable efficacy between omeprazole and quecertin suggests that quercetin might have a similar mechanism as omeprazole in protecting gastric injury. To the best of our knowledge, this is the first report in which L-012 is applied in the site of stomach to evaluate reactive oxygen species level around the damaged gastric tissue for in vivo imaging. It should be noted here that there seems to be a good correlation between in vivo chemiluminescent imaging and the histological and macroscopic results which are indicating the extent of gastric mucosal injury. Generally, L-012-associated chemiluminescence is considered as a marker of cell oxidant stress (Pizzolla et al., 2012). In view of the central role of reactive oxygen species in inflammatory pathologies, it has been also shown that L-012-associated chemiluminescence imaging can be used to identify and to quantify the location and degree of inflammatory responses surrounding inflamed tissue (Kielland et al., 2009; Zhou et al., 2012). Because inflammatory responses are also implicated in the pathogenesis of ethanol-induced gastric mucosal injury (Hiraishi et al., 1999; Rahim et al., 2014), as presented in our study that ethanol ingestion caused inflamed gastric tissue, which typically exhibiting inflammation infiltration, edema and hemorrhage, it is reasonable to assume that L-012-associated chemiluminescence imaging might indirectly reflect the extent of gastric injury provoked by the non-specific irritant, ethanol in this case. However, whether L-012-mediated chemiluminescence could be a

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useful avenue for evaluating the effect of gastroprotective agents on gastric mucosal injury still needs further investigation. In conclusion, this is the first evidence showing quercetin could protect H2O2-induced oxidative damage in gastric epithelial GES-1 cells and ameliorate reactive oxygen species production in acute gastric injury in mice. The protective effect of quercetin was related to inhibition of reactive oxygen species production, regulation of calcium, upregulation of PGC-1α, inhibition of mitochondrial membrane potential and subsequent apoptosis. Accordingly, quercetin and quercetin-rich diets appear to be a candidate as food supplement in the prevention of early pathologic changes in gastric disorders.

Acknowledgments We would like to thank Dr. Su Chen for his helpful advice on designing and plotting figures, as well as manuscript writing. We would also like to thank Min Lu for her technical support in flow cytometry. This work was supported by the National Natural Science Foundation of China (Grant number 81072682) and the State Key Laboratory of Pharmaceutical Biotechnology of Nanjing University (Grant number 02ZZYJ-201302). References Alarcon de la Lastra, C., Martin, M.J., Motilva, V., 1994. Antiulcer and gastroprotective effects of quercetin: a gross and histologic study. Pharmacology 48, 56–62. Alvarez-Suarez, J.M., Dekanski, D., Ristic, S., Radonjic, N.V., Petronijevic, N.D., Giampieri, F., Astolfi, P., Gonzalez-Paramas, A.M., Santos-Buelga, C., Tulipani, S., Quiles, J.L., Mezzetti, B., Battino, M., 2011. Strawberry polyphenols attenuate ethanol-induced gastric lesions in rats by activation of antioxidant enzymes and attenuation of MDA increase. PLoS One 6, e25878. Arsic, I., Zugic, A., Antic, D.R., Zdunic, G., Dekanski, D., Markovic, G., Tadic, V., 2010. Hypericum perforatum L. hypericaceae/guttiferae sunflower, olive and palm oil extracts attenuate cold restraint stress-induced gastric lesions. Molecules 15, 6688–6698. Austin, S., St-Pierre, J., 2012. PGC1 alpha and mitochondrial metabolism-emerging concepts and relevance in ageing and neurodegenerative disorders. J. Cell Sci. 125, 4963–4971. Bhattacharyya, A., Chattopadhyay, R., Mitra, S., Crowe, S.E., 2014. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol. Rev. 94, 329–354. Biswas, K., Bandyopadhyay, U., Chattopadhyay, I., Varadaraj, A., Ali, E., Banerjee, R.K., 2003. A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical. J. Biol. Chem. 278, 10993–11001. Blandizzi, C., Natale, G., Gherardi, G., Lazzeri, G., Marveggio, C., Colucci, R., Carignani, D., Del Tacca, N., 2000. Gastroprotective effects of pantoprazole against experimental mucosal damage. Fundam. Clin. Pharmacol. 14, 89–99. Boland, C.R., Luciani, M.G., Gasche, C., Goel, A., 2005. Infection, inflammation, and gastrointestinal cancer. Gut 54, 1321–1331. Boligon, A.A., de Freitas, R.B., de Brum, T.F., Waczuk, E.P., Klimaczewski, C.V., de Ávila, D.S., Athayde, M.L., de Freitas Bauermann, L., 2014. Antiulcerogenic activity of Scutia buxifolia on gastric ulcers induced by ethanol in rats. Acta Pharm. Sin. B 4, 358–367. Boots, A.W., Haenen, G.R.M.M., Bast, A., 2008. Health effects of quercetin: from antioxidant to nutraceutical. Eur. J. Pharmacol. 585, 325–337. Boots, A.W., Li, H., Schins, R.P.F., Duffin, R., Heemskerk, J.W.M., Bast, A., Haenen, G., 2007. The quercetin paradox. Toxicol. Appl. Pharmacol. 222, 89–96. Brookes, P.S., Yoon, Y.S., Robotham, J.L., Anders, M.W., Sheu, S.S., 2004. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol.: Cell Physiol. 287, C817–C833. Chiu, C.J., McArdle, A.H., Brown, R., Scott, H.J., Gurd, F.N., 1970. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch. Surg. 101, 478–483. Choi, M.H., Lee, I.K., Kim, G.W., Kim, B.U., Han, Y.H., Yu, D.Y., Park, H.S., Kim, K.Y., Lee, J.S., Choi, C.H., Bae, Y.S., Lee, B.I., Rhee, S.G., Kang, S.W., 2005. Regulation of PDGF signalling and vascular remodelling by peroxiredoxin II. Nature 435, 347–353. Davis, J.M., Murphy, E.A., Carmichael, M.D., Davis, B., 2009. Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance. Am. J. Physiol.: Regul. Integr. Comp. Physiol. 296, R1071–R1077. Ferreira, I.L., Resende, R., Ferreiro, E., Rego, A.C., Pereira, C.F., 2010. Multiple defects in energy metabolism in Alzheimer's disease. Curr. Drug Targets 11, 1193–1206. Gibellini, L., Pinti, M., Nasi, M., Montagna, J.P., De Biasi, S., Roat, E., Bertoncelli, L., Cooper, E.L., Cossarizza, A., 2011. Quercetin and cancer chemoprevention. Evid. Based Complement. Altern. Med., 1–15.

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