Protective effects of Artemisia campestris extract against gastric acid reflux-induced esophageal mucosa injuries

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Protective effects of Artemisia campestris extract against gastric acid reflux-induced esophageal mucosa injuries Mohamed-Amine Jabri a,∗ , Haifa Tounsi b , Afifa Abdellaoui b , Lamjed Marzouki a , Hichem Sebai a a Laboratoire de Physiologie Fonctionnelle et Valorisation des Bio-Ressources, Institut Supérieur de Biotechnologie de Béja, Université de Jendouba, Avenue Habib Bourguiba, B.P. 382, 9000 Béja, Tunisia b Laboratoire d’anatomie pathologique humaine et expérimentale, Institut Pasteur de Tunis, 13, Place Pasteur, Tunis 1002, BP-74, Tunisia

a r t i c l e

i n f o

Article history: Received 17 August 2017 Received in revised form 21 October 2017 Accepted 1 January 2018 Available online xxx Keywords: Artemisia campestris Gastroesophageal reflux Lipoperoxidation Thiol groups Intracellular mediators

a b s t r a c t Artemisia campestris L. has been widely used in alternative medicine to treat digestive system diseases, particularly gastroesophageal disorders. In the present investigation, we studied the putative protective effect of Artemisia campestris aqueous extract (ACAE) against gastro-esophageal reflux (GER)-induced esophagitis in rats. The experimental ophagitis was induced by the ligation of the pylorus as well as the junction between the forestomach and the corpus. We firstly found that ACAE administration at 100, 200 and 400 mg/kg, b.w., p.o. significantly protected GER-induced macroscopic and histological injuries in the esophagus tissue. Our extract also counteracted GER-induced esophagus lipoperoxidation, restored the depletion of antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) as well as thiol groups levels. Furthermore, we showed that acute GER provoked an increase in esophagus mucosa hydrogen peroxide (H2 O2 ), free iron and calcium levels, whereas ACAE treatment reversed all GER-induced intracellular mediators’ disturbances. In conclusion, we suggested that ACAE had potent protective effects against esophagitis due, in part, to its antioxidant properties as well as its opposite effect on some intracellular mediators. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Gastro-Esophageal Reflux (GER) designates the permanent or intermittent reflux of gastric contents to the esophagus through the esophageal hiatus [1,2]. The physiological GER that exists in all subjects, essentially after meals, is not accompanied by symptoms or esophageal mucosal lesions [3], whereas pathological GER is characterized by the presence of those symptoms and/or lesions known as esophagitis. The reflux of the gastric contents is then frequent and/or prolonged [4,5]. The most aggressive elements are the concentrations of H+ ions and pepsin. However, the harmful effects depend not only on the concentration, but also on the exposure duration of esophageal mucosa [3,4]. The reflux of pancreatic enzyme probably has no relative importance, except after total gastrectomy or in case of achlorhydria [2]. Several biochemical mechanisms have been proposed to be involved in the pathophys-

∗ Corresponding author at: Laboratoire de Physiologie Fonctionnelle et Valorisation des Bio-Ressources, Institut Supérieur de Biotechnologie de Béja, Avenue Habib Bourguiba, B.P. 382, 9000 Béja, Tunisia. E-mail address: [email protected] (M.-A. Jabri).

iology of esophagitis, the most important of which is oxidative stress which is a cellular imbalance between the antioxidant and the prooxidant systems in favor of high production of reactive oxygen species (ROS) [6,7]. The oxidative stress may later have different cellular sources and the most important one is mitochondria [8]. The superoxide radicals produced by the NADPH oxidase route can then give rise, by successive reductions to other species, such as hydrogen peroxide (H2 O2 ) and the hydroxyl radical (OH• ) which are characterized by high reactivity [7,9]. Several investigations have reported that oxidative lesions are involved in numerous digestive diseases such as ulcerative colitis [10], gastric ulcer [11] and esophagitis [12,13]. Special importance is given to medicinal plants which act on the esophageal and gastric mucosa to treat esophagitis [14]. Artemisia campestris is a perennial herb belonging to Compositae genus and Asteraceae family, from 30 to 80 cm in height. This plant has very small flowerheads, narrow (1–1.5 mm), ovoid or conical, with scarous involucre and it contains only 3–8 yellow flowers bordered of red, and with peduncle provided with whitish to brownish hairs. It is distributed in Europe, Siberia, Asia Minor and Africa, and it grows particularly in the steppe and desert. Flowering takes place from August to October [15]. From the clinical and experimental

https://doi.org/10.1016/j.pathophys.2018.01.001 0928-4680/© 2018 Elsevier B.V. All rights reserved.

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Table 1 Characterisation of phenolic compounds of ACAE by HPLC-DAD-ESI–MS/MS. compound

tR (min)

UV ␭max (nm)

[M−H]− (m/z)

Fragment ion (m/z)

composition (%)

Tentative identification

1 2 3 4 5 6 7 8 9 10 11

16.5 17.2 21.4 23.9 24.1 24.6 25.1 25.7 27.7 30.03 32.88

242, 298 (sh), 325 242, 296 (sh), 323 259, 357 241, 298 (sh), 327 241, 298 (sh), 329 241, 298 (sh), 329 265, 349 267, 337 267, 339 253, 270 (sh), 347 267, 337

353 179 477 515 515 515 477 431 577 285 269

191; 179

4.3 2.15 5.37 0.24 0.78 0.93 0.05 6.42 15.52 2.74 62.42

chlorogenic acid caffeic acid isorhamnetin hexoside 3,4-dicaffeoylquinic acid 3,5-dicaffeoylquinic acid 4,5-dicaffeoylquinic acid quercetin-3-O-glucuronide apigenin-7-O-hexoside apigenin-6-C-glucuronide-8-C-pentoside kaempferol apigenin

315 353, 191, 179 353, 191, 179 353, 191, 179 301 269 517, 401, 269

studies performed on Artemisia campestris, it seems that most of its beneficial effects are related to its antioxidant properties which are mainly due to its ability to scavenge reactive oxygen species and/or inhibit lipid peroxidation [16,17]. For this reason, the Atemisia extracts are known to exhibit many beneficial health effects such as renoprotective [18], antidiabetic [19] and gastroprotective [11] effects. All these pharmacological effects are related to the wide range of biologically active compounds identified in the artemisia, particularly phenolic compounds such as isochlorogenic acid and 3,5-dicaffeoylquinic acid [20], rhamnetin, quercetin and Petunidin3-O-acetyl glucoside [21], as well as kaempferol and apigenin [14]. The literature shows that most of these compounds had potent pharmacological properties. In fact, the isochlorogenic acid is known for its hepatoprotective and antiinflammatory effects [22]. 3,5-dicaffeoylquinic acid has been shown to mitigate inflammatory mediator production [23]. Moreover, Zhong et al. [24] reported that kaempferol and apigenin exhibited a strong ROS scavenging activity. In addition, they have been shown to alleviate intestinal inflammation [25] as well as gastric ulcer [26]. Accordingly, this leads us to think of the effect of ACAE on esophageal injuries. In view of these biological activities, we sought to investigate on how artemisia extract could prevent the development of esophageal injuries in rat models. Currently, some of the experimental animal models are used to study the pathogenesis and pathophysiology of the esophagitis and gastroesophageal reflux. A model of GER in rats is one of the common models in the esophagus diseases research and it resembles human esophagitis in histology, such as the infiltration of inflammatory cells and the appearance of edematous zones in the esophageal mucosa [3]. To test our hypothesis, the present study was undertaken to determine the protective effect of an aqueous extract of A. campestris against gastric acid reflux-induced esophageal mucosal injury. We also studied the implication of oxidative stress and some intracellular mediators in such esophageal protection.

and then ground in an electric mixer. The plant powder was subsequently dissolved in distilled water and incubated at room temperature for 24 h under magnetic stirring. The sample was then centrifuged at 10.000g for 10 min and the supernatant was lyophilized, aliquoted and stored at −80 ◦ C until use. The chemical composition of ACAE (Table 1 and Fig. 1) was determined according to Sebai et al. [14]. 2.3. Animals Adult male Wistar rats (200–220 g, 15 weeks old) were provided by Pasteur Institute of Tunis and used in accordance with the Tunis University ethics committee for the use and care of Laboratory animals and in accordance with the NIH recommendations [27]. They were provided with food and water ad libitum and maintained at room temperature of 22–25 ◦ C. 2.4. Experimental model The rats were divided into six groups of 10 animals each. GER was induced to all used animals except control group. Following light ether anesthesia, rats were laparotomised to ligate the junction between the forestomach and the corpus as well as the pylorus [28] and they were then deprived of food and water. However, Groups I and II served respectively as normal and GER controls and had per orally (p.o.) a physiological solution. Groups III, IV, and V were treated with different doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.), while Group VI received famotidine (20 mg/kg, b.w., p.o.). Six hours later, animals were autopsied; their esophageal portion of the digestive tract was rapidly excised, cleaned, macroscopically examined and homogenized in phosphate buffer saline to measure the biochemical parameters such as MDA levels, H2 O2 , calcium, free iron, protein and −SH groups, as well as antioxidant enzyme activities. 2.5. Esophagitis severity evaluation

2. Materials and methods 2.1. Chemicals Butylated hydroxytoluene (BHT), bovine catalase, Epinephrine, trichloroacetic acid and 2-Thio-barbituric acid (TBA) were from Sigma chemicals Co (Germany). All other chemicals used were of analytical grade. 2.2. Sampling and Artemisia campestris aqueous extract (ACAE) preparation Artemisia campestris L. was collected during March 2014 from Béja governorate (Tunisia). The artemisia leaves (10%, weight/volume) were dried in an incubator at 40 ◦ C during 72 h

The severity of esophagitis was macroscopically scored, using an ulcer index. The following scale was used: 0, no injury; 1, erosion of mucosal epithelium; 2, the length of hemorrhagic ulcer area520 mm; 3, the length of hemorrhagic ulcer area 20–30 mm; 4, the length of hemorrhagic ulcer area 30–40 mm; 5, the length of hemorrhagic ulcer area440 mm or perforation [12,13]. 2.6. Histopathological analysis Immediately after sacrifice, the esophageal segments were harvested and washed with ice-cold saline. Tissue fragments were then fixed in a 10% neutral buffered formalin solution, embedded in paraffin, and used for histopathological examination. From this, 5 ␮m thick sections were cut, deparaffinized, hydrated, and stained with hematoxylin plus eosin (H + E). Tissue preparations

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Fig. 1. Phenolic compounds found in Artemisia campestris aqueous extract (ACAE).

were observed and micro-photographed under a light BH2 Olympus microscope. 2.7. Lipid peroxidation determination The lipid peroxidation is detected by the determination of malondialdhyde (MDA) production using the thiobarbituric acid method [29]. Briefly, aliquots from esophageal tissues homogenates were mixed with BHT-trichloroacetic acid (TCA) solution containing 1% BHT (w/v) dissolved in 20% TCA (w/v) and centrifuged at 1000 × g for 5 min at 4 ◦ C. Then, the supernatant was mixed with 0.5 NaCl and 120 mM TBA (thiobarbituric acid) in 26 mM Tris and heated in water bath at 80 ◦ C for 10 min. After cooling, the absorbance of the resulting chromophore was determined at 532 nm. MDA levels were determined by using an extinction coefficient for MDA-TBA complex of 1.56 × 105 M−1 cm−1 . 2.8. Sulfhydryl groups (-SH) measurement Sulfhydryl groups (-SH) were performed according to Ellman’s method [30]. Briefly, esophageal homogenates were mixed with 100 ␮L of 20 mM EDTA (ethylene tetra acetic acid) pH 8.2. Then, the reaction mixture was vortexed and its optical density was measured at 412 nm (A1). Thereafter, 100 ␮L of 10 mM DTNB were added, incubated during 15 mins and the absorbance of the sample was measured at 412 nm (A2). The sulfhydryl groups concentration was calculated using this expression: (A2–A1–B) × 1.57 Mm. The results were expressed as ␮mol of thiol groups per mg of protein.

homogenate was added to 2 mL reaction mixture containing 20 ␮L of epinephrine (5 mg/mL), 10 ␮L of bovine catalase (0.4 U/␮l) and 62.5 mM of sodium carbonate/bicarbonate buffer pH 10.2. Changes in absorbance were measured at 480 nm. Catalase activity was determined according to the method described by Aebi [32]. The reaction mixture contained 33 mM H2 O2 in 50 mM phosphate buffer pH 7.0 and CAT activity was calculated using the extinction coefficient of 40 mM-1cm-1 for H2 O2 . The GPx activity was quantified following the method described by Flohé and Günzler [33]. Briefly, 1 mL of reaction mixture containing 0.2 mL of esophageal supernatants, 0.4 mL of H2 O2 (5 mM), 0.2 mL of phosphate buffer 0.1 M pH 7.4 and 0.2 mL of GSH (4 mM) was incubated at 37 ◦ C for 1 min and the reaction was stopped by addition of 0.5 mL TCA (5%, w/v). After centrifugation at 1500 × g for 5 min, aliquot (0.2 mL) from supernatant was combined with 0.5 mL DTNB (10 mM) and 0.5 mL of phosphate buffer 0.1 M pH 7.4 and absorbance was measured at 412 nm. The GPx activity was expressed as nmol of GSH consumed/min/mg protein. 2.10. H2 O2 determination Hydrogen peroxide levels were determined according to the method described by of Dingeon et al. [34]. Briefly, in the presence of peroxidase, the hydrogen peroxide reacts with p-hydroxybenzoic acid and 4-aminoantipyrine leading to a quantitative formation of a quinoneimine which has a pink color detected at 505 nm. 2.11. Iron measurement

2.9. Antioxidant enzyme activities assays The method of Misra and Fridovich was used to determine the activity of Superoxide dismutase (SOD) [31]. Briefly, esophageal

Esophageal non haem iron was measured using ferrozine method as described by Leardi et al. [35]. Briefly, the iron dissociated from transferrin-iron complex by a solution of guanidine

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acetate and reduced by ascorbic acid reacts with ferrozine to give a pink complex measured at 562 nm. 2.12. Calcium determination The esophageal tissue calcium level was performed using a colorimetric method according to Stern and Lewis [36]. Briefly, at alkaline medium, calcium reacted with cresolphtalein leading to a colored complex measurable at 570 nm. 2.13. Protein determination Protein concentration was determined by the method of Hartree [37] which is a slight modification of the Lowry method. Using bovine serum albumin as standard.

Table 2 Effect of ACAE and famotidine (FAM) on GER-induced esophageal injury. Animals were treated with vehicle (H2 O), various doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.) or FAM (20 mg/kg, b.w., p.o.) during 6 h after esophagitis induction. The data are expressed as mean ± SEM (n = 10).). a: p < 0.05 compared to the control group, b: p < 0.05 compared to the esophagitis group c: p < 0.05 compared to ACAE-100 group and d: p < 0.05 compared to ACAE-200 (ANOVA test). Groups

Esophageal injury index

Protection (%)

Control GER GER + ACAE-100 GER + ACAE −200 GER + ACAE −400 GER + FAM

4.6 ± 0.24 3.20 ± 0.37a 1.80 ± 0.37b 1.20 ± 0.20bc 1.00 ± 0.31bcd

30.43 60.86 73.91 78.26

ACAE showed a significant and dose-dependent ulcer inhibition up to 78.26% with the high dose (400 mg/kg, b.w., p.o.).

2.14. Statistical analysis 3.2. Histological evaluation All the data were expressed as mean ± standard error of the mean (S.E.M.). Differences between the experimental groups were assessed by one-way ANOVA followed by Duncan’s test. Values were considered statistically significant when p < 0.05. 3. Results 3.1. Effect of ACAE treatment on esophageal injury index The macroscopic examination of the esophageal segment was carried out immediately after the sacrifice. Animals with GERinduced injury showed extensive elongated thick, dark red and black bands of hemorrhagic lesions throughout the esophagus. However, treatment with ACAE and famotidine protected esophageal epithelium from GER-induced injury (data not shown). However, as shown in Table 2, the induction of GER led to the formation of esophageal injuries with a high ulcer index (4.6 ± 0.24).

The histological examination of the esophageal walls in GERinduced esophagitis showed a disruption and an exfoliation of the superficial esophageal epithelium, a small alteration of surface coating, the appearance of some inflammatory infiltrates and hyperemia of epithelial layers (Fig. 2). Whereas, following ACAE treatment, the esophageal pathological injuries were significantly and dose-dependently attenuated. 3.3. Effect of ACAE treatment on esophageal lipoperoxydation Rats inflicted with esophagitis showed noticeable (P < 0.05) higher esophagus MDA levels compared to the control group. As shown in Fig. 3, ACAE at the dose range (100–400 mg/kg, b.w., p.o) has effectively inhibited this lipoperoxidation compared to the esophagitis values. In the same way, famotidine (20 mg/kg, b.w., p.o), used as a reference, significantly reduced the MDA level (Fig. 3).

Fig. 2. Esophageal histology showing the protective effects of Artemisia campestris aqueous extract (ACAE) and (FAM) famotidine, and gallic acid (GA) on GER-induced esophageal. Animals were treated with vehicle (H2 O), various doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.) or FAM (20 mg/kg, b.w., p.o.) during 6 h after esophagitis induction. (A) Control; (B) GER; (C, D and E) GER + ACAE (100, 200 and 400 mg/kg, b.w., p.o.) respectively and (F) GER + FAM (20 mg/kg, b.w., p.o.).

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Fig. 3. Effect of ACAE and famotidine (FAM) on GER-induced esophageal lipoperoxydation. Animals were treated with vehicle (H2 O), various doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.) or FAM (20 mg/kg, b.w., p.o.) during 6 h after esophagitis induction. The data are expressed as mean ± SEM (n = 10). a: p < 0.05 compared to the control group, b: p < 0.05 compared to the esophagitis group c: p < 0.05 compared to ACAE-100 group and d: p < 0.05 compared to ACAE-200 (ANOVA test).

Fig. 4. Effect of ACAE and famotidine (FAM) on GER-induced disturbance in esophageal thiol groups level. Animals were treated with vehicle (H2 O), various doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.) or FAM (20 mg/kg, b.w., p.o.) during 6 h after esophagitis induction. The data are expressed as mean ± SEM (n = 10). a: p < 0.05 compared to the control group, b: p < 0.05 compared to the esophagitis group c: p < 0.05 compared to ACAE-100 group and d: p < 0.05 compared to ACAE-200 (ANOVA test).

3.4. Effect of ACAE treatment on esophageal thiol groups Results shown in Fig. 4 clearly revealed the effects of Artemisia campestris extract and famotidine on esophageal thiol groups in GER-induced esophagus injury. As expected, esophagitis induced by GER caused a significant decrease in −SH groups content of esophageal tissues (P < 0.05). ACAE treatment significantly increased thiols towards the normal value (P < 0.05). 3.5. Effect of ACAE treatment on esophageal antioxidant enzyme activities As shown in Fig. 5, the induction of GER significantly decreased the esophagus antioxidant enzyme activities such as SOD (A), CAT (B) and GPx (C). ACAE treatment significantly restored the depletion of these enzymes in a dose-dependent manner. Fig. 4 also showed a positive effect of famotidine as compared to GER group.

Fig. 5. Effect of ACAE and famotidine (FAM) on GER-induced disturbance in esophageal antioxidant enzyme activities: (A) superoxide dismutase (SOD), (B) catalase (CAT), and (C) glutathione peroxidase (GPx). Animals were treated with vehicle (H2 O), various doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.) or FAM (20 mg/kg, b.w., p.o.) during 6 h after esophagitis induction. The data are expressed as mean ± SEM (n = 10). a: p < 0.05 compared to the control group, b: p < 0.05 compared to the esophagitis group c: p < 0.05 compared to ACAE-100 group and d: p < 0.05 compared to ACAE-200 (ANOVA test).

load in the esophageal free iron, H2 O2 and calcium (P < 0.05) level was observed in esophagitis group when compared to the control. In contrast, ACAE (100, 200 and400 mg/kg, b.w., p.o) and famotidine (100–400 mg/kg, b.w., p.o) significantly and dose-dependently protected against GER-induced esophagus intracellular mediators deregulation. 4. Discussion

3.6. Effect of ACAE treatment on esophageal intracellular mediators We also approached the putative involvement of intracellular mediators in GER-induced esophagitis (Table 3). Significant over-

Amongst the different models of experimental esophagitis, the ligation of the pylorus as well as the junction between the forestomach and the corpus are one of the standardized and extensively used methods [12,13,38]. We firstly showed in the present work

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Table 3 Effect of ACAE and famotidine (FAM) on GER-induced disturbance in esophageal H2 O2 , free iron, and calcium levels. Animals were treated with vehicle (H2 O), various doses of ACAE (100, 200 and 400 mg/kg, b.w., p.o.) or FAM (20 mg/kg, b.w., p.o.) during 6 h after esophagitis induction. The data are expressed as mean ± SEM (n = 10). a: p < 0.05 compared to the control group, b: p < 0.05 compared to the esophagitis group c: p < 0.05 compared to ACAE-100 group and d: p < 0.05 compared to ACAE-200 (ANOVA test). Groups

Free iron (␮mol/mg prot)

Hydrogen peroxide (mmol/mg prot)

Calcium (mmol/mg prot)

Control GER GER + ACAE-100 GER + ACAE −200 GER + ACAE −400 GER + FAM

34.28 ± 4.07 61.14 ± 4.35a 45.73 ± 2.46b 39.41 ± 3.16bc 36.48 ± 5.07bc 35.98 ± 4.37b

0.44 ± 0.11 1.13 ± 0.23a 0.82 ± 0.14b 0.65 ± 0.12bc 0.57 ± 0.18bc 0.48 ± 0.09b

0.82 ± 0.12 2.01 ± 0.06a 1.65 ± 0.08b 1.34 ± 0.11bc 1.05 ± 0.14bcd 1.01 ± 0.03b

that GER induced clear macroscopic and histological changes in the esophageal mucosa. ACAE treatment has effectively mitigated all the histopathological signs related to the GER as assessed by the decrease of esophageal injury index, the exfoliation of the superficial esophageal epithelium, the infiltration of inflammatory cells as well as the alteration of surface coating in the esophageal mucosa. In fact, a GER phenomenon is highly related to transient relaxation episodes and/or the increase in inadequate lower esophageal sphincter (LES) tone during increased abdominal pressure [39]. The contact between the esophageal epithelium and the refluxing gastric contents is a central event in the development of esophageal lesions either by decreasing production of esophageal mucus with an opposite effect in the pepsin diffusion, or by the loss of resistance of the non-keratinized squamous epithelium which has a high resistance against chlorhydropeptic aggressions [1]. According to the literature, our findings are in accordance with many other reports demonstrating that GER-induced esophageal injuries can be attenuated by several medicinal plants such as Morinda citrifolia [12], Curcuma longa [14] and Myrtus communis [13] as well as by some pharmacological agents such as beeswax alcohols (D-002) [40] and the natural immunomodulator, lactoferrin [41]. In the present study, we also showed that the GER-induced esophageal injuries are accompanied by a disruption in the esophageal redox balance, resulting in lipid peroxidation as evidenced by increased levels of malondialdehyde and the antioxidant enzyme activity depletion as well as decreased levels of the sulfhydryl groups. In this respect, many previous studies have demonstrated that esophagitis can be highly associated to the oxidative stress status in the esophagus [13,42,43]. However, in the physiological state, the ROS are modulators of signal transduction pathways and the expression of genes that participate in cellular functioning. Thus, the GER seems to be a cause of imbalance of ROS production particularly by the activation of NADPH oxidases and mitochondrial respiratory chain, which leads to the release of proinflammatory factors and promotes the process of apoptosis and/or necrosis in the esophagus mucosa cells [7,42,43]. All the oxidative damage induced by GER seems to be significantly and dosedependently restored by the administration of ACAE. However, our extract is very rich in bioactive compounds such as caffeic acid, quercetin-3-O-glucuronide, kaempferol, 3,4-dicaffeoylquinic acid, apigenin, 4,5-dicaffeoylquinic acid, chlorogenic acid, isorhamnetin hexoside, 3,5-dicaffeoylquinic acid, apigenin-6-C-glucuronide-8C-pentoside and apigenin-7-O-hexoside [11]. These molecules are powerful antioxidants that can reduce the oxidative damage induced by acidic gastric juice, because they have a structure that allows them to inhibit the ROS by neutralizing them, which prevents them from reaching their biological targets [8]. We further examined the effects of GER and ACAE on certain intracellular mediators related to oxidative stress system. As expected, GER-induced esophagitis significantly increased free iron, hydrogen peroxide and calcium levels in esophageal tissue. Indeed, the spontaneous dismutation of superoxide radicals led to the production of hydrogen peroxide which contributed via the Fenton reactions with free iron in the hydroxyl radicals generation

[44], leading to membranes lipoperoxidation and the enhancement of its permeability to calcium [45,46]. It seems that the loss of calcium homeostasis has a primal role in GER pathophysiology. More importantly, ACAE administration significantly and dose dependently mitigated GER-induced intracellular mediator deregulation. The same mechanism concerning ACAE beneficial effects has been previously described for aspirin-induced gastric injury [11]. 5. Conclusion In conclusion, our data confirm that the oral administration of Artemisia compestris aqueous extract exerts protective effect in GER-induced esophagitis in rats which are mediating via the modulation of oxidant/anti-oxidant balance in the esophageal tissue, the inhibition of the production of intracellular mediators including free iron, hydrogen peroxide and ionizable calcium. Indeed, the presence of phytochemicals such as caffeic acid, chlorogenic acid, quercetin-3-O-glucuronide, apigenin and kaempferol might have multifunctional targets on the improvement of the antioxidant defense system, the chelation of free iron and the trapping of free radicals, thereby inhibiting esophagitis. Ethical consideration All procedures on animals in this study were compiled with the NIH recommendations for the use and care of animals. Competing interests The authors declare that they have no competing interests. Financial disclosures None declared. Acknowledgements Financial support of the Tunisian Ministry of Higher Education and Scientific Research is gratefully acknowledged. We thank Nariman Kamel, English teacher at the Higher Institute of Biotechnology of Beja for his assistance. References [1] P. Ducrotté, U. Chaput, Pathophysiology of gastro-oesophageal reflux, EMC-Hepato-Gastroenterol. 2 (2005) 362–369. [2] C.T. Ferreira, E.d. Carvalho, V.L. Sdepanian, M.B. Morais, M.C. Vieira, L.R. Silva, Gastroesophageal reflux disease: exaggerations, evidence and clinical practice, J. Pediatr. (Rio J) 90 (2014) 105–118. [3] P.W. Weijenborg, A.J. Bredenoord, How reflux causes symptoms: reflux perception in gastroesophageal reflux disease, Best Pract. Res. Clin. Gastroenterol. 27 (2013) 353–364. [4] A. Meining, M. Classen, The role of diet and lifestyle measures in the pathogenesis and treatment of gastroesophageal reflux disease, Am. J. Gastroenterol. 95 (2000) 2692–2697.

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[5] M.E. Kavanagh, K.E. O’Sullivan, C. O’Hanlon, J.N. O’Sullivan, J. Lysaght, J.V. Reynolds, The esophagitis to adenocarcinoma sequence; the role of inflammation, Cancer Lett. 10 (2014) 182–189. [6] H. Kawahara, A. Kubota, T. Hasegawa, N. Phdoongsombut, P. Ratanasuwon, S. Kasiwong, Effects of rikkunshito on the clinical symptoms and esophageal acid exposure in children with symptomatic gastroesophageal reflux, Pediatr. Surg. Int. 23 (2007) 1001–1005. [7] A. Favier, Le stress oxydant: oxidative stress: conceptual and experimental interest in the understanding of disease mechanisms and therapeutic potential, L’Act Chim. 270 (2003) 108–115. [8] M. Rigoulet, E.D. Yoboue, A. Devin, Mitochondrial ROS generation and its regulation: mechanisms involved in H2 O2 signaling, Antioxid. Redox Signal. 14 (2011) 459–468. [9] M. Hurtado-Nedeleca, P.M.C. Dang, C. Renato Monteiro, R.C. Monteiroa, Physiology of human neutrophils, French Med. Mag. 462 (2014) 25–38. [10] M.A. Jabri, K. Rtibi, H. Tounsi, K. Hosni, A. Souli, J. El-Benna, L. Marzouki, M. Sakly, H. Sebai, Myrtle berries seeds aqueous extract inhibits in vitro human neutrophils myeloperoxidase and attenuates acetic acid-induced ulcerative colitis in rat, RSC Adv. 5 (2015) 64865–64877. [11] H. Sebai, M.A. Jabri, A. Souli, K. Hosni, S. Selmi, H. Tounsi, O. Tebourbi, S. Boubaker, J. El-Benna, M. Sakly, Protective effect of Artemisia campestris extract against aspirin-induced gastric lesions and oxidative stress in rat, RSC Adv. 4 (2014) 49831–49984. [12] S. Mahattanadul, W. Ridtitid, S. Nima, N. Phdoongsombut, P. Ratanasuwon, S. Kasiwong, Effects of Morinda citrifolia aqueous fruit extract and its biomarker scopoletin on reflux esophagitis and gastric ulcer in rats, J. Ethnopharmacol. 134 (2011) 243–250. [13] M.A. Jabri, H. Tounsi, K. Rtibi, L. Marzouki, M. Sakly, H. Sebai, Ameliorative and antioxidant effects of myrtle berry seed (Myrtus communis) extract during reflux-induced esophagitis in rats, Pharm. Biol. 54 (2016) 1575–1585. [14] P. Goetz, Traitement du reflux gastro-oesophagien, Phytothérapie 3 (2005) 107–109. [15] E. Le Floc’h, Contribution à une étude ethnobotanique de la Flore Tunisienne, Imprimerie Officielle de la République Tunisienne, Tunisia, 1983. [16] Z. Ghlissi, N. Sayari, R. Kallel, A. Bougatef, Z. Sahnoun, Antioxidant, antibacterial, anti-inflammatory and wound healing effects of Artemisia campestris aqueous extract in rat, Biomed. Pharmacother. 84 (2016) 115–122. [17] A. Akrout, L.A. Gonzalez, H. El Jani, P.C. Madrid, Antioxidant and antitumor activities of Artemisia campestris and Thymelaea hirsuta from southern Tunisia, Food Chem. Toxicol. 49 (2011) 342–347. [18] M. Sefi, A. Troudi, F.B. Hamida, N. Soudani, T. Boudawara, N. Zeghal, Protective effects of Artemisia campestris upon fenthion-induced nephrotoxicity in adult rats and their progeny, Gen. Physiol. Biophys. 32 (2013) 577–588. [19] M. Sefi, H. Fetoui, M. Makni, N. Zeghal, Mitigating effects of antioxidant properties of Artemisia campestris leaf extract on hyperlipidemia, advanced glycation end products and oxidative stress in alloxan-induced diabetic rats, Food Chem. Toxicol. 48 (2010) 1986–1993. [20] M. Dib, L. Tits, J.N. Angenot, A. Wauters, H. Assaidi, M. Mekhfi, M. Aziz, A. Bnouham, M. Legssyer, A. Ziyyat Frederich, Antihypertensive and vasorelaxant effects of aqueous extract of Artemisia campestris L. from Eastern Morocco, J. Ethnopharmacol. 206 (2017) 224–235. [21] W. Megdiche-Ksouri, N. Trabelsi, K. Mkadmini, S. Bourgou, A. Noumi, M. Snoussi, R. Barbria, O. Tebourbi, R. Ksouri, Artemisia campestris phenolic compounds have antioxidant and antimicrobial activity, Ind. Crops Prod. 63 (2015) 104–113. [22] B.J. Hao, Y.H. Wu, J.G. Wang, S.Q. Hu, D.J. Keil, H.J. Hu, J.D. Lou, Y. Zhao, Hepatoprotective and antiviral properties of isochlorogenic acid A from Laggera alata against hepatitis B virus infection, J. Ethnopharmacol. 144 (2012) 90–94. [23] M.D. dos Santos, G. Chen, M.C. Almeida, D.M. Soares, G.E. de Souza, N.P. Lopes, R.C. Lantz, Effects of caffeoylquinic acid derivatives and C-flavonoid from Lychnophora ericoides on in vitro inflammatory mediator production, Nat. Prod. Commun. 5 (2010) 733–740. [24] Y. Zhong, C. Jin, J. Gan, X. Wang, Z. Shi, X. Xia, X. Peng, Apigenin attenuates patulin-induced apoptosis in HEK293 cells by modulating ROS-mediated mitochondrial dysfunction and caspase signal pathway, Toxicon 137 (2017) 106–113.

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[25] F. Jailani, G. Williamson, Effect of edible oils on quercetin, kaempferol and galangin transport and conjugation in the intestinal Caco-2/HT29-MTX co-culture model, Food Funct. 5 (2014) 653–662. [26] A.P. Beber, P. de Souza, T. Boeing, L.B. Somensi, L.N. Mariano, B.J. Cury, L.M. Burci, C.B. da Silva, E. Simionatto, S.F. de Andrade, L.M. da Silva, Constituents of leaves from Bauhinia curvula Benth. exert gastroprotective activity in rodents: role of quercitrin and kaempferol, Inflammopharmacology (2017), http://dx.doi.org/10.1007/s10787-017-0313-8, in press. [27] National Research Council, Guide for the Care and the Use of Laboratory Animals, vol. 20, National Institute of Health, Bethesda, 1985, p 85. [28] K. Nakamura, Y. Osawa, Y. Furuta, H. Miyazaki, Effects of sodium polyacrylate (PANa) on acute esophagitis by gastric juice in rats, Jpn. J. Pharmacol. 32 (1982) 445–456. [29] H.H. Draper, M. Hadley, Malondialdehyde determination as index of lipid peroxidation, Methods Enzymol. 186 (1990) 421–431. [30] H.P. Misra, I. Fridovich, The role of superoxide anion in the autoxidation of epinephrine and simple assay for superoxide dismutase, J. Biol. Chem. 247 (1972) 3170–3175. [31] H.H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [32] L. Flohé, W.A. Günzler, Assays of glutathione peroxidase, Methods Enzymol. 105 (1984) 114–121. [33] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77. [34] B. Dingeon, J.P. Ferry, A. Roullet, Automatic assay of blood sugar by Trinder’s method, Ann. Biol. Clin. 33 (1975) 3–13. [35] A. Leardi, M. Caraglia, C. Selleri, S. Pepe, C. Pizzi, R. Notaro, A. Fabbrocini, S. De Lorenzo, M. Musicò, A. Abbruzzese, A.R. Bianco, P. Tagliaferri, Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic, cells, Br. J. Haematol. 102 (1998) 746–752. [36] J. Stern, W.H. Lewis, The colorimetric estimation of calcium in serum with ocresolphthalein complexone, Clin. Chim. Acta 2 (1957) 576–580. [37] E.F. Hartree, Determination of protein. a modification of the Lowry method that gives a linear photometric response, Anal. Biochem. 48 (1972) 422–427. [38] L. Yang, H. Cai, J. Tou, W. Gu, X. Shu, T. Zhang, X. Yang, Z. Shen, M. Jiang, The role of the 5-hydroxytryptamine pathway in reflux-induced esophageal mucosal injury in rats, World J. Surg. Oncol. 10 (2012) 219. [39] S.J. Spechler, E. Lee, D. Ahnen, R.K. Goyal, I. Hirano, F. Ramirez, J.P. Raufman, R. Sampliner, T. Schnell, S. Sontag, Z.R. Vlahcevic, R. Young, W. Williford, Long-term outcome of medical and surgical therapies for gastroesophageal reflux disease. Follow-up of a randomized controlled trial, JAMA 285 (2001) 2331–2338. [40] Z. Zamora, V. Molina, R. Mas, Y. Ravelo, Y. Perez, A. Oyarzabal, Protective effects of D-002 on experimentally induced gastroesophageal reflux in rats, World J. Gastroenterol. 20 (2014) 2085–2090. [41] M. Inamori, J. Togawa, S. Matsumoto, K. Harad, M. Matsuura, H. Iida, K. Akimoto, H. Endo, T. Nonaka, H. Takahashi, T. Koide, W. Shibata, T. Ida, A. Kusakabe, H. Nagase, S. Tominaga, K. Tanaka, E. Gotoh, A. Nakajima, S. Maeda, Protective effect of lactoferrin on acute acid reflux-induced esophageal mucosal damage, Hepatogastroenterology 61 (2014) 1595–1600. [42] T.Y. Oh, J.S. Lee, B.O. Ahn, H. Cho, W.B. Kim, Y.B. Kim, Y.J. Surh, S.W. Cho, K.B. Hahm, Oxidative damages are critical in pathogenesis of reflux esophagitis: implication of antioxidants in its treatment, Free Radic. Biol. Med. 30 (2001) 905–915. [43] X. Chen, Y.W. Ding, G. y. Yang, F. Bondoc, M.J. Lee, C.S. Yang, Oxidative damage in an esophageal adenocarcinoma model with rats, Carcinogenesis 21 (2002) 257–263. [44] S. Osuna, M. Swart, M. Sola, On the mechanism of action of fullerene derivatives in superoxide dismutation, Chem. Eur. J. 16 (2010) 3207–3214. [45] M.A. Jabri, N. Aissani, H. Tounsi, M. Sakly, L. Marzouki, H. Sebai, Protective effect of chamomile (Matricaria recutita L.) decoction extract against alcohol-induced injury in rat gastric mucosa, Pathophysiology 24 (2017) 1–8. [46] G. Ermak, K.J. Davies, Calcium and oxidative stress: from cell signaling to cell death, Mol. Immunol. 38 (2002) 713–721.

Please cite this article in press as: M.-A. Jabri, et al., Protective effects of Artemisia campestris extract against gastric acid reflux-induced esophageal mucosa injuries, Pathophysiology (2017), https://doi.org/10.1016/j.pathophys.2018.01.001