Tamarix gallica phenolics protect IEC-6 cells against H2O2 induced stress by restricting oxidative injuries and MAPKs signaling pathways

Biomedicine & Pharmacotherapy 89 (2017) 490–498

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Original article

Tamarix gallica phenolics protect IEC-6 cells against H2O2 induced stress by restricting oxidative injuries and MAPKs signaling pathways Jamila Bettaiba,b,c,* , Hélène Talarminc , Mickaël Droguetc, Christian Magnéd, Mondher Boulaabab , Marie-Agnès Giroux-metgesc, Riadh Ksourib a

Université de Tunis El Manar, Faculté des Sciences de Tunis, 2092, Tunis, Tunisie Centre de Biotechnologie de Borj-Cédria, LR15CBBC06 Laboratoire des Plantes Aromatiques et Médicinales, BP 901, 2050 Hammam-lif, Tunisie Université de Bretagne Occidentale, Faculté de Médecine et des Sciences de la Santé, EA 1274 Laboratoire de Physiologie, 29200, Brest, France d Université de Bretagne Occidentale, UFR Sciences et Techniques, EA 2219 Géoarchitecture, CS 93837, 29238 Brest Cedex 3, France b c

A R T I C L E I N F O

Article history: Received 21 November 2016 Received in revised form 12 February 2017 Accepted 15 February 2017 Keywords: Antioxidants Cytoprotection H2O2-induced oxidative stress IEC-6 cells Tamarix gallica

A B S T R A C T

Polyphenolic compounds gained interest in the pharmaceutical research area due to their beneficial properties. Herein, antioxidant and cytoprotective capacities of T. gallica extract on H2O2-challenged rat small intestine epithelial cells were investigated. To set stress conditions, IEC-6 cultures were challenged with numerous H2O2 doses and durations. Then, 40 mM H2O2 during 4 h were selected to assess the cytoprotective effect of different T. gallica extract concentrations. Oxidative parameters, measured through CAT and SOD activities as well as MDA quantification were assessed. In addition, the expression of possibly involved MAPKs was also valued. Main results reported that T. gallica was rich in polyphenols and exhibited an important antioxidant activity (DPPH
Assay, IC50 = 6 mg mL1; ABTS
+ test, IC50 = 50 mg mL1; Fe-reducing power, EC50 = 100 mg mL1). The exposure of IEC-6 cultures to 40 mM H2O2 during 4 h caused oxidative stress manifested by (i) over 70% cell mortality, (ii) over-activity of CAT (246%), (iii) excess in MDA content (18.4 nmol mg1) and (iiii) a trigger of JNK phosphorylation. Pretreatment with T. gallica extract, especially when used at 0.25 mg mL1, restored cell viability to 122%, and normal cell morphology in H2O2-chalenged cells. In addition, this extract normalized CAT activity and MDA content (100% and 14.7 nmol mg1, respectively) to their basal levels as compared to control cells. Furthermore, stopping cell death seems to be due to dephosphorylated JNK MAPK exerted by T. gallica bioactive compounds. In all, T. gallica components provided a cross-talk between regulatory pathways leading to an efficient cytoprotection against harmful oxidative stimulus. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Abbreviations: BSA, bovine serum albumin; CAT, catalase; DMEM, Dulbecco’s Modified Eagle Medium; DMSO, dimethyl sulfoxide; DW, dry weight; EC50, effective concentration at which the absorbance was 0.5; FBS, fetal bovine serum; GAE, gallic acid equivalent; IC50, inhibition concentration at 50%; PBS, phosphate buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MAPKs, mitogen-activated protein kinases; MDA, malondialdehyde; NBT, Nitrotetrazolium blue chloride; IEC-6 cells, rat small intestine epithelial cells; SOD, superoxide dismutase; TgE, Tamarix gallica extract; TBARS, thiobarbituric acid reactive substances. * Corresponding author at: Centre de Biotechnologie de Borj-Cédria, LR15CBBC06 Laboratoire des Plantes Aromatiques et Médicinales, BP 901, 2050 Hammam-lif, Tunisie. E-mail address: [email protected] (J. Bettaib). http://dx.doi.org/10.1016/j.biopha.2017.02.047 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

Over-expression of reactive oxygen species (ROS) induces severe oxidative threats to cell components including: protein, lipid, DNA and RNA associated with cell structural damage, tissue injury and gene mutation [1]. ROS excess is harmful for transcriptions factors, ion channels, phosphatases activity and can lead to inappropriate cell signaling. The mitogen-activated protein kinases (MAPKs) pathways are triggered in response to stressassociated stimuli, resulting in growth arrest or even apoptosis [2]. These disorders play a causative role in aging and are often associated with several physiological disruptions such as cognitive dysfunction, cancer, atherosclerosis, heart disease, and inflammation injury [3]. To deal with detrimental effects of ROS, cells possess a number of compensatory mechanisms such as the induction of superoxide

J. Bettaib et al. / Biomedicine & Pharmacotherapy 89 (2017) 490–498

dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) [4]. In vivo as well as in vitro, superoxide anion, a major free radical generated in oxidative stress, is catalyzed into H2O2 by SOD. Consecutively, the produced H2O2 is detoxified into water and molecular oxygen by CAT and GPx. It is commonly assumed that ROS–antioxidant balance and membrane integrity are maintained when the activities of these detoxifying enzymes are privileged [5]. Otherwise, phenolics, flavonoids, tannins and anthocyanidins are proved to be natural, efficient and safe antioxidants [3]. These bioactive compounds can delay the lipids and proteins oxidation by inhibiting the initiation or/and propagation of oxidative chain reaction. Thus, they may prevent or repair cell damage caused by oxygen [6]. In recent years, natural biophenols, such as resveratrol, caffeic and rosmarinic acids were highlighted as efficient protectors against the oxidative cytotoxicity of hydrogen peroxide by the regulation of the endogenous antioxidant defense system and the modulation of signaling pathways [7]. In this context, bioactive compounds such as stilbene and resveratrol were described for their capacity in regulating CAT, SOD and GPx activities as well as for their ability to restrict JNK and p38 MAPKs phosphorylation induced by H2O2 stress [5,7,8]. In Tunisia, a considerable diversity of halophytic species of multiple interests including therapeutic practices occurs, such as Tamarix gallica. This medicinal halophyte was studied by our laboratory for its important phenolic content; high antioxidant and antimicrobial activities and it even showed efficient antitumoral capacity [6,9,10]. Following these works schedule, this species is further investigated for its cytoprotective effect against oxidative stress. In that framework, antioxidant capacities of T. gallica were further evaluated. Then, cytoprotective effect of several T. gallica extract concentrations on H2O2-induced insult within IEC-6 cells was explored. SOD and CAT activities as well as MDA levels were assessed and the possible underlying mechanism involving JNK and p38 MAPKs was investigated. 2. Materials and methods 2.1. Reagents IEC-6 cells (rat-derived intestinal epithelial cell line) were purchased from Public Health England (88071401). DMEM (Dulbecco’s Modified Eagle Medium), fetal bovine serum (FBS), antibiotics (100 mg/mL of streptomycin and 100 UI/mL of penicillin) and trypsin/EDTA were obtained from Lonza (France). Hydrogen peroxide 3% (w/w) was procured by Laboratoire Gilbert (France). Phosphate buffered saline (PBS) was furnished by Dominique Dutscher (France). Solvents (ethanol, DSMO), MTT, protease and phosphatase inhibitor cocktail, riboflavin, methionine, NBT, hydrochloric acid, trichloroacetic acid, thiobarbituric acid, BSA and primary antibodies b-actin were purchased from Sigma Aldrich (Germany). Anti-p38, pp38 and horseradish peroxidise-conjugated secondary antibody were acquired from Abcam (England). SAPK/JNK and phospho SAPK/JNK were from Cell Signaling Technology. ECL detection agents were acquired from Amersham International. 2.2. Extract preparation Extract of T. gallica shoots were obtained by maceration of powdered samples (3 g) during 30 min in 30 mL of water–ethanol solvent (1:1, v/v) at room temperature. After 24 h in obscurity at 4  C, supernatants were recovered by centrifugation at 1250  g for 5 min at 4  C. T. gallica extract (TgE) was stored at 4  C until use [6].

491

For the cytoprotective effect analysis, TgE was prepared as described above and then concentrated by vacuum-evaporation until dryness. Dry extracts were dissolved in DMSO to get 10% stock concentration. Obtained extract was stored at 20  C until analysis. 2.3. Characterization of phenolic levels and antioxidant activities in plant extract 2.3.1. Determination of total polyphenol content The amount of total phenolics in TgE was determined with the Folin–Ciocalteu reagent [11]. An aliquot of 125 mL of diluted extract was added to 500 mL of distilled water and 125 mL of the Folin–Ciocalteu reagent. The mixture was shaken, before adding 1250 mL of Na2CO3 (7%) and adjusting with distilled water to a final volume of 3 mL. After incubation for 90 min at 23  C in the dark, the absorbance versus prepared blank was read at 760 nm. Total phenolic content was expressed as mg GAE (Gallic Acid Equivalent)/g DW (Dry Weight) using a calibration curve with gallic acid, ranged from 0 to 400 mg mL1. The sample was analyzed in triplicate. 2.3.2. Total antioxidant capacity The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH [12]. An aliquot of sample extract was combined in an eppendorf tube with 1 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were incubated in a thermal block at 95  C for 90 min. After the mixture had cooled to room temperature, the absorbance of each solution was measured at 695 nm against a blank. The antioxidant capacity was expressed as mg gallic acid equivalent per gram of dry weight (mg GAE/g DW). The sample was analyzed in three replications. 2.3.3. DPPH scavenging activity One milliliter of the extract at known concentrations was added to 0.5 mL of a DPPH methanolic solution. The mixture was shaken vigorously and left standing at room temperature in the dark for 30 min. The absorbance was then measured at 517 nm and corresponds to the extract ability to reduce the radical DPPH
to the yellow-colored diphenylpicrylhydrazine [13]. The ability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging effect (%) = (A0  A1)/A0*100

(1)

Where A0 is the absorbance of the control at 30 min, and A1 is the absorbance of the sample at 30 min. The antiradical activity was expressed as IC50 (mg mL1), the antiradical dose required to cause a 50% inhibition. The sample was analyzed in triplicate. 2.3.4. Scavenging ability on ABTS test The ABTS
+ was produced by the reaction between 5 ml of 14 mM ABTS solution and 5 ml of 4.9 mM potassium persulfate solution, stored in the dark at room temperature for 16 h. Before usage, this solution was diluted with ethanol to get an absorbance of 0.700  0.020 at 734 nm. In a final volume of 1 ml, the reaction mixture comprised 950 mL of ABTS
+ solution and 50 mL of the TgE at various concentrations. These mixtures were homogenised and its absorbance was recorded at 734 nm. All measurements were done after at least 6 min. Similarly, the reaction mixture of standard group was made with 950 mL of ABTS
+ solution and 50 mL of BHT [14]. As for the antiradical activity, ABTS scavenging ability was expressed as IC50 (mg mL1). The inhibition percentage of ABTS radical was calculated using formula (1).

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2.3.5. Iron reducing power The sample extract was mixed with 2.5 mL of sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide, and the mixture was incubated at 50  C for 20 min. After that, 2.5 mL of 10% trichloroacetic acid was added, and the mixture was centrifuged at 650  g for 10 min. The upper layer fraction (2.5 mL) was mixed with deionised water and 0.5 mL of ferric chloride. The absorbance was measured at 700 nm in a spectrophotometer and ascorbic acid was used as positive control. A higher absorbance indicates a higher reducing power [15]. EC50 value (mg mL1) is the effective concentration at which the absorbance was 0.5 for reducing power and was obtained from linear regression analysis.

2.7.2. Enzymatic assays CAT activity was measured according to a previously published method [18]. SOD activity was assayed as previously described [19]. Enzyme activities were normalized by protein content (U mg1). 2.7.3. Biomarker of lipid oxidative damage As an index of the lipid peroxidation, the formation of TBARS during an acid-heating reaction, was used as previously described [20]. Results were expressed as nmoles MDA equivalents per mg protein, using the molar extinction coefficient of the MDA (1.56 105 M1 cm1). 2.8. Western-blot analysis

2.4. Cell maintenance IEC-6 (Public Health England-88071401) cells were maintained in DMEM and supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic and incubated at 37  C in a humidified incubator under 5% CO2. When cells were 80–90% confluent, they were harvested with trypsin/EDTA. For all experiments, cells were seeded at a concentration of 2  104 cells/well in 96-well plates. 2.5. Cell treatment protocols 2.5.1. Establishing of H2O2 optimal effective concentration A stock solution of stabilized hydrogen peroxide 3% (w/w) was diluted in DMEM lacking of FBS and sodium pyruvate. Serial concentrations ranging from 10 to 200 mM of H2O2 were dispensed per well. IEC-6 cells were exposed during 4, 24 and 48 h.

IEC-6 cells (4  105 cells mL1) were pre-incubated with 0.25 mg mL of TgE during 1 h and then exposed to 40 mM H2O2 for 4 h. Then, cells were washed in PBS and lysed by RIPA buffer with protease inhibitor cocktail. Cell lysate was clarified by centrifugation at 14,000  g for 20 min at 4  C. The protein-containing supernatant was kept. Proteins (40 mg) were loaded and run by electrophoresis for 90 min at 110 V, before being transferred onto nitrocellulose membranes. After blocking with 5% BSA, membrane was incubated at 4  C overnight under shaking with primary antibodies b-actin, anti-p38, pp38, SAPK/JNK and phospho SAPK/ JNK. The bands were detected by horseradish peroxidise-conjugated secondary antibody using enhanced chemiluminescence system ECL. Protein content was assayed as previously described [17]. 1

2.9. Statistical analysis 2.5.2. Cytotoxicity of plant extract TgE was used at various dilutions ranging between 0.01 and 50 mg mL1 prepared in FBS and sodium pyruvate-free DMEM. For all concentrations, exposure time was fixed to 4 h. 2.5.3. Cytoprotective effect T. gallica extract against H2O2 oxidative insult IEC-6 cells were pretreated with a rage of concentration (0.01–50 mg mL1) of TgE, for 1 h prior to H2O2 exposure. Then, cells were challenged with 40 mM of H2O2 for 4 h. The control group was treated with DMEM medium alone in the same manner. 2.6. IEC-6 cell morphology and MTT cell viability assay In order to evaluate morphological modifications of the IEC-6 cells in the presence of H2O2 with or without TgE, cells were observed with inverted microscope (Olympus optical CO-LTD. BH2-RFL-T3) after each treatment. At the end of each treatment, 100 mL of MTT solution (0.05%) were added to the culture medium. After 2 h incubation, the formazan produced was dissolved using 100 mL of DMSO. Absorbance was measured at 540 nm on a multidetection microplate reader. Results shown represent the mean of three independent experiments. 2.7. Antioxidant enzyme activities and lipid peroxidation assay 2.7.1. Cell lysis and protein quantification IEC-6 cells were lysed in 0.1 M Tris-HCl (pH 7.4) solution, supplemented with protease inhibitor cocktail. After lysis, cells were scraped, collected and then homogenized thoroughly. The resulting suspension was centrifuged for 10 min at 13,000  g and 4  C [16]. Once recovered, the supernatant was stored at 80  C until use. Protein content was assayed as previously described [17].

For all parameters, all samples were analysed in three replications. Data are shown as mean  SD. A one-way analysis of variance (ANOVA) using the post hoc analyse with Duncan’s test was carried out to test any significant differences at p  0.05. 3. Results and discussion 3.1. Phytochemical investigation Results concerning total phenolic content and antioxidant capacities were given in Table 1. T. gallica extract had important phenol level that was equal to 132.5 mg GAE g1 DW and a strong total antioxidant activity (107.7 mg GAE g1 DW). Thus, TgE displayed interesting IC50 values for DPPH
and ABTS
+ tests (6 and 50 mg mL1, respectively) as well as for Fe-reducing power (EC50 = 100 mg mL1). Interestingly, T. gallica was richer on total phenolic content than some other medicinal halophytes like Limoniastrum monopetalum and Limonium densiflorum (15.85 and 48.04 mg GAE g1 DW, respectively) [1,21]. Generally, such richness in phenolic components contributes significantly to the efficient antioxidant activity. Accordingly, a close relationship may Table 1 Total phenolic contents and antioxidant activities. The antioxidant activities presented were: total antioxidant, antiradical and reducing abilities. Values represent averages of at least three replications. T. gallica shoots

Value

Unit

Polyphenol content Total antioxidant activity DPPH
test ABTS
+ Reducing power

132.41 107.72 6 50 100

mg GAE g1 DW mg GAE g1 DW IC50, mg mL1 IC50, mg mL1 EC50, mg mL1

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493

120

IEC-6 Viability (% of the Control)

100 # *

80

*

4H

#

60

*

+

24H 48H # #

+

40

+

20

*

+ *

#

+

*

#

+

* #

+

0 CTR

DMSO 0.5%

10

20

µM

µM

30 µM

40 µM

50 µM

100 µM

200 µM

H2O2 doses (µM) Fig. 1. H2O2 cytotoxic effect on IEC-6 cell viability IEC-6 rat intestinal. Cells (2  104 cells/mL) were exposed to oxidative stress through H2O2 applied as a range of dilutions (10–200 mM) during 4, 24 and 48 h. Error bars represent standard deviations of three replications. Bars marked with asterisks are significantly different from the control at p  0.05, according to ANOVA test: *4 h, #24 h, +48 h.

be suggested between phenolic amounts and antioxidant capacities in T. gallica aerial parts [6,9]. 3.2. Cytotoxic effect of H2O2 on IEC-6 cells Typical dose/time-dependent inhibitory effects of H2O2 against IEC-6 cells were plotted in Fig. 1.

survival rates of IEC-6 and C6 glioma cells were tightly dependent on the H2O2-dose and the incubation time [22,23]. Actually, H2O2 crosses membranes and interferes with cells attachment to initiate immediately cellular damages. Those damages include, principally but not exclusively, cell shape changes and mitochondrial dysfunction leading to metabolic alterations [24]. 3.3. IEC-6 cell viability in the presence of T. gallica extract

3.2.1. Effect of H2O2 treatment duration The decline in cell viability after 4 h of induced oxidative stress was spectacular (over 70%, since 40 mM H2O2). In addition, 48 h duration decreased statistically cell viability (inferior to 40%) as compared to 4 and 24 h exposures (Fig. 1). 3.2.2. Effect of H2O2 treatment concentration As for H2O2 exposure duration, increasing H2O2 concentrations significantly altered cell viability (Fig. 1). Actually, cell growth was markedly inhibited since 40 mM H2O2 by 70% and dropped to 90% at 100 and 200 mM H2O2. In the current study, 4 h exposure to 40 mM H2O2 were selected as stress conditions, to obtain a decrease of at least 70% of cell viability, in order to detect the potential cytoprotective capacity of TgE. 3.2.3. Cell morphological aspect In line with MTT data, photomicrographs of IEC-6 cells (Fig. 2b) demonstrated impressive changes in cell shape (arrows indicated a round shape) and an elevated number of dead cells in the supernatant, in response to H2O2-stress. In previous studies,

In the absence of exogenous H2O2, adding TgE (0.01–5 mg mL1) had no significant effect on cell growth (Fig. 3) and the viability rates ranged from 93 to 102%. At higher concentration (50 mg mL1), TgE might cause damages to the cell and resulted in lowered cell viability to 62%. Our findings are in line with those of [25] who reported that high concentrations (superior to 50 mg mL1) of effective extract may reduce notably IEC-6 cell viability. As for their antioxidant capacities, and under certain conditions such as over doses, phenolic compounds beneficial potency may be inversed and become actually lethal for the cells [26]. 3.4. T. gallica extract promoted IEC-6 cell viability Different additives in the culture medium, such as pyruvate and serum albumin [27,28] protect cells from harmful action of active oxygen. In the current study, IEC-6 cells were exposed under serum and pyruvate-free conditions. Thus, cell growth, lowered by H2O2 insult, occurred only due to TgE pretreatment. As shown in Fig. 4, H2O2-induced cell mortality was obviously

Fig. 2. Photomicrographs of rat IEC-6 cells demonstrating cellular morphology in response to H2O2-induced stress and in combination with 0.25 mg/mL TgE (100); a Control, b. Exposed to H2O2 (40 mM, 4 h): changes in cell shape are indicated by arrows, c. Pretreated with TgE (0.25 mg/mL, 1 h) and then exposed to H2O2 (40 mM, 4 h).

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120

IEC-6 viability (% of the control)

100 80 60 * 40 20 0 CTR

DMSO 0.5%

0.01

0.1

0.25

0.5

5

50

TgE concentrations (µg mL-1) Fig. 3. IEC-6 cell viability in the presence of TgE. Cells at 2  104 cells/mL were cultured in the presence of different concentrations of TgE ranging from 0.01 and 50 mg/mL for 4 h. Values represent the means of three independent experiments  standard deviation. The symbol * corresponds to the statistical significance (p  0.05) in comparison to control cultures.

IEC-6 Viability (% of the control)

140

*

120 100 80 60

40 20 0 CTR

H2O2 40 µM

0.1

0.01

0.25

0.5

5

TgE concentrations (µg/mL) Fig. 4. Effect of TgE pretreatment on cell viability of H2O2-challenged IEC-6Cells at 2  10 cells/mL were pretreated with a range of TgE concentrations (0.01–5 mg mL1) for 1 h, and then exposed to 40 mM H2O2 during 4 h. Data are mean  SD obtained from three independent experiments. The symbol * corresponds to the statistical significance (p  0.05) comparing to H2O2-treated cultures alone. 4

alleviated by a pretreatment with TgE in a dose dependent manner. In fact, IEC-6 cells became less sensitive to the cytolethal action of H2O2, especially with the use of TgE at 0.25 mg mL1, for which the viability was restored to the extent of 122% of control (Fig. 4). Moreover, as shown in Fig. 2c photo, the normal shape was recovered. Thus, it was possibly suggested that TgE, mainly in low concentrations, had cytoprotective effect on IEC-6 cells owing to its stimulatory action on cell proliferation. These data point toward T. gallica phenolics as the origin of cellular anti-oxidative effect. As previously described, antioxidant phenolics act as reducing agents, hydrogen donors, singlet oxygen quenchers, free radical scavengers and chelating agents of pro-oxidants metals. Indeed, plant extracts rich in phenolic compounds are capable of complexing with and stabilizing transition metal ions, rendering them unable to initiate the oxidative chain reaction [6]. It has to be taken into account that quercetin, major phenolic component in T. gallica, was very potent in protecting Caco-2, HepG2 cells and human leukaemia cell line against H2O2 [10,29]. Additionally, resveratrol (another major compound in T. gallica) has the capability to quench H2O2 to a certain extent in glioma cells

[10,29]. The bioactivity of quercetin and resveratrol is probably due to their reliable interaction with cells; as bioactive compounds association with cellular membranes is a major factor in their dispersion into cellular compartments and therefore their efficacy [29]. Taken together, pretreatment with 0.25 mg mL1 TgE for 1 h then with 40 mM H2O2 during 4 h were chosen as experimental settings for the rest of investigations. 3.5. T. gallica extract supported enzymatic defense SOD and CAT are the first and the major line of defense against oxidative stress in cells controlling free radical pathways [5]. After 4 h exposure to 40 mM H2O2, CAT activity of IEC-6 cells increased by 146% comparing to control group (Fig. 5). Interestingly, 0.25 mg mL1 TgE lowered significantly (p  0.05) CAT activity by the same rate, in comparison to the TgE free-group. Accurately, the prophylactic effect of T. gallica might be due to H2O2 neutralization to H2O and O2 before the end of the experiment most probably by its phenolics such as resveratrol and kaempferol previously

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495

A 350

* CAT

CAT, SOD activities (% of the control)

300

SOD

250 200 150 100 50 0 CTR

DMSO 0.5%

H2O2 40 µM

TgE + H2O2

Treatments

B 8 7

SOD/CAT Ratio

6 5 4 3 2 1 0

CTR

DMSO

H2O2 40 µM Treatment

TgE + H2O2

Fig. 5. The oxidative state of rat small intestine, following TgE pretreatment (0.25 mg/mL, 1 h) and H2O2 exposure (40 mM, 4 h), was determined by measuring SOD and CAT activities (A) and SOD/CAT ratio assessment (B). Data of three independent experiments is presented as mean  SD. Bars marked with asterisk * are significantly different from the control at p  0.05, according to ANOVA test.

characterized for their efficacy to scavenge H2O2 [5,10,29]. Indeed, it was reported that the pretreatment with kaempferol maintained CAT activity in normal level when erythrocytes were attacked by 2,20 -azobis (2-amidinopropane) [30]. At elevated concentrations of H2O2, catalase displays spectacular efficiency in reducing it and this activity decreases at low concentrations of this ROS. In fact, catalase requires the reaction of two H2O2 molecules to carry out its reduction and this is more unlikely to occur as the concentration of the substrate falls under antioxidant treatment [29]. Our results revealed no significant variation in SOD activity (Fig. 5A). SOD/CAT ratio decreased considerably after 40 mM H2O2 exposure, but trended to return to basal level in the TgE pretreatedgroup (Fig. 5B). These data occurred because SOD levels are unresponsive to H2O2 exposure [31]. Indeed, SOD had no direct affinity to hydrogen peroxide as it scavenges superoxide radicals to hydrogen peroxide, which is then handled by CAT [8]. These findings suggest that the protective effect of antioxidant treatment was mainly attributed to CAT activity regulation (Fig. 6). 3.6. T. gallica phenolics reduced lipid peroxidation in IEC-6 cells Table 2 showed that incubation of IEC-6 cells with H2O2 caused a marked MDA production, which attempted 18.4 nmol mg1. TgE was effective to protect IEC-6 cells against H2O2-induced lipid damage and lowered MDA content to 14.7 nmol mg1 (p  00.5). It

is worthy to mention that in absence of TgE, the MDA content and SOD/CAT ratio were at their maximum. High MDA level and SOD/CAT imbalance, biomarkers of oxidative stress, damage the fluidity and permeability of the cell membrane. These disruptions are harmful to cell’s structure and function. Interestingly, phenolics and especially flavonoids, were reported for their ability to delay the processes of lipid peroxidation [6]. For instance, catechin was closely correlated to the inhibition of lipid peroxidation [32]. As a matter of fact, kaempferol, ()-epicatechin and its related procyanidins, previously described as major phenolics in T. gallica, adsorb to membranes through associations with the polar head-groups of phospholipids [6,30]. Thus, the created flavonoid-rich environment limit the access of oxidants to the bilayer and control the propagation rate of free radical chain reactions into the cell membrane [33]. 3.7. T. gallica extract suppress JNK MAPK phosphorylation MAPKs are capable of regulating a large number of cellular processes, including cell survival, apoptosis, adaptation, differentiation, and proliferation in response to stress [2]. As observed in Fig. 5(A and B), in H2O2 group, the expression of pJNK1 protein was significantly increased by 150%, although JNK1 isoform decreased by 63% in comparison to control cells. Moreover, in H2O2 challenged cells and in TgE-pretreated group the level of pJNK1 expression was higher than pJNK2 one. The

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Fig. 6. Western analysis of the JNK1/2 and p38 MAPK proteins. IEC-6 cells at (4  105 cells/ml) were pre-incubated with 0.25 mg/ml of TgE during 1 hour and then exposed to 40 mM H2O2 for 4 h. After protein extraction, the same blot was incubated with the appropriate antibodies (a). Results shown are typical of 3 independent experiments. (b) Relative intensities of detected bands of JNK1/2, pJNK1/2, p38 and pp38. Values represent averages of three replications  SD. *Statistical significance (p  0.05) in comparison to the control. #Statistical significance (p  0.05) in comparison to H2O2-group.

Table 2 TBARS levels in IEC-6 cells stressed with 40 mM H2O2 during 4 h, and in pretreated ones by 0.25 mg mL1TgE for 1 h. Results are expressed as nmoles MDA equivalents per mg protein. Values represent averages of at least three replications  SD. The symbol * corresponds to the statistical significance (p  0.05) in comparison to the control (CTR).

MDA levels

CTR

H2O2 (40 mM)

TgE + H2O2

15.98  0.8

18.40  0.6*

14.72  0.2

phosphrylation rate of p38 MAPK was not altered in all the groups (Fig. 5). In contrary to H2O2 group, JNK1 expression increased within TgE pretreated cells and reached 88%, while pJNK1 MAPK was clearly down-regulated by 72% with TgE. ROS have been reported to activate both JNK and p38 MAPK pathways or to act only on one MAPK protein [34]. In accordance with our data, shoot extract of T. gallica did not activate p38 MAPK, while leaf and flower extracts enhanced its phosphorylation in Caco2-cells [9]. Exposure to H2O2 leads to the thiol groups oxidation of cysteine residues in protein tyrosine phosphatases (PTPs) rendering them

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susceptible to oxidation and therefore inactivation [34]. Inactivated PTPs increase the level of MAPKs phophorylation resulting in cell death [2]. Subsequent to cell stress-stimulation, pJNK1 isoform is more efficient in phosphorylating c-Jun (which is required for efficient cell cycle progression) rendering it transcriptionally active which leads to cell death. Although, in unstimulated cells, JNK2 preferentially bound to c-Jun, limiting the duration of its signaling [35]. The current data with pretreated IEC-6 cells indicated that TgE act as H2O2 scavenger, to inhibit PTPs phosphorylation and down-regulate the JNK pathway. In agreement with our notion, other studies indicated that JNK pathway down-regulation and CAT normalized activity prevent cell death in several cell models: transformed lung epithelial cells, primary type II lung cells, human arterial endothelial cells and C2 skeletal myoblasts [36,37]. Moreover, T. gallica regulate JNK signaling pathway by its phytochemical compounds. Notably, resveratrol abrogated JNK1/2 phosphorylation in HT22 neuronal cells [7]. In addition, kaempferol inhibited JNK MAPK signaling pathway to attenuate LPS-induced acute lung injury in mice [38]. Additionally, quercitin alleviated the JNK phosphorylation to protect H9c2 cardiomyocytes from hypoxia/reoxygenation [2]. Overall, a tight relationship between the reducing effect T. gallica antioxidants on PTPs, the regulation of CAT and MAPKs can be established. 4. Conclusions In summary, T. gallica may play an important protective role in IEC-6 cells to avoid the detrimental effect of H2O2. The protective action of T. gallica extract may occur by scavenging H2O2, competing CAT, inhibiting lipid peroxidation and avoiding JNK phosphorylation to prevent cell death. Thereby, medicine might benefit from current investigations to prevent some pathologies involving oxidative stress. Funding This work was supported by the Tunisian Ministry of Higher Education and Scientific Research (LR15CBBC06). Competing interest The authors declare that they have no competing interests. References [1] F. Medini, S. Bourgou, K. Lalancette, M. Snoussi, K. Mkadmini, I. Coté, C. Abdelly, J. Legault, R. Ksouri, Phytochemical analysis, antioxidant, anti-inflammatory and anticancer activities of the halophyte Limonium densiflorum extracts on human cell lines and murine macrophages, S. Afr. J. Bot. 99 (2015) 158–164. [2] C. Li, T. Wang, C. Zhang, J. Xuan, C. Su, Y. Wang, Quercetin attenuates apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways, Gene 577 (2) (2015) 275–280. [3] M. Saada, H. Falleh, I. Jalleli, M. Snoussi, R. Ksouri, Phenolic profile, biological activities and fraction analysis of the medicinal halophyte Retama raetam, S. Afr. J. Bot. 94 (2014) 114–121. [4] J. Chen, J. Xu, J. Li, L. Du, T. Chen, P. Liu, S. Peng, M. Wang, H. Song, Epigallocatechin-3-gallate attenuates lipopolysaccharide-induced mastitis in rats via suppressing MAPK mediated inflammatory responses and oxidative stress, Int. Immunopharmacol. 26 (1) (2015) 147–152. [5] S. Salla, R. Sunkara, S. Ogutu, L.T. Walker, M. Verghese, Antioxidant activity of papaya seed extracts against H2O2 induced oxidative stress in HepG2 cells, LWT Food Sci. Technol. 66 (2016) 293–297. [6] R. Ksouri, H. Falleh, W. Megdiche, N. Trabelsi, B. Mhamdi, K. Chaieb, A. Bakrouf, C. Magné, C. Abdelly, Antioxidant and antimicrobial activities of the edible medicinal halophyte Tamarix gallica L. and related polyphenolic constituents, Food Chem. Toxicol. 47 (8) (2009) 2083–2091. [7] M. Fukui, H.J. Choi, B.T. Zhu, Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death, Free Radic. Biol. Med. 49 (5) (2010) 800–813.

497

[8] L. Zhai, P. Zhang, R.Y. Sun, X.Y. Liu, W.G. Liu, X.L. Guo, Cytoprotective effects of CSTMP, a novel stilbene derivative, against H2O2-induced oxidative stress in human endothelial cells, Pharmacol. Rep. 63 (6) (2011) 1469–1480. [9] M. Boulaaba, S. Tsolmon, R. Ksouri, J. Han, K. Kawada, A. Smaoui, C. Abdelly, H. Isoda, Anticancer effect of Tamarix gallica extracts on human colon cancer cells involves Erk1/2 and p38 action on G2/M cell cycle arrest, Cytotechnology 5 (6) (2013) 927–936. [10] M. Boulaaba, M. Snoussi, M. Saada, K. Mkadmini, A. Smaoui, C. Abdelly, R. Ksouri, Antimicrobial activities and phytochemical analysis of Tamarix gallica extracts, Ind. Crops Prod. 76 (2015) 1114–1122. [11] V. Dewanto, X. Wu, K.K. Adom, R.H. Liu, Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity, J. Agric. Food Chem. 50 (2002) 3010–3014. [12] P. Prieto, M. Pineda, M. Aguilar, Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E, Anal. Biochem. 269 (2) (1999) 337–341. [13] T. Hatano, H. Kagawa, T. Yasuhara, T. Okuda, Two new flavonoids and other constituents in licorice root: their relative astringency and radical scavenging effects, Chem. Pharm. Bull. 36 (1988) 2090–2097. [14] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med. 26 (9) (1999) 1231–1237. [15] M. Oyaizu, Studies on products of the browning reaction prepared from glucose amine, Jpn. J. Nutr. 44 (1986) 307–315. [16] M. Gargouri, C. Magné, X. Dauvergne, R. Ksouri, A. El Feki, M.A. Giroux-Metges, H. Talarmin, Cytoprotective and antioxidant effects of the edible halophyte Sarcocornia perennis L. (swampfire) against lead-induced toxicity in renal cells, Ecotoxicol. Environ. Saf. 95 (2013) 44–51. [17] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1–2) (1976) 248–254. [18] H. Aebi, Catalase in vitro, Method Enzymol. 105 (1984) 121–126. [19] K. Asada, S. Kanematsu, Reactivity of thiols with superoxide radicals, Agric. Biol. Chem. 40 (9) (1976) 1891–1892. [20] K. Yagi, A simple fluorometric assay for lipoperoxide in blood plasma, Biochem. Med. 15 (2) (1976) 212–216. [21] N. Trabelsi, W. Megdiche, R. Ksouri, H. Falleh, S. Oueslati, B. Soumaya, H. Hajlaoui, C. Abdelly, Solvent effects on phenolic contents and biological activities of the halophyte Limoniastrum monopetalum leaves, LWT Food Sci. Technol. 43 (4) (2010) 632–639. [22] H. Shoji, S. Oguchi, S. Fujinaga, K. Shinohara, K. Kaneko, T. Shimizu, Y. Yamashiro, Effects of human milk and spermine on hydrogen peroxideinduced oxidative damage in IEC-6 cells, J. Pediatr. Gastroenterol. Nutr. 41 (4) (2005) 460–465. [23] M. Gülden, A. Jess, J. Kammann, E. Maser, H. Seibert, Cytotoxic potency of H2O2 in cell cultures: impact of cell concentration and exposure time, Free Radic. Biol. Med. 49 (8) (2010) 1298–1305. [24] S. Whitehouse, P.L. Chen, A.L. Greenshields, M. Nightingale, D.W. Hoskin, K. Bedard, Resveratrol, piperine and apigenin differ in their NADPH-oxidase inhibitory and reactive oxygen species-scavenging properties, Phytomedicine 23 (12) (2016) 1494–1503. [25] K.Y. Cheah, G.S. Howarth, R. Yazbeck, T.H. Wright, E.J. Whitford, C. Payne, R.N. Butler, S.E. Bastian, Grape seed extract protects IEC-6 cells from chemotherapy-induced cytotoxicity and improves parameters of small intestinal mucositis in rats with experimentally-induced mucositis, Cancer Biol. Ther. 8 (4) (2009) 382–390. [26] L.J. McGaw, E.E. Elgorashi, J.N. Eloff, 8-Cytotoxicity of African medicinal plants against normal animal and human cells, in: V. Kuete (Ed.), Toxicological Survey of African Medicinal Plants, Elsevier, 2014, pp. 181–233. [27] K. Sasaki, S. Bannai, N. Makino, Kinetics of hydrogen peroxide elimination by human umbilical vein endothelial cells in culture, BBA Gen. Subj. 1380 (2) (1998) 275–288. [28] S. Carballal, R. Radi, M.C. Kirk, S. Barnes, B.A. Freeman, B. Alvarez, Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite, Biochemistry 42 (33) (2003) 9906–9914. [29] E. Cemeli, A. Baumgartner, D. Anderson, Antioxidant and the comet assay, Mutat. Res. 68 (2009) 151–167. [30] W. Liao, L. Chen, X. Ma, R. Jiao, X. Li, Y. Wang, Protective effects of kaempferol against reactive oxygen species-induced hemolysis and its antiproliferative activity on human cancer cells, Eur. J. Med. Chem. 114 (2016) 24–32. [31] J.A. Imlay, S. Linn, DNA damage and oxygen radical toxicity, Science 240 (4857) (1988) 1302–1309. [32] M.C. Tsai, T.Y. Song, P.H. Shih, G.C. Yen, Antioxidant properties of watersoluble polysaccharides from Antrodia cinnamomea in submerged culture, Food Chem. 104 (2007) 1115–1122. [33] V. Ugartondo, M. Mitjans, J.L. Torres, M.P. Vinardell, Biobased epicatechin conjugates protect erythrocytes and nontumoral cell lines from H2O2-induced oxidative stress, J. Agric. Food Chem. 57 (10) (2009) 4459–4465. [34] E.A. Veal, A.M. Day, B.A. Morgan, Hydrogen peroxide sensing and signaling, Mol. Cell 26 (1) (2007) 1–14. [35] Z.E. Ronai, JNKing revealed, Mol. Cell 15 (6) (2004) 843–844. [36] Y. Arita, S.H. Harkness, J.A. Kazzaz, H.C. Koo, A. Joseph, J.A. Melendez, J.M. Davis, A. Chander, Y. Li, Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells, Am. J. Physiol. Lung Cell. Mol. Physiol. 290 (5) (2006) 978–986.

498

J. Bettaib et al. / Biomedicine & Pharmacotherapy 89 (2017) 490–498

[37] M. Peleli, I.K. Aggeli, A.N. Matralis, A.P. Kourounakis, I. Beis, C. Gaitanaki, Evaluation of two novel antioxidants with differential effects on curcumininduced apoptosis in C2 skeletal myoblasts: involvement of JNKs, Bioorg. Med. Chem. 23 (3) (2015) 390–400.

[38] X. Chen, X. Yang, T. Liu, M. Guan, X. Feng, W. Dong, X. Chu, J. Liu, X. Tian, X. Ci, H. Li, J. Wei, Y. Deng, X. Deng, G. Chi, Z. Sun, Kaempferol regulates MAPKs and NFkB signaling pathways to attenuate LPS-induced acute lung injury in mice, Int. Immunopharmacol. 14 (2) (2012) 209–216.